Abstract

Background: The angiotensin converting enzyme insertion-deletion (ACE I/D) polymorphism located on chromosome 17q23 (287 bp in intron 16) is associated with cardiovascular risk factors (CRFs), but results vary among populations, which is thought to be the cause of ethnic differences. This study explored the role of the ACE I/D polymorphism and its correlation with CRF determinants among urban and rural groups.

Methods: A total of 182 male and female participants were recruited in the age range of 20 – 55 years for CRF determinant examination and ACE gene polymorphism (n = 140).

Results: Most samples examined for polymorphic ACE genes showed increased CRF determinants in the two groups. For genotype II and urban group ID, the risk was increased 5 – 8 times for the CRF of obesity. The frequency of genotype II significantly increased the incidence of CRFs of smoking and sedentary by 1 – 3 times in both groups.

Conclusions: The ACE I/D polymorphism has a differential effect on both urban and rural groups. Smoking, sedentary behavior, and obesity were risk factors for CRF in both groups. Therefore, an overall design strategy for health policies is needed to mitigate the burden of cardiovascular disease, which ends in death in both groups.

Introduction

The current public health challenge is the prevention and control of cardiovascular risk factors (CRFs)1. Coronary heart disease, often known as coronary artery disease (CAD), contributes to 31% of deaths worldwide. The trend of disease is increasing throughout Southeast Asia2. Coronary heart disease attacks people aged 3. A paradigm shift in lifestyle increases the prevalence of this disease. Various lifestyle changes include unbalanced diets, excessive carbohydrate consumption, progressive economic growth, and urbanization4. Based on population analysis, it has been reported that on the Asian continent, including in Indonesia, this disease is increasing exponentially in the population, especially in vulnerable urban groups5.

The migration of several rural to urban community groups occurs due to employment factors that have effects on society and health. This is certainly detrimental to the population, including the potential to increase the incidence of CRF determinants6. One challenge that is very serious in developing countries, especially in Indonesia, is urban health issues, including CAD and CRF7.

One way CAD is affected in urban communities is via extreme lifestyle changes, including lack of physical activity, smoking, and unhealthy eating habits8.

Previous studies have reported the involvement of genes in various populations, particularly in urban and rural populations, but other studies have shown that urban groups are correlated with CRFs in contrast to rural groups9.

Other studies have focused on ethnicity and ethnic groups and their CRFs, such as nutritional deficits, obesity, and hypertension (HTN). Furthermore, Pena et al. (2016) studied the complex correlation between genes and the environment triggered by CRFs10.

Thus, it is important to trace the role of genetics in different urban and rural populations, especially gene polymorphisms of populations.

The angiotensin converting enzyme (ACE) gene, which is located on chromosome 17q23, contains many polymorphisms that are collections of different phenotypes composed of DNA strands. The insertion‒deletion (I/D) polymorphism consists of 287 bp in intron 1611. The ACE I/D polymorphism is one of the main enzymes that control the renin–angiotensin system (RAS) pathway. Some researchers have reported that this polymorphism is associated with several diseases, especially non-communicable and generative diseases, such as metabolic syndrome, HTN, dyslipidemia, diabetes, obesity, and hypertriglyceridemia12.

The population distribution among urban and rural populations has shown that they have the same risk of developing CRF diseases. However, no reports have compared the two populations. Thus, it is important to provide an overview of the population distribution for those at risk of CAD and CRFs and the role of genetics among urban and rural populations, especially gene polymorphisms in South Sulawesi populations.

Methods Study design

A population-based design with a cross-sectional study approach was used in this study. Data were collected via a purposive random sampling method. The samples were from urban and rural community groups.

Sample size

In total, 182 participants were successfully recruited in the age range of 20 – 50 years and included both males and females. Of these, 42 subjects were excluded from the study because they refused to provide consent to participate in the study, even though the informed consent form had been explained in detail. The ACE I/D polymorphism was examined and identified, and samples were separated according to population location (n = 60 for the rural group and n = 80 for the urban group). CRF determinant tests included smoking, sedentary, obesity, HTN, abnormal heart rate, and poor VO2max capacity (n = 140) for both groups. We prepared anamnesis schedules and initial examinations to obtain basic participant data, including each participant’s name, age, gender, medical history, and occupation. Three milliliters of blood were collected using a standard biomedical laboratory protocol from the biomolecular Hasanuddin University - Medical Research Center (HUM-RC). All materials and tools used in this study were approved by the Health Ethics Committee of the Makassar Health Polytechnic.

Anthropometric measurement and CRF determinants

Anthropometric measurements included age (years), height (cm), weight (kg), body mass index (BMI = BB/TB2) in kg/m2, systolic blood pressure (sbp) in mmHg, diastolic blood pressure in mmHg, heart rate, and VO2max. Specific standardization of BMI refers to Southeast Asian populations according to WHO standards (2000)13. Blood pressure (BP) measurements were taken on the left arm of participants while they laid on the examination room bed using a BP digital instrument (OMROM-7080), with standardization of measurements according to JNC VII guidelines. Measurements were performed at one time to minimize measurement errors14.

Polymorphism and genotyping

DNA was extracted according to the Rigat et al. (1990) protocol, and we performed allele-specific polymerase chain reaction (PCR) of ACE I/D polymorphism using specific primer sequences via standardpro kol from Rigat et al., 199015.

Forward primer: 5′-CTGGAGACCACTCCCATCCTTTCT-3′ and

Reverse primer: 5′-GATGTGGCCATCACATTCGTCAGAT-3′

DNA was amplified for 30 cycles with denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 2 min using an Applied Biosystems thermal cycler. Genotyping of the PCR products [in base pairs (bp): DD (190 bp), ID (490 bp, 190 bp), and II (490 bp)] was performed via 2% agarose gel electrophoresis. As per Perna et al. 1992 and Shanmugam et al. 1993, there is a possibility of ID genotypes being mistyped as DD. To avoid this, 51 samples with DD genotypes were reanalyzed using the protocol suggested by Shanmugam et al. 1993 in which 5% dimethyl sulfoxide (DMSO) was added to the PCR mixture16.

Statistical analysis

Using SPSS software application version 22.0, we conducted statistical testing using the chi-square (X2) test to identify general characteristics of the population and CRF among the urban and rural groups. We also used the chi-square test to determine the differences in genotype distributions according to the CRF determinants. Meanwhile, Hardy–Weinberg equilibrium (HWE) was carried out to observe the frequency distribution of ACE I/D polymorphism genotypes among the two population groups. Furthermore, the variables, including age, sex, education, occupation, smoking, and alcohol consumption, were adjusted to understand the role of the ACE I/D polymorphism in the CRF determinants using logistic regression and interaction tests (moderated regression analysis), with a significance of p

Table 1.

General characteristics of the population in urban and rural settings

General characteristics Urban Rural χ2 P Age (years) 39.04 ± 9.5 38.78 ± 9.4 .025 (.875) Sex Female 67 (83.75) 30 (50) 20.819 (.0001) Male 13 (16.25) 30 (50) Education Illiterate 56 (72.73) 32 (53.33) 20.819 (.012) literate 21 (27.27) 28 (46.67) Occupation Sedentary 52 (65) 25 (41.67) 7.858 (.006) Active 28 (35) 35 (58.33) Alkohol No 67 (83.75) 50 (83.33) .004 (.948) Yes 13 (16.25) 10 (16.67) Smoking No 53 (66.25) 51 (85) 6.514 (.012) Yes 27 (33.75) 9 (15)

Table 2.

Distribution of cardiovascular risk factors among urban and rural settings