Introduction

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among the Asian population [1]. According to World Health Organization, the number of deaths from CVDs in 2004 were 17.1 million and it is predicted that by 2030, about 23.6 million deaths will be reported due to CVDs, mainly from coronary artery disease (CAD) and stroke [2]. Multiple risk factors such as environmental, genetic and clinical risk factors seems to play principle role in predisposition of CADs [3]. The development of CAD is well known to be controlled by smoking, alcohol intake, physical inactivity, diabetes, dyslipidemia, hypertension, obesity, and psychosocial conditions [4–7]. These modifiable risk factors, account for more than 90 % of myocardial infarction (MI) cases and more than 80 % of stroke cases worldwide [8, 9]. Over the past few decades, regional changes in dietary intake and habits have led to a compelling decrease in serum cholesterol levels in North American and western Europe, counterbalanced by an increase everywhere in developing regions in Southeast Asia and the Pacific [10]. Current studies have also proposed that various risk factors conventionally thought as solely environmental, might have a genetic predisposition [11–15]. The interactions of genetic and modifiable risk factors with each other effect cardiovascular risk [16].

The renin-angiotensin system (RAS) is a hormonal signaling mechanism implicated in the regulation of blood pressure and cardiovascular homeostasis. Through modulation of gene expression, growth, fibrosis, and inflammatory response, it also plays an important role in the pathological changes preceding kidney damage [17]. The angiotensin-1–converting enzyme (ACE) gene, consists of 26 exons and spans 21 kb, located on chromosome 17q23. A very common polymorphism in ACE gene was identified by Rigat et al. [18]. The 287–base pair Alu insertion/deletion (I/D) polymorphism in intron 16 is the most studied polymorphism and has been related to ACE levels [19]. Serum ACE concentrations were significantly higher in homozygotes with the “shorter” deletion allele (D/D) than in heterozygotes (I/D) or in homozygotes with the “longer” insertion allele (I/I) [18]. Previous studies demonstrated the association of this polymorphism with susceptibility to hypertension [20], central obesity [21], elevated glucose [22] and hypertriglyceridemia [23]. Therefore, ACE I/D polymorphism might be involved in the pathogenesis of Metabolic syndrome (MetS). However, results have been inconsistent [24–32]. The present study was planned to investigate the association of angiotensin converting enzyme (insertion/deletion) gene polymorphism with CVD in north Indian population.

Research Design and Methodology

Human Subjects

A total of 662 subjects (330 CAD patients and 332 healthy controls) recruited from North Indian population. This sample is a part of our ongoing study, started in 2013 with the objectives to investigate the risk factors associated with coronary artery disease in Asian Indian population. All the subjects were originated from similar geographical locations. The diagnosis of the occurrence of CAD was performed by cardiologists, based on the clinical symptoms, characteristic ECG changes, cardiac enzyme levels, and the findings in coronary angiography and/or echocardiography. Informed written consent was obtained from all the study participants. The study was approved by the ethical committee of the Post Graduate Institute of Medical Education and Research, Chandigarh, India.

Clinical and Biochemical Measurements

Standard anthropometric measurements were performed including height, weight, waist and hip circumferences and blood pressure. Body Mass Index (BMI) was calculated as [weight (kg)/height (meter)2] and Waist Hip Ratio (WHR) as ratio of waist to hip circumferences. According to WHO Expert Consultation 2004, the following BMIs have been proposed to indicate risk of developing weight-related diseases in Asian populations: <23 kg/m2 for low risk, 23–27.5 kg/m2 for increased risk, and >27.5 kg/m2 for high risk. Abdominal obesity was measured according to the new cutoffs proposed for South Asian Indians (WHR >0.89 for men and WHR >0.81 for women).

Blood samples were drawn in EDTA coated and plain vials. All the biochemical measurements such as Glucose, Total Cholesterol (TC), Triglycerides (TG), High Density Lipoprotein (HDL) and Creatinine levels were done in serum using standard kits. Body fat percentage was calculated in all the subjects using a previous method [33].

Amplification of Insertion/Deletion Polymorphism of ACE Gene

The genomic DNA was extracted from peripheral blood leukocytes using phenol/chloroform method. PCR amplification of ACE (I/D) polymorphism was done using the following primer sequences. Forward primer: 5′-CTGGAGACCACTCCCATCCTTTCT-3′ and reverse primer: 5′-GATGTGGCCATCACATTCGTCAGAT-3′. The PCR was carried out in a final volume of 25 µl containing 1xPCR buffer, 100–200 ng of genomic DNA, 1.5 mmol/l MgCl2, 0.2 mmol/l of each dNTP (Eppendorf, Germany), 250 pmol of each primer (IDT, USA), 1.25 units Taq DNA polymerase (NEB, Boverly, MA) and 5 % of DMSO to avoid mistyping. The cycling conditions were as follows: an initial denaturation at 94 °C for 5 min, followed by 30 cycles of annealing at 55 °C for 1 min, extension at 72 °C for 1.5 min, denaturation at 94 °C for 1 min, and final extension at 72 °C for 10 min. PCR amplified products were then separated on 2 % agarose-gel containing ethidium bromide (Metaphor agarose, FMC Bioproducts, Rockland, ME). The wild type homozygote, II yielded only the 490 bp fragment and mutant type homozygote, DD yielded only the 190 bp fragment. PCR product from a heterozygote yielded both the 190 and 490 bp fragments. The assays were performed in blind to the phenotype. Mistyping of ID heterozygotes as D homozygotes may occur due to the preferential amplification of the D allele and inefficiency in the amplification of the I allele [34]. To increase the specificity of DD genotyping, PCR amplifications were also performed with an insertion specific primer pair (5′-TGGGACCACAGCGCCCGCCACTAC-3′ and 5′-TCGCCAGCCCTCCCATGCCCATAA-3′) in all samples with DD genotype. Briefly, insertion-specific amplification was carried out in a final volume of 25 µl containing 1xPCR buffer, 100–200 ng of genomic DNA, 1.5 mmol/l MgCl2, 0.2 mmol/l of each dNTP, 250 pmol of each primer, 1.0 units Taq DNA polymerase and 5 % of DMSO. The cycling conditions were as follows: an initial denaturation at 94 °C for 5 min, followed by 30 cycles of annealing at 55 °C for 1 min, extension at 72 °C for 1.5 min, denaturation at 94 °C for 1 min, and final extension at 72 °C for 10 min. PCR amplified products were then separated on 2 % agarose-gel containing ethidium bromide. Only “I” allele produced a 335 bp fragment. The reaction yields no products in samples with DD genotype.

Statistical Analysis

Results were expressed as mean ± SD. Chi square analysis was applied to test the significance of differences in genotypic and allelic frequencies. Comparisons between CAD patients and control subjects were done using Chi square tests, and group comparisons were done using unpaired t tests. One-way ANOVA test was used for comparison of normally distributed variables among genotypes. All the p values <0.05 (two-tailed) were considered as significant difference. Logistic regression analysis was carried out to correlate various clinical parameters with genotypes. Statistical analysis was performed using the Statistical Package of Social Sciences (SPSS) for Windows, version 16.0 (SPSS, Inc., Chicago, IL).

Results

Baseline Characteristics of the Study Subjects

A total of 662 subjects (330 CAD patients and 332 healthy controls) were examined in the present case control study. The baseline characteristics of all the participants are summarized in Table 1. The mean age of the CAD patients and control subjects was 60.53 ± 8.6 years and 56.55 ± 7.7 years, respectively (p = 0.000). All the CAD patients were regularly taking lipid lowering drugs under the guidance of cardiologist. The mean BMI values was significantly higher among CAD patients and control subjects (26.98 ± 4.9 vs 24.04 ± 4.7, p = 0.000). CAD patients and control subjects did not significant difference in abdominal adiposity as reflected by their waist circumference and WHR (Table 1). Following stratification of the data based on the BMI cutoffs by WHO Expert Consultation (2004) for Asians, we observed pronounced central obesity (measured by WHR) in both CAD patients and controls, even at the lowest BMI values (<23 kg/m2). No significant difference in blood pressure (systolic and diastolic) was observed in CAD patients and control subjects. The biochemical parameters are shown in Table 2. There were significantly higher levels of total cholesterol, triglycerides, LDL and VLDL were observed in control subjects compared to CAD patients. However, no significant difference was observed in HDL and creatinine level among the CAD patients and controls (Table 2).

Table 1 Anthropometric measurements of the study subjects stratified by sex and disease status
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Table 2 Clinical parameters of the study subjects stratified by sex and disease status
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Genotype Distribution and Allele Frequencies

The distribution of genotypes and allele frequencies of insertion/deletion polymorphism of ACE gene are shown in Table 3. Both patients and control populations were in Hardy–Weinberg equilibrium. Figure 1 shows the II, ID and DD genotypes of ACE gene polymorphism. The distribution of II genotype was higher in control subjects than the CAD patients (32.5 vs 25.5 %). Significantly high frequency of DD genotype (40 %) was observed in CAD patients compared to control subjects (28.3 %). The frequency of D allele was significantly higher in CAD subjects (p = 0.001). No significant difference was observed in the distribution of ID genotypes between CAD patients and control subjects. Logistic regression analysis of data demonstrate that DD genotype was associated with 1.8 fold increased risk of development of CAD in north Indians (OR 1.8; 95 % CI 1.22–2.66; p = 0.003). The clinical characteristics of the CAD patients and control subjects according to the genotypes of ACE (I/D) polymorphism are shown in Table 4. Although significant differences were observed in various metabolic characters in CAD patients and control subjects as shown in Tables 1 and 2, but no significant differences were observed amongst all the genotypes (II, ID and DD) of ACE gene in both CAD patients and control subjects and metabolic traits in our population when the data was stratified according to the genotypes of ACE gene.

Table 3 Genotype and allele frequencies of insertion/deletion polymorphism of ACE gene
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Fig. 1
figure 1

Agarose gel electrophoresis of PCR amplified product of ACE (I/D) gene polymorphism. M represents 100 bp DNA ladder; Lanes 8, 9, 13 and 14 as II genotype (490 bp); Lanes 1–4 and 11 as DD genotype (190 bp) and Lanes 5, 6, 7, 10, 12 and 15 as ID genotypes (490 and 190 bp) of ACE gene

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Table 4 Clinical characteristics of CAD patients and control subjects stratified by genotypes of ACE (I/D) polymorphism
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Discussion

The incidence and prevalence of CAD is rising in developing countries. It is the single major cause of mortality in the developed countries and a leading cause of death and disability in developing countries [35]. It is a multifactorial disorder in which both genetic and environmental factors play a pivotal role in its pathogenesis [36]. Conventional risk factors such as hypertension and dyslipidemia are associated with the risk of CAD in Asian Indians [37]. The present case–control study was undertaken to establish the risk factors associated with CAD in an urban population of North India. Asian-Indians have been identified as one of the ethnic groups with a high prevalence of cardiovascular diseases. The findings described in this paper confirm and extend our knowledge of the dynamics of the present epidemic of CVD in North Indians [38]. The improved socioeconomic conditions in India have resulted in a decrease in physical activity and an increase in obesity, which has led to the increase in the prevalence of CVD and T2DM in urban Indians [16]. The present study subjects depict an unusual clinical picture of uneven distribution of adiposity and dyslipidemia in CAD patients as well as control subjects. The mean age of onset of CAD in north Indians was a decade earlier than the Caucasians but is consistent with the age of onset in other Asian populations.

Abdominal obesity can be considered one of the key factors for causing insulin resistance and progression to the development of CAD, T2DM and its complications [38, 39]. Our data showed a strong tendency toward upper body adiposity (waist circumference and WHR), even in control groups. Compared to western population, North Indian diabetic subjects were leaner and had relatively lower BMI. Body fat percentage at a given BMI is comparatively higher in Asian Indians. It has been suggested that thrifty genes that provided a survival advantage in previous eras now result in central obesity and T2DM and CAD in populations living in the rapidly modernizing environment of India. Dyslipidemia is not very much prominent in our study population because most of the CAD patients were taking lipid lowering drugs in routine. Therefore, the upper body adiposity along with physical inactivity and abnormalities in the HDL-C level may induce insulin resistance in diabetic subjects, which in turn results in increased prevalence of CAD, T2DM and other complications in North Indians.

In the present case–control study, ACE (I/D) gene polymorphism is significantly associated with the risk of CAD in Asian Indian population. Our findings were in line with the findings of previous studies carried out in different populations. [40–43]. The ethnic background seems to influence the allele frequencies in ACE (I/D) gene polymorphism worldwide. However, some epidemiological studies did not show association of ACE (I/D) gene polymorphism with the risk of CAD [44, 45]. Some studies have shown the association of ACE (I/D) gene polymorphism with CVD in type 2 diabetic subjects [41–43, 46, 47], while the association of the I/D polymorphism with CVD has not been confirmed in other studies [48, 49]. This polymorphism also affects the level of circulating ACE, but there is great individual variation, even between those with the same genotype [44]. The CAD subjects carrying DD genotype have higher levels of ACE, relative to II individuals, while ID subjects have intermediate ACE levels. Apart from CAD, ACE (I/D) gene polymorphism was found to be associated with Type 2 diabetes and its complications [50–58]. ACE (I/D) is also a risk factor for hypertension [52]. The ethnic background appears to influence the ACE (I/D) gene polymorphism globally. It demonstrates the importance of using a homogeneous population in the selection of the study samples, making possible the identification of more exact distributions of the ACE genotypes among racial populations. In conclusion, our data suggested that the DD genotype of ACE gene might be a significant risk factor for the development of CAD in Asian Indian population.