Association of the insertion allele of the common ACE gene polymorphism with type 2 diabetes mellitus among Kuwaiti cardiovascular disease patients

Abstract

Background and objectives:

The D allele of the common angiotensin-converting enzyme (ACE) I/D gene polymorphism (rs4646994) predisposes to type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). However, results on which allele predisposes to disease susceptibility remain controversial in Asian populations. This study was performed to evaluate the association of the common ACE I/D gene polymorphism with both T2DM and CVD susceptibility in an Arab population.

Methods:

We genotyped the ACE I/D polymorphisms by direct allele-specific PCR in 183 healthy controls and 400 CVD patients with diabetes (n=204) and without (n=196). Statistical analysis comparing between the different groups were conducted using R statistic package “SNPassoc”.

Results:

Two genetic models were used: the additive and co-dominant models. The I allele was found to be associated with T2DM (OR=1.84, p=0.00009) after adjusting for age, sex and body mass index. However, there was no association with CVD susceptibility (p>0.05).

Conclusion:

The ACE I allele is found to be associated with T2DM; however, no association was observed with CVD. The inconsistency between studies is suggested to be attributed to genetic diversity due to the existence of sub-populations found in Asian populations.

Keywords

diabetes T2DM cardiovascular disease CVD Kuwait genetic polymorphism

Introduction

Cardiovascular disease (CVD) is one of the leading causes of mortality worldwide. The prevalence is 40% in groups aged 40–59 years old according to a recent report from the American Heart Association.¹ Type 2 diabetes mellitus (T2DM) is a common risk factor of CVD, with studies showing the risk of CVD higher in patients with T2DM compared with those without.² It has also been reported that about 50% of acute myocardial infarction (AMI) patients have diabetes, with the risk of death of AMI with diabetes 3-fold higher compared with AMI without diabetes.³ The prevalence of T2DM has increased 30% between 2001 to 2009,¹ and according to the International Diabetes Federation, Kuwait is ranked third among the top 10 countries with highest diabetes prevalence worldwide.² This is alarming, especially considering that the prevalence of obesity, another well-known risk factor for CVD, is 48% in the Kuwaiti population.⁴

ACE is involved in converting angiotensin I into angiotensin II, a potent vasoconstrictor.^5,6 The insertion/deletion (I/D) polymorphism (rs4646994) of the angiotensin-converting enzyme gene (ACE) is a 287-bp ALU repeat DNA sequence found on intron 16.⁷ The DD and ID genotypes have been associated with 60% and 30% higher ACE plasma levels compared with the II genotype.^7,8 The I/D polymorphism has been found to be associated with increased risk of CVD⁹ and T2DM;¹⁰ however, results are still inconsistent, with studies finding no association¹¹ or an inverse association.^12,13 This observed inconsistency seems to be attributed to gene–environment interactions and population differences. Therefore we aimed to investigate the association between the ACE I/D (rs4646994) gene polymorphism and the risk of both CVD and T2DM in patients from Kuwait.

Materials and methods

Study subjects

A total of 583 Kuwaiti nationals including both patients (n=400) and controls (n=183) were recruited for this study; they were asked to sign an informed consent for the purpose of the study. The samples were collected from various hospitals in Kuwait during the period February to April 2010. Relevant phenotypic data was recorded and documented for each sample (Table 1). This study has been approved by the Local Ethical Committee at Kuwait University as well as the Ministry of Health Ethical Board Kuwait following the Declaration of Helsinki guidelines.

Table 1.

Demographic characteristics and distribution of risk factors in CVD patients and healthy controls.

Variable	Controls (n=183)	Cases (n=400)	p-value
Sex (females)	54(29.1%)	137(34.3%)	0.255
Age (years)	49.94±12.88	55.25±3.07	<0.0001
BMI (kg/m²)	28.67±6.45	29.58±6.68	0.123
Cholesterol (mmol/l)	4.76±0.89	4.47±1.43	0.012
Triglyceride (mmol/l)	1.38±0.77	2.01±1.68	<0.0001
VLDL (mmol/l)	0.57±0.31	0.91±0.76	<0.0001
HDL-C (mmol/l)	0.93±0.3	1.12±0.36	<0.0001
LDL-C (mmol/l)	3.25±0.92	2.87±0.87	<0.0001
Diabetes (yes)	0	204(51%)	<0.0001
Hypertension (yes)	0	218(54%)	<0.0001

Data are represented as mean ± SD or percentage. BMI: body mass index; VLDL: very low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein.

Clinical diagnosis and blood biochemical analysis

The CVD patient samples (n=400) were recruited mainly from the Chest Hospital, Al-Amiri Hospital and Mubarak Hospitals. The mean age of patients was 55 years old, including 263 males and 137 females. Clinical diagnosis was provided from the medical profile according to the presence of typical chest pain, echocardiogram and previous history of myocardial infarction, coronary angioplasty or percutaneous transluminal coronary angioplasty and coronary artery bypass graft. The CVD patient samples were further divided into two groups: with confirmed T2DM (n=204), based on the criteria suggested by American Diabetes Association,¹⁴ and those without T2DM (n=196). The control group included a randomized sample of Kuwaiti nationals who are confirmed to be free of CVD based on angiography indicating a normal coronary artery with no evidence of plaque, smooth and no evidence of peripheral vascular disease, and medical records.

A peripheral venous whole-blood sample (10 ml) was collected from all the subjects under complete aseptic conditions by a certified hospital laboratory staff following a minimum of 12 hours of fasting. Quantitative measurements of blood plasma total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were determined in 3 ml of blood by enzymatic methods with commercially available kits (Synchron System Reagents, Beckman Coulter, USA) analyzed on a UniCel DxC 800 Synchron Clinical Systems (Beckman Coulter, USA) in the clinical chemistry laboratories at the relevant hospital, Ministry of Health. The normal values of serum TC, TG, HDL-C, LDL-C and VLDL used as reference were 3.0–5.2, 0.40–2.50, 0.91–1.81, 1.8–3.3 mmol/l, 0.0–40 mmol/l, respectively. The lipid profile for patients who were under medication and treatment was not provided, since their lipid profiles were post treatment and hence were of normal values; however, the medical history of dyslipidemic patients was recorded and, if available from the medical records, old profiles were used.

Genotyping

Total genomic DNA was extracted from 7 ml of whole blood by the salting-out method as previously described by Miller et al.¹⁵ The final volume of the extracted DNA was 1 ml, to yield a final concentration of 10 ng/1 µl. All extracted DNA was analyzed prior to genotyping using a nanodrop 1000 spectrophotometer (ND 1000, Thermo Fisher Scientific Inc., Wilmington, Delaware, USA).

Genotyping of the ACE I/D gene polymorphism (rs4646994) was achieved by amplification of the target sequence by the polymerase chain reaction (PCR).¹⁶ Two specific primers flanking the polymorphic site in intron 16 of the ACE gene were used (forward: 5’-GATGTGGCCATCACATTCGTCAGAT-3’ and reverse: 5’-CTGGAGACCACTCCCATCCTTTCT-3’). The expected PCR products of 490 bp and 190 bp correspond to the I and D alleles, respectively. The D allele has been reported to have a higher chance for amplification than the I allele, rendering potential mistyping of ID heterozygous, and in some cases its suppression yielding mistyping as DD.¹⁷ Although the mechanism for this suppression is not fully understood, the mistyping is considered a serious concern. Therefore, all heterozygous ID and homozygous DD were re-genotyped by a second round of amplification using an insertion-specific primer pair (forward: 5’-TGGGACCACAGCGCCCGCCACTA C-3’ and reverse: 5’-TCGCCAGCCCTCCCATGC CCATAA-3’) that recognizes the inserted sequence.¹⁸ The amplified PCR products were analyzed on a 1.2% agarose gel (routine use-Sigma; A9539, USA). A 50 bp DNA ladder was used to estimate the size of the PCR products. Electrophoresis was carried out at a constant voltage of 200 V with 250 mA for about 2 h, or until the dye reached the edge of the gel.

Statistical analysis

Statistical analysis was performed using SPSS software (version 22; SPSS Inc, Chicago, IL, USA). Results were expressed as mean ± SEM and percentages where appropriate. Logarithmic transformation was applied where appropriate. Hardy–Weinberg Equilibrium (HWE) was tested using the web-based calculator available at www.tufts.edu/, which confirmed the population to be in equilibrium. The genetic association was analyzed and controlled for age, sex and body mass index (BMI) using the SNPassoc package from R software.¹⁹ The results are expressed as odds ratio (OR) with 95% confidence intervals (CI). A power > 80% at alpha = 0.05 was achieved in this study assuming an average OR of 2.5 and minor allele frequency of 11% using the statistical software package StatCalc (version 7.1.2.0; Epi Info^TM, Atlanta GA, USA).

Results

The clinical and demographic characteristics of controls and CVD patients are reported in Table 1. Among the 400 patients with CVD, 204 were classified as CVD with T2DM and 196 CVD without T2DM. The mean age of CVD patients was higher than that in controls and therefore was controlled for in all statistical analysis along with BMI and sex. The frequency of genotypes were found in HWE in the control samples (p=0.78) whereas it deviated in the CVD samples (p=0.007). The minor allele (I) frequency in controls was 31% compared with 42% in CVD.

A positive association of the ACE I allele with CVD was observed (Table 2). Association of the I allele was observed in all genetic models as well with p<0.01 after adjusting for BMI, age and sex (Table 2). The frequency of the II genotype was higher in CVD patients compared with controls (20.2% compared with 10.9%, respectively) and with an OR 1.5 (95% CI 1.16–1.94; p=0.001) in the additive model (Table 2). However, there was no association observed after controlling for T2DM (p>0.05). The results remained significant in all genetic models after controlling for hypertension (p<0.05).

Table 2.

Genotypic distribution of the ACE polymorphism and statistic comparison between controls and CVD patients.

SNP	Model	Controls	CVD	OR (95% CI)	p-value
ACE-rs4646994	Co-dominant				0.007
	DD	90 (49.2%)	151 (37.8%)	1
	ID	73 (39.9%)	168 (42%)	1.42 (0.96–2.09)
	II	20 (10.9%)	81 (20.2%)	2.33 (1.33–4.11)
	Dominant				0.009
	DD	90 (49.2%)	151 (37.8%)	1
	ID+II	93 (50.8%)	249 (62.2%)	1.62 (1.13–2.32)
	Recessive				0.009
	DD+ID	163 (89.1%)	319 (79.8%)	1
	II	20 (10.9%)	81 (20.2%)	1.97 (1.15–3.36)
	Additive	183 (30.4%)	400 (68.6%)	1.5 (1.16–1.94)	0.001

CVD: cardiovascular disease; OR: odds ratio; CI: confidence interval.

Association was diminished after controlling for T2DM, and therefore we compared the association of the ACE rs4646994 polymorphism between controls and CVD patients with T2DM (Table 3). The I allele was found to be associated with CVD in patients with T2DM in all genetic models (Table 3). The frequency of the II genotype was higher in the patients group compared with controls (24% compared with 10.9%, respectively) with an OR of 2.39 (95% CI 1.3–4.39; p=0.00009) in the additive model (Table 3). The lack of association between controls and CVD patients without T2DM is summarized in Table 4 in which no association was observed in all studied genetic models (p>0.05) (Table 4). Although the frequency of II genotype was higher in CVD patients without T2DM compared with controls (16.3% compared with 10.9%, respectively), results failed to reach significance (p>0.05).

Table 3.

Genotypic distribution of the ACE polymorphism and statistic comparison between controls and CVD patients with diabetes.

SNP	Model	Controls	CVD patients with T2DM	OR (95% CI)	p-value
ACE-rs4646994	Co-dominant				0.0004
	DD	90 (49.2%)	65 (31.9%)	1
	ID	73 (39.9%)	90 (44.1%)	1.9 (1.17–3.07)
	II	20 (10.9%)	49 (24%)	3.31 (1.71–6.41)
	Dominant				0.0004
	DD	90 (49.2%)	65 (31.9%)	1
	ID+II	93 (50.8%)	139 (68.1%)	2.22 (1.41–3.47)
	Recessive				0.004
	DD+ID	163 (89.1%)	155 (76%)	1
	II	20 (10.9%)	49 (24%)	2.39 (1.3–4.39)
	Additive	183 (47.3%)	204 (52.7.6%)	1.84 (1.34–2.51)	0.00009

CVD: cardiovascular disease; T2D: type 2 diabetes; OR: odds ratio; CI: confidence interval.

Table 4.

Genotypic distribution of the ACE polymorphism and statistic comparison between controls and CVD patients without diabetes.

SNP	Model	Controls	CVD patients without T2D	OR (95% CI)	p-value
ACE-rs4646994	Co-dominant				0.26
	DD	90 (49.2%)	86 (43.9%)	1
	ID	73 (39.9%)	78 (39.8%)	1.14 (0.74–1.77)
	II	20 (10.9%)	32 (16.3%)	1.69 (0.89–3.19)
	Dominant				0.26
	DD	90 (49.2%)	86 (43.9%)	1
	ID+II	93 (50.8%)	110 (56.1%)	1.26 (0.84–1.90)
	Recessive				0.13
	DD+ID	163 (89.1%)	164 (83.7%)	1
	II	20 (10.9%)	32 (16.3%)	1.58 (0.87–2.89)
	Additive	183 (48.3%)	196 (51.7%)	1.25 (0.94–1.68)	0.12

CVD: cardiovascular disease; T2D: type 2 diabetes; OR: odds ratio; CI: confidence interval.

Furthermore we compared the distribution of the ACE rs4646994 polymorphism among CVD patients with and without T2DM (Table 5). The association between the ACE I allele and CVD was found associated with CVD patients with T2DM compared with CVD without T2DM in both the dominant and additive genetic models (p>0.05) (Table 5). The frequency of the ACE II genotype was higher in CVD patients with T2DM compared with those without T2DM (24% compared with 16.3%, respectively) and with an OR of 1.39 (95% CI 1.05–1.85; p=0.02) in the additive model (Table 5).

Table 5.

Genotypic distribution of the ACE polymorphism and statistic comparison between CVD without diabetes and CVD with diabetes.

SNP	Model	CVD without T2D	CVD patients with T2D	OR (95% CI)	p-value
ACE-rs4646994	Co-dominant				0.06
	DD	86 (43.9%)	65 (31.9%)	1
	ID	78 (39.8%)	90 (44.1%)	1.52 (0.96–2.40)
	II	32 (16.3%)	49 (24%)	1.89 (1.06–3.36)
	Dominant				0.02
	DD	86 (43.9%)	65 (31.9%)	1
	ID+II	110 (56.1%)	139 (68.1%)	1.62 (1.06–2.49)
	Recessive				0.11
	DD+ID	164 (83.8%)	155 (76%)	1
	II	32 (16.2%)	49 (24%)	1.52 (0.90–2.56)
	Additive	196 (49%)	204 (51%)	1.39 (1.05–1.85)	0.02

CVD: cardiovascular disease; T2D: type 2 diabetes; OR: odds ratio; CI: confidence interval.

Discussion

We observed a positive association between the ACE I allele and T2DM. The observed association between the ACE I allele and CVD was diminished once controlled for T2DM. Our findings support that the initial association observed with CVD is mediated through the presence of T2DM. This was confirmed when a significant association of the ACE I allele in CVD patients with T2DM compared with those without T2DM was detected. Recent findings have shown the association between the ACE D allele and risk of developing CVD and T2DM.^20–23 On the other hand, a lack of association between the ACE I/D polymorphism with CVD and T2DM has also been reported.^11,24 However, an association of the ACE I allele conferring an increase in disease risk is rarely observed, as in the case of this study. A recent meta-analysis on Asian populations demonstrated the D allele and the II genotype was associated with the risk of T2DM patients developing T2DM nephropathy (T2DN).¹³ Another study, by Vijayan et al., demonstrated an association of the ACE I allele with an increased risk of ischemic stroke among males in south India.¹² However, a meta-analysis on Arab populations demonstrated the D allele to be associated with T2DM.²³ Inconsistency can be attributed to the heterogeneity among these studies, and therefore differences in ethnicity and study design are the most likely explanation according to various authors.^12,13 In the current study all samples were of Kuwaiti nationality, suggesting homogeneity. However, three genetic subgroups have been previously defined in the Kuwaiti population, and therefore future studies may need to be stratified accordingly.^25,26

Our association of the ACE I allele with T2DM can be explained by understanding the complexity of the rennin–angiotensin–aldosterone system (RAAS) as reviewed by Rahimi et al.²⁷ Angiotensin II is converted by ACE, resulting in aldosterone secretion and elevated blood pressure.²⁷ A study using intravenous infusion of angiotensin II showed an enhanced blood flow resulting in insulin sensitivity.²⁸ This is consistent with previous findings showing that homozygous carriers of the I allele of the ACE gene have greater insulin resistance compared with carriers of the DD genotype.^29–31 A study by Katsuya et al. demonstrated that subjects with the DD genotype were more insulin sensitive.³² Another study demonstrated that DD and ID genotypes were associated with lower insulin resistance compared with II genotypes,³¹ and that doses of angiotensin II increase insulin sensitivity in healthy subjects.³³ The DD and ID genotypes show 60% and 30% higher ACE plasma levels compared with the II genotype, supporting the association with insulin resistance.^7,8 Such findings suggest a potential risk to T2DM in subjects carrying the I allele of the ACE gene, which is consistent with our findings. Due to the inconsistency in findings and complexity of the RAAS it is possible that gene–gene interactions play a major role within this system,³⁴ and therefore it would be useful to investigate the interactions of other genetic markers in the RAAS in patients with T2DM.

The inconsistency in association studies observed with the ACE I/D polymorphism may also be attributed to epigenetic mechanisms, which are alterations in gene expression due several modifications that include DNA methylation, histone modifications and RNA-based mechanisms that are not caused by changes in DNA sequences.^35,36 A recent study by Rangel et al. demonstrated an interaction between DNA methylation and the ACE I/D polymorphism.³⁷ The DD genotype was found to be associated with a decrease in DNA methylation, which was associated with higher ACE activity.³⁷ Environmental factors such as nutrition and physical activity have been associated with modifying a gene’s epigenetic status.^38,39 Differences in nutrition and levels of physical activity differ between populations. Therefore such interaction between epigenetic modifications and polymorphisms describes the complexity of the genetic architecture which can underlie the inconsistency in association studies observed with the ACE I/D polymorphism in different populations.

Conclusion

In conclusion, the present study is the first to find an association of the ACE I allele and T2DM in an Arab population. More association studies with a stratified population are required to confirm this finding. Our results suggest that the inconsistency observed with the ACE I/D polymorphism is highly dependent on the population being studied and epigenetic mechanisms. The limitation of our study is the absence of measurement of ACE plasma level and its correlation with the ACE genotype.

Footnotes

Acknowledgements

The authors extend their deepest appreciation and gratitude to all the participants in this study, Mrs. Babitha Annice for her technical support, and Al-Sabah Chest Hospital, Al-Amiri and Mubarak Hospitals in Kuwait for their assistance with the blood collection and lipid profile analysis.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was partially supported and funded by Kuwait University Research Administration, Project YS 06/09.

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