Association of Angiotensin Converting Enzyme (Insertion/Deletion) Gene Polymorphism with Essential Hypertension in Northern Indian Subjects

Abstract

Objective:

Essential hypertension is a multifactorial disease in which genetic and environmental factors play an important role. The renin-angiotensin system (RAS) is known to play a critical role in the homeostasis of blood pressure. Angiotensin-I converting enzyme (ACE) is a significant component of RAS, and an insertion/deletion (I/D) polymorphism in its gene has been implicated in predisposition to hypertension. The purpose of the current study is to investigate the association of I/D polymorphism of the ACE gene with essential hypertension in northern Indians.

Method:

Two hundred twenty-two patients with essential hypertension and 252 controls were recruited for the study. DNA samples were isolated from peripheral blood by using a kit. Polymerase chain reaction was used for genotyping.

Result:

All the genotypes and allele distribution in study subjects were in the Hardy-Weinberg equilibrium. There was a significant difference in the distribution of DD, II, and ID genotypes of ACE polymorphism in patients and controls. In the subjects having an I allele, the odds ratio is 2.08 [1.6-2.58] at 95% confidence interval, thus suggesting an association of ACE I/D gene polymorphism with essential hypertension.

Conclusion:

Our findings suggest that the I allele of ACE I/D polymorphism is associated with essential hypertension in our population.

Introduction

Cardiovascular diseases are becoming a major health burden in developing countries. About 2.6 million Indian people are estimated to die due to coronary heart disease (CHD) alone by the year 2020 (Nistar, 2002). Essential hypertension is an important risk factor for the development of CHD. It is a multifactorial and polygenic disorder in which the interaction between several candidate genes and environmental factors play a role in which the blood pressure is elevated. The renin-angiotensin system (RAS) has a potential role in the development of essential hypertension by regulating blood pressure and metabolism of fluid and electrolytes and also by influencing the proliferation of the vascular smooth muscle cells (MacGregor et al., 1981). Angiotensin converting enzyme (ACE), an important regulatory enzyme of the RAS that plays a critical role in maintaining blood pressure homeostasis, is widely distributed in the kidney (Salem and Batzer, 2009). The ACE catalyzes the conversion of angiotensin I to the biologically active peptide, angiotensin II, a vasoconstrictor that results in a vasopressor response and inactivates bradykinin and kallidin (vasodilators) which is involved in the control of fluid electrolyte balance and systemic blood pressure (Wang and Staessen, 2000). The ACE gene is 21 kilo bases (kb) long comprising of 26 exons and 25 introns and is located on the long arm of chromosome 17(17q23.3). I/D polymorphism is located in a non-coding region (namely the intron) of the ACE gene (Rigat et al., 1990); this polymorphism is characterized by the presence (insertion) or absence (deletion) of a 287 bp AluYa5 element inside intron 16. The D allele is related to an increased activity of ACE in serum, as the highest serum ACE activity was seen in the DD genotype, whereas the lowest was seen in the II genotype (Sakuma et al., 2004). Several studies carried out around the world suggested genetic predisposition associated with the ACE I/D polymorphism with several diseases including CHDs, stroke, hypertension, and diabetes mellitus (Kennon et al., 1999; Zee et al., 1999; Obineche et al., 2001; Gesang et al., 2002). However, conflicting results have been reported regarding the association between ACE polymorphism and disease (Moleda et al., 2007; Taal, 2000). Moreover, various reports were published suggesting inter-ethnic variations in the frequency of allelic forms of the ACE genes (Barley and Jeffery et al., 1994; Saha et al., 1996). The ACE (I/D) polymorphism is presumed to be in linkage disequilibrium with functional variants that determine enzyme activity (Zhu et al., 2000) such that the D allele is associated with increased ACE activity in white (Rigat et al., 1990) and Asian (Nakai et al., 1994) subjects but not in black subjects (Bloem et al., 1996). However, many other studies have failed to identify such associations (Barley et al., 1994; Duru et al., 1994; O'Donnell et al., 1998). The prevalence of hypertension in India varies from less than 30% to as much as 61% (Kutty et al., 1993; Swami et al., 2002; Gupta et al., 2004). The few studies available from India reported conflicting results and have failed to identify such associations (Gupta et al., 2009; Sameer et al., 2010). These controversial findings prompted us to study the polymorphism of the ACE gene in patients with essential hypertension in Asian Indians. Therefore, we carried out a case-control study in our population to determine whether ACE I/D polymorphism is associated with essential hypertension in northern Indian subjects.

Methodology

The present proposal is a prospective study in patients with essential hypertension and normal healthy volunteers.

Study subjects and their selection criteria

Group 1

The patients (N=222, Age >25 <60 years) with essential hypertension (systolic pressure >140mmHg and/or diastolic pressure >90 and secondary causes of hypertension ruled out) were selected from a series of consecutive outpatients of Department of Cardiology, All India Institute of Medical Sciences (AIIMS). They were not on antihypertensive treatment, had no metabolic or endocrine disorder or any acute illness.

Group 2

The controls (n=252) age and sex matched from the similar population group were recruited from the staff of AIIMS and residents of Delhi and surrounding areas.

Previous informed consent from both the study groups was obtained. An approval of study protocol by the ethical committee of AIIMS, New Delhi, was obtained before the start of the study.

Collection of blood samples

Approximately 5 mL of peripheral blood samples were collected in a screw cap tube that contained 5% EDTA. The specimen was capped and transported to the laboratory on dry ice. It was then stored at −20°C if not immediately assayed.

Detection of ACE genotype

Genomic DNA extraction was carried out by using the QIAamp^® DNA Blood Mini Kit by QIAGEN^®. To determine the ACE genotype, genomic DNA was initially amplified by a polymerase chain reaction (PCR) by using a primer pair that specifically recognized the insertion specific sequence. The sense oligonucleotide primer was 5′-CTG GAG ACC ACT CCC ATC CTT TCT-3′, and the antisense primer was 5′-GAT GTG GCC ATC ACA TTC GTC AGA TTT−3′ determined by the programme gene runner 3.05. The PCR mixture contained 50 ng genomic DNA, 10 pmol of each primer (Sigma-Aldrich), 10 mmol dNTP (SBS Genetech), 2.5 μL of 10× PCR buffer (Geneaid), and 1.0 unit Taq DNA polymerase (Geneaid) in a final volume of 25 μL. The amplification cycle was performed on a Bio Rad thermal cycler. After initial denaturation at 94°C for 5 min, the DNA was amplified through 35 cycles: denaturation for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, followed by a final elongation at 72°C for 7 min. Amplification products were separated by electrophoresis on a 1.5% agarose gel, and visualized under ultraviolet light after ethidium bromide staining. The PCR product is a 190 bp fragment in the presence of a deletion (D) allele, and a 490 bp fragment in the absence of a deletion (I) allele. Thus, each DNA sample revealed one of three possible patterns after electrophoresis: a 490 bp band (II genotype), a 190 bp band (DD genotype), or both 490 and 190 bp bands (I/D genotype).

Statistical analysis

All computations were carried out with STATA program, version 8. Chi-square goodness of fit was used to verify the agreement of observed genotype frequencies with those expected (Hardy-Weinberg equilibrium). Allele and genotype frequencies were compared by standard contingency table analysis by using a chi-square test (Yates corrected). Odds ratios at (95% confidence intervals [CI]) were calculated as an index of the association of the gene with the disease. Statistical significance was defined as a p-value <0.05.

Results

The two study groups were well matched for sex, age, and sample size. The mean systolic (SBP) and diastolic (DBP) blood pressures were significantly higher among hypertensive subjects than in control subjects. However, there is no significant mean age difference between both groups. The baseline characteristics of the patients and controls are shown in Table 1. The age, total cholesterol, triglyceride, high-density lipoprotein-cholesterol, and low-density lipoprotein were comparable in patients and controls. The number of men was higher in both the study subjects. SBP and DBP were higher in patients as compared with controls.

Table 1.

Baseline Characteristics of the Study Subjects

		Patients	Controls
1.	Number (N)	222	252
2.	Sex (M/F)	142/80	194/58^a
3.	Age (years)	51.6+7.2	49.7+10.4
4.	LDL cholesterol (mg/dL)	85.3+41.6	95.3+37.0
5.	HDL cholesterol (mg/dL)	40.5+7.2	38.6+7.2
6.	Total cholesterol (mg/dL)	177.3+40.3	168+20
7.	Triglyceride (mg/dL)	140+108	160+54
8.	SBP (mm Hg)	145.5±14.05	120±3.47^a
9.	DBP (mm Hg)	93.6±8.09	80.49±2.5^a

Patients groups were compared with controls with t-test of significance or by chi-square test.

p-Value<0.01, significant.

LDL, low-density lipoprotein; HDL, high-density lipoprotein; SBP, systolic blood pressure; DBP, diastolic blood pressure.

The ACE genotype and allele frequencies distribution of control and hypertensive subjects are presented in Table 2. The frequencies of II, ID, and DD genotypes among the control group were 6.34%, 38.88%, and 54.76%, respectively; whereas, in the hypertensive group, the same were found to be 18.91%, 47.7%, and 33.33%, respectively. There was no detectable deviation from Hardy-Weinberg equilibrium in either data set. There is significant difference (p<0.05) observed in the distribution of ACE genotype polymorphism between the two groups. Genotype percentage was in the order of DD>ID>II in controls, whereas it was ID>DD>II in patients. In the subjects having an I allele, the calculated odds ratio for hypertension is 2.08 [1.6-2.58] at 95% CI, thus suggesting an association of ACE I/D gene polymorphism with essential hypertension (Yates corrected chi=29.01, p=0.000000) after full adjustment of age and sex (Table 3).

Table 2.

Genotype Frequencies of Angiotensin Converting Enzyme (Insertion/Deletion) Gene Polymorphism in Study Subjects

	Genotypes
Group	II (%)	ID (%)	DD (%)
Controls (n=252)	16 (6.34)	98 (38.88)	138 (54.76)	χ²=0.06, p>0.05, ns
Patients (n=222)	42 (18.91)	106 (47.7)	74 (33.33)	χ²=0.07, p>0.05, ns
Unadjusted odds ratio=2.42 [1.64-3.58]			Odds ratio=2.39 [1.62-3.52]^a
Unadjusted relative risk=1.64 [1.32-2.04]			Relative risk=1.6 [1.2-2.02]^a
Unadjusted Yates corrected chi=21.06, p=0.0000044			Yates corrected chi=20.01, p=0.00006^a

Adjusted for age and sex; patients' groups were compared with controls with chi-square (χ²) test.

ns, not significant.

Table 3.

Allele Frequencies of Angiotensin Converting Enzyme (Insertion/Deletion) Gene Polymorphism in Study Subjects

	Allele frequency
Group	I	D
Controls (n=252)	0.26	0.74
Patients (n=222)	0.43	0.57
Unadjusted odds ratio=2.15 [1.62-2.86]		Odds ratio=2.08 [1.6-2.58]^a
Unadjusted relative risk=1.3 [1.18-1.43]		Relative risk=1.1 [1.2-1.5]^a
Unadjusted Yates corrected chi=29.75, p=0.000000		Yates corrected chi=29.01, p=0.000000^a

Adjusted for age and sex; patients' groups were compared with controls with chi-square (χ²) test.

Discussion

Essential hypertension is a multifactorial disease with significant contribution of a genetic component. Cardiovascular diseases are rapidly emerging as a major health concern in most developing countries, and hypertension is one of the major preventable causes of morbidity and mortality from cardiovascular disease. A polymorphic marker that is found on intron 16 of the ACE gene was correlated with circulating concentrations of ACE (Rigat et al., 1990).

Some studies have proposed that the DD genotype increases the incidence of essential hypertension (Higaki et al., 2000), whereas others have not found a significant association (Mondry et al., 2005). This inconsistent association may be due to the fact of different ethnicity of the population groups and environmental heterogeneity (Barley et al., 1994; Stassen et al., 1997; O'Donnell et al., 1998).

With regard to our data from patients, the frequency of the I allele was 0.43, which was comparatively lower than Tibetans (Gesang et al., 2002), Japanese (Higaki et al., 2000), and German (Mondry et al., 2005) populations, where the range of I allele frequency was in the range of 0.48 to 0.65. The frequency of I allele in patients of previously reported populations from India varied between 0.38 and 0.54. Although many of these studies have not reported any association possibly because of small sample size (Gupta et al., 2009), an association was reported in the Kashmiri population between the D allele and essential hypertension (Sameer et al., 2010). In the current study, we have found that the individuals with the I allele of the ACE gene were strongly associated with essential hypertension (p<0.00000) in Asian Indians having a similar socio-economic-geographical background. This information will be of immense use in personalization of drug therapy to patients with hypertension based on the ACE I/D genotypes.

Our findings also provide the direct evidence of gene disease association of this region and higher predisposition of Asian Indians to the essential hypertension.

Footnotes

Acknowledgments

This work is supported by a grant from Department of Science and Technology, India, to Dr. Kamna Srivastava.

Disclosure Statement

No competing financial interests exist.

References

Barley

, Blackwood

, Carter

et al. 1994. Angiotensin converting enzyme insertion/deletion polymorphism: association with ethnic origin. J Hypertens, 12:955-957.

Bloem

, Manatunga

, Pratt

. 1996. Racial difference in the relationship of an angiotensin I-converting enzyme gene polymorphism to serum angiotensin I-converting enzyme activity. Hypertension, 27:62-66.

Duru

, Farrow

, Wang

. 1994. Frequency of a deletion polymorphism in the gene for angiotensin converting enzyme is increased in African-American with hypertension. Am J Hypertens, 7:759-762.

Gesang

, Liu

, Cen

et al. 2002. Angiotensin-converting enzyme gene polymorphism and its association with essential hypertension in a Tibetan population. Hypertens Res, 25:481-485.

Gupta

, Sarna

, Thanvi

et al. 2004. High prevalence of multiple coronary risk factors in Punjabi Bhatia community: Jaipur Heart Watch-3. Indian Heart J, 56:646-652.

Gupta

, Agrawal

, Goel

et al. 2009. Angiotensin-converting enzyme gene polymorphism in hypertensive rural population of Haryana. India J Emerg Trauma Shock, 2:150-154.

Higaki

, Baba

, Katsuya

et al. 2000. Deletion allele of angiotensin-converting enzyme gene increases risk of essential hypertension in Japanese men. Circulation, 101:2060-2065.

Kennon

, Petrie

, Small

et al. 1999. Angiotensin- converting enzyme gene and diabetes mellitus. Diabet Med, 16:448-458.

Kutty

, Balakrishnan

, Jayasree

et al. 1993. Prevalence of coronary heart disease in the rural population of Thiruvananthapuram district, Kerala, India. Int J Cardiol, 39:59-70.

10.

MacGregor

, Markandu

, Roulston

et al. 1981. Maintenance of blood pressure by the renin-angiotensin system in normal man. Nature, 28:329-331.

11.

Moleda

, Majkowska

, Safranow

et al. 2007. Relationship between I/D polymorphism of angiotensin I converting enzyme gene and microvascular complications in type 2 diabetic patients. Przegl Lek, 64:134-139.

12.

Mondry

, Loh

, Liu

et al. 2005. Polymorphisms of the insertion/deletion ACE and M235T AGT genes and hypertension: surprising new findings and meta-analysis of data. BMC Nephrol, 6:1.

13.

Nakai

, Itoh

, Miura

et al. 1994. Deletion polymorphism of the angiotensin I-converting enzyme gene is associated with serum ACE concentration and increased risk for CAD in the Japanese. Circulation, 90:2199-2202.

14.

Nistar

. 2002. Prevention of coronary heart disease in South Asia. Lancet, 360:1015-1018.

15.

Obineche

, Frossard

, Bokhari

. 2001. An association study of five genetic loci and left ventricular hypertrophy amongst Gulf Arabs. Hypertens Res, 24:635-639.

16.

O'Donnell

, Lindpainter

, Larson

et al. 1998. Evidence for association and genetic linkage of the angiotensin-converting enzyme locus with hypertension and blood pressure in man but not women in the Framingham Heart Study. Circulation, 97:1766-1772.

17.

Rigat

, Hubert

, Alhenc-Gelas

et al. 1990. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Inves, 86:1343-1346.

18.

Saha

, Talmud

, Tay

et al. 1996. Lack of association of angiotensin-converting enzyme (ACE). Gene insertion/deletion polymorphism with CAD in two Asian populations. Clin Genet, 50:121-125.

19.

Sakuma

, Hirata

. 2004. Five polymorphisms in gene candidates for cardiovascular disease in Afro- Brazilian individuals. J Clin Lab Anal, 18:309-316.

20.

Salem

, Batzer

. 2009. High frequency of the D allele of the angiotensin-converting enzyme gene in Arabic populations. BMC Res Notes, 2:99.

21.

Sameer

, Syeed

, Tak

et al. 2010. ACE I/D Polymorphism in Hypertensive Patients of Kashmiri Population. Cardiol Res, 1:1-7.

22.

Stassen

, Wang

, Ginocchio

et al. 1997. The deletion/insertion polymorphism of the angiotensin converting enzyme gene and cardiovascular-renal risk. J Hypertens, 15:1579-1592.

23.

Swami

, Bhatia

, Gupta

et al. 2002. Population based study of hypertension among the elderly in northern India. Public Health, 116:45-49.

24.

Taal

. 2000. Angiotensin-converting enzyme gene polymorphisms in renal disease: clinically relevant? Curr Opin Nephrol Hypertens, 9:651-657.

25.

Wang

, Staessen

. 2000. Genetic polymorphisms in the renin-angiotensin system: relevance for susceptibility to cardiovascular disease. Eur J Pharmacol, 410:289-302.

26.

Zee

, Ridker

, Stampfer

et al. 1999. Prospective evaluation of the angiotensin-converting enzyme insertion/deletion polymorphism and the risk of stroke. Circulation, 99:340-343.

27.

Zhu

, McKenzie

, Forrester

et al. 2000. Localization of a small genomic region associated with elevated ACE. Am J Hum Genet, 67:1144-1153.