Association of angiotensin-converting enzyme and angiotensin-converting enzyme-2 gene polymorphisms with essential hypertension in the population of Odisha, India.

Background: Hypertension is a serious health issue worldwide and essential hypertension, which includes 90–95% of the cases, is influenced by both genetic and environmental factors. Identification of these factors may help in control of this disease. The Insertion/Deletion (I/D) polymorphism in Angiotensin-Converting Enzyme (ACE) gene and rs2106809 (C > T) polymorphism in Angiotensin-Converting Enzyme 2 (ACE2) gene have been reported to be associated with essential hypertension in different populations. Aim: To investigate the association of ACE I/D and ACE2 rs2106809 polymorphisms with essential hypertension in the population of Odisha, an eastern Indian state. Subjects and methods: A total of 246 hypertensives (159 males and 87 females) and 274 normotensives (158 males and 116 females) were enrolled in the study. Detailed anthropometric data, tobacco, alcohol and food habits were recorded and 2 ml of venous blood was collected for biochemical and genetic analysis. Results: The DD genotype of ACE and TT genotype of ACE2 were significantly high among female hypertensives, while T allele of ACE2 was linked to male hypertensives. In the male population, alcohol was also identified as a potential risk factor. Conclusion: Among females, ACE I/D and ACE2 rs2106809 polymorphisms, while among males, ACE2 rs2106809 polymorphism and alcohol consumption are associated with essential hypertension in the study population.

Keywords: gene polymorphism; hypertension; risk factors; ACE

Essential hypertension is a complex multi-factorial condition influenced by genetic and environmental factors (Lifton et al., [17]). The Renin--Angiotensin--Aldosterone system (RAAS) is an important regulatory system for maintaining normal blood pressure and electrolyte balance. Since angiotensin-converting enzyme (ACE) plays a key role in the RAAS pathway, the gene expressing ACE has been studied extensively as a potential marker for essential hypertension (Jandeleit-Dahm et al., [16]).

ACE is encoded by a 21 Kb long gene that has been mapped to chromosome 17q23. It consists of 26 exons and 25 introns (Mattei et al., [18]). Insertion/deletion (I/D) polymorphism of a 287 bp Alu repeat sequence in intron 16 of the ACE gene is associated with altered levels of ACE and its activity in plasma. The DD genotype has been shown to be associated with high serum ACE production and activity while II and ID genotypes relate to low and intermediate levels and activities, respectively (Rigat et al., [28]; Suehiro et al., [35]). While several studies have shown a link between ACE I/D polymorphism and hypertension (Choudhury et al., [8]; Nakano et al., [20]), many studies have failed to detect this association (Gupta et al., [13]; Harrap et al., [14]).

Angiotensin-converting enzyme 2 (ACE2), a recently described RAAS component that shares 42% identity with the catalytic domain of somatic ACE, has been found to play a protective role in regulation of BP homeostasis and cardiac function. The ACE2 gene contains 18 exons that encode a 805 amino-acid polypeptide and maps to the chromosome Xp22. ACE2 hydrolyses angiotensin II to angiotensin 1–7 which is a vasodilator and partially hydrolyses angiotensin I (Tipnis et al., [36]). ACE2 rs2106809 mutation (C→T) has been reported to be associated with clinical manifestation of hypertension (Chen et al., [5]; Fan et al., [12]). However, no report in this aspect is available in India. Therefore, an attempt has been made to investigate the association of ACE I/D and ACE2 rs2106809 gene polymorphisms and some environmental factors with the clinical manifestation of hypertension among the native population of Odisha.

Patients attending the medical out patients department of the Capital Hospital, Bhubaneswar and VSS Medical College and Hospital, Burla during January 2011–August 2012 for treatment of various ailments were recruited for the study. A total 246 hypertensive patients and 274 normotensive individuals (controls) were enrolled for investigation based on inclusion/exclusion criteria. Informed consent was obtained from all individuals before enrolment. The study was approved by the ethical committee of the Regional Medical Research Centre, Bhubaneswar, India.

The inclusion criteria for patients were systolic blood pressure (SBP) ≥ 140 mmHg and/or mean diastolic blood pressure (DBP) ≥ 90 mmHg (Chobanian et al., [7]) or current anti-hypertensive medication. The exclusion criteria were persons with secondary hypertension (hypertension due to secondary causes such as renovascular disease, renal failure, pheochromocytoma, aldosteronism or other causes of secondary hypertension), diabetes or under lipid-lowering drugs. The controls had no history of hypertension, diabetes or any other cardio-vascular disease and were not under any lipid-lowering drugs. Data on age, sex, family history, education, intake of additional salt during any sort of food intake, diet (vegetarian/non-vegetarian), tobacco and alcohol habits were recorded. Ex-tobacco consumers and alcoholics were excluded from the study.

Blood pressure was measured by sphygmomanometer in a sitting position on the right arm. Height was measured in centimetres and weight in kilograms. Body Mass Index was calculated using the formula (weight in Kg)/(height in metres)2 and individuals with BMI ≥ 23 kg/m2 and ≥25 kg/m2 were classified as overweight and obese, respectively (WHO, [41]).

At least 2 ml of venous blood was collected aseptically from each study subject after overnight fasting and stored in EDTA vials. Blood was transferred to the laboratory under cold conditions. From 1 ml of blood, plasma was separated within 3 hours by centrifuging the blood at 3000 rpm for 3 minutes and stored at −20 °C for biochemical analysis.

The Adult Treatment Panel-III (National Cholesterol Education Program), 2002 criteria was used for stratifying the biochemical data. The lipid profile (total cholesterol, high density lipoprotein: HDL, low density lipoprotein: LDL, triglycerides) and the indicators of kidney function status (urea and creatinine) were analysed by an automatic analyser (Cobas Integra 400, Roche Diagnostics, Germany) using the commercially available reagent kits supplied by the company.

The genomic DNA was extracted from the whole blood using the standard phenol–chloroform method (Sambrook & Russell, [31]). The extracted DNA was re-suspended in 100 µl of DNase-free water and kept at −20 °C until use.

To determine the I/D polymorphism of ACE gene, a flanking primer pair 5′-CTGGAGACCACTCCCATCCTTTCT-3′ and 5′-GATGTGGCCATCACATTCGTCACGAT-3′ was used to amplify the segment of the ACE gene containing the mutation (Ramachandran et al., [26]). All the PCR amplifications were performed in a 20 µl reaction mixture containing 5 picomoles each of forward and reverse primer, 1.9 nM of each dNTP, 10 mM Tris-HCL, 50 mM KCl, 2.75 mM MgCl2, 0.01% Gelatin, 1.5 U Taq DNA polymerase (Bangalore Genei) and 3 µl of template DNA. The PCR cycling conditions were carried out with an initial denaturation for 10 minutes at 96 °C, followed by 35 cycles of denaturation at 94 °C for 1 minute, annealing at 66 °C for 1 minute and extension at 72 °C for 1 minute, followed by a final extension for 10 minutes at 72 °C. The products were separated by electrophoresis on 2% agarose gels and visualized after staining with ethidium bromide (0.5 µg/ml).

The ACE2 rs2106809 polymorphism was detected using the forward primer 5′-GAAAGCCAGATGCTTTAACAAG-3′ and the reverse primer 5′-TTTTTCCATATCTCTATCTGATCG-3′ (Fan et al., [11]). The reaction mixture composition was the same as ACE, with the only exception that the MgCl2 concentration was dropped to 1.5 mM. The cycling conditions included an initial denaturation at 95 °C for 10 minutes, followed by 35 cycles of denaturation at 95 °C for 50 seconds, annealing at 55 °C for 50 seconds and elongation at 72 °C for 30 seconds and a final extension at 72 °C for 10 minutes. The products were digested with 2.5 units of TaqI overnight and separated using 3% agarose gels and visualized after EtBr (0.5 µg/ml) staining.

Unpaired t-test or chi-square test or Fisher's exact test was used to compare the characteristics of the two groups and to compare the characteristics according to different genotypes in females one-way ANOVA was used. Genotypes and alleles were compared using chi-square test or Fisher's exact test as applicable. Graph Pad version 5 was used for the above analysis. Logistic regression analysis was carried out to identify the independent risk factors using SPSS version 17.

A total number of 246 hypertensive (males: 159 and females: 87) and 274 normotensive (males: 158 and females: 116) individuals were included in the study. The characteristics of the enrolled individuals have been depicted in Table 1. BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP) and triglyceride levels were significantly higher among the patients as compared to controls and HDL levels were significantly higher in controls. Similarly the proportion of people being overweight/obese and with the habit of alcohol consumption was significantly higher among the hypertensives. Interestingly, mean BMI values in both groups were higher than the normal cut-off levels.

Table 1. Characteristics of patients and controls.

9 *p < 0.05. Continuous variables are expressed in Mean ± Standard Deviation and frequency data are expressed in percentages. BMI, Body Mass Index; SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure; TC, Total Cholesterol; HDL, High Density Lipoprotein; LDL, Low Density Lipoprotein; TG, Triglycerides; HDL/LDL, High Density Lipoprotein/Low Density Lipoprotein ratio; mmol/L, millimoles/Litre. #Females were not smokers or alcoholics.

Gender-wise analysis of the variables between hypertensive and normotensive cases indicates that SBP and DBP and frequency of overweight/obesity were significantly higher in both male and female patients compared to respective controls. The level of triglycerides and alcohol consumption were higher only in male patients, whereas BMI, creatinine levels and hyperlipidemia were higher only in female patients compared to controls.

The banding pattern of ACE I/D revealed three genotypes such as the 490 bp band for the homozygous ancestral genotype (Insertion/Insertion, II), 190 bp band for the homozygous derived genotype (Deletion/Deletion, DD) and both 490 and 190 bp bands for the heterozygous genotype (Insertion/Deletion, ID). The distribution of the genotypes in the studied population showed no significant deviation from Hardy-Weinberg equilibrium. On analysing the genotypes according to different genetic models, a significant association of the mutation with hypertension was found in additive (DD versus II) (p = 0.006, OR = 2.47, CI = 1.24–4.74) and recessive (II/ID versus DD) (p = 0.003, OR = 2.50, CI = 1.35–4.64) models. When gender-specific analysis was carried out associations were observed in females in co-dominant (DD versus ID, p = 0.04, OR = 3.46, CI = 1.05–11.41; ID versus II, p = 0.019, OR = 2.46, CI = 1.15–5.24), additive (p < 0.001, OR = 8.50, CI = 2.41–29.95), dominant (ID/DD versus II) (p = 0.002, OR = 3.06, CI = 1.47–6.37) as well as recessive (II/ID versus DD) (p = 0.006, OR = 4.86, CI = 1.53–15.49) models. In males, no association could be observed. When frequencies of both the alleles were compared, D allele was significantly higher in patients than in controls in the total (p = 0.04, OR = 1.34, CI = 1.01–1.77) as well as female population (p < 0.001, OR = 2.36, CI = 1.46–3.84).

In the case of ACE2 rs2106809, production of 183 bp and 24 bp byproducts after overnight digestion at 65 °C of the 207 bp amplicon with Taq I indicates the presence of T allele. The polymorphism was analysed separately among males and females because of its localization on the X chromosome. The genotype distribution among female hypertensives showed deviation from Hardy--Weinberg equilibrium (χ2 = 11.6), but no association could be found either in females or males on analysing different genetic models. The results have been summarized in Tables 2–4 and the gel photographs illustrating the banding patterns have been shown in Figures 1 and 2.

Graph: Figure 1. Banding pattern of ACE I/D polymorphism: 1st and 11th lanes: 100bp ladder, 2nd, 3rd and 4th lanes: II, 5th, 6th and 7th lanes: ID, 8th, 9th and 10th lanes: DD.

Graph: Figure 2. Banding pattern of ACE2 rs2106809 polymorphism 1st, 5th and 9th lanes: 100 bp ladder, 2nd and 6th lanes: CC/C, 3rd and 7th lanes: CT, 4th and 8th lanes: TT/T.

Table 2. Genotype and allele distribution of ACE and ACE2.

10 OR, Odds Ratio; 95% CI, 95% Confidence Interval. OR, CI and p values have been derived using Chi-square test or Fisher's exact test.

Table 3. Association of different genotypes of ACE with hypertension.

11 OR, Odds Ratio; 95% CI, 95% Confidence Interval. OR, CI and p values have been derived using Chi-square test or Fisher's exact test.

Table 4. Association between different genotypes of ACE2 (females) with hypertension.

12 OR, Odds Ratio; 95% CI, 95% Confidence Interval. OR, CI and p values have been derived using Chi-square test or Fisher's exact test.

Logistic regression analysis (Table 5) was performed in order to find out the independent influencing factors. In the total population, three factors were observed to be independent risk factors for hypertension, viz., ACE I/D polymorphism, ACE2 rs2106809 polymorphism and alcohol consumption. In males, ACE2 rs2106809 polymorphism and alcohol consumption were linked to hypertension. However, low HDL/LDL ratio was associated with normotensives. In females, however, the polymorphisms, ACE I/D and ACE2 rs2106809 polymorphisms only, were identified as independent risk factors.

Table 5. Results of logistic regression analysis.

13 *Females were not smokers or alcoholics. OR, Odds Ratio; 95% CI, 95% Confidence Interval. Abbreviations used are the same as in Table 1.

Because it is likely that the polymorphisms in different genes may have a joint effect on the risk of the disease, we tested various combinations of genotypes of both the genes separately in males and females. The genotype frequency of ACE DD + ACE2 rs2106809 TT was found to be higher in female subjects (p = 0.048) but, after logistic regression analysis, no association could be observed (Table 6).

Table 6. Combined genetic analysis.

14 OR, Odds Ratio; 95% CI, 95% Confidence Interval. OR, CI and p value computed as DD + TT versus II + CC/CT.

The characteristics of the subjects according to different genotypes were analysed separately in males and females. In males two different types of analysis were carried out, in the first one, subjects with ID/DD genotype and T-allele were combined into one group and compared with those carrying the II genotype and C allele and, in the second, subjects with II/ID genotype and C allele were taken as one group and compared with those having the DD genotype and T allele. In the first case, BMI, HDL and triglyceride levels were significantly different among the groups and, in the second case, total cholesterol, LDL and triglyceride levels were significantly different. In females the homozygotes having the ancestral genotypes, the heterozygotes and the derived genotype homozygotes were grouped separately and their characteristics were compared. The pattern of BMI was different but no specific gradation was observed. No other factor showed any difference in females. The results have been summarized in Tables 7 and 8.

Table 7. Characteristics of male subjects according to different genotypes.

15 OR, Odds Ratio; 95% CI, 95% Confidence Interval. Continuous variables are expressed in Mean ± Standard Deviation and frequency data are expressed in percentages. Abbreviations used are the same as in Table 1.

Table 8. Characteristics of female subjects according to different genotypes.

16 Continuous variables are expressed in Mean ± Standard Deviation and frequency data are expressed in percentages. Abbreviations used are the same as in Table 1.

Knowledge of genetic predisposition to hypertension is essential for the study of pharmocogenetics of the disease. Therefore, many genes have been studied worldwide to find out the candidate genes which can be targeted for its therapy and this would also help in identifying individuals at an increased risk of developing this disease to initiate appropriate actions to avoid development or delay the onset of disease (Gupta et al., [13]). To our knowledge, this is the first study regarding ACE I/D polymorphism in relation to hypertension in the state of Odisha (India) and ACE2 rs2106809 in India.

ACE and ACE2 both play an important role in BP regulation and act in a counteracting fashion to maintain a normal blood pressure level in the human body (Fan et al., [12]). Therefore, studies have been conducted to analyse mutations in these two genes which may predispose to hypertension. In our study we have observed a significant association between DD genotype essential hypertension, which still remained a significant risk factor (p < 0.001) after adjustment for confounding factors. Similar to our findings, the ACE D allele has been documented to be positively associated with hypertension in African Americans (Duru et al., [10]), Chinese (Chiang et al., [6]), Japanese populations (Nakano et al., [20]) and among the natives of Bangladesh (Morshed et al., [19]). In India, DD genotype and D allele has been found to be linked to high BP in Andhra Pradesh (Bhavani et al., [4]) and significant high frequency of D/D genotype has been detected among hypertensive patients compared to the control group in Chennai (Choudhury et al., [8]). However, no associations of ACE I/D with BP have been found in Haryana (Gupta et al., [13]). Contrary to this finding, I allele was found to be associated with the increased risk of hypertension in Pakistan (Ismail et al., [15]) and among a Kashmiri population of India (Sameer et al., [32]). This type of contradictory result might be due to gender bias, different ethnicity of the population groups and environmental heterogeneity (Barley et al., [2]; Staessen et al., [33], Sagnella et al., [29]). Moreover, the ACE I/D polymorphism is possibly only a marker that is in tight linkage disequilibrium with a functional polymorphism that strongly influences circulating as well as tissue enzyme activity (Rigat et al., [28]; Tiret et al., [37]) and presumably the degree of linkage disequilibrium between the two may vary from population to population (Samani et al., [30]).

When we analysed the genetic data according to gender, the ACE I/D gene polymorphism was found to be linked with expression of hypertension in females even after adjusting for other risk factors (p = 0.005). The persons with DD genotype were at greatest risk followed by those with ID and those with II genotype were at least risk. In males, however, no association could be observed. Similar to our observation, gender-specific association have also been observed in other populations. A highly significant association has been reported between the D allele and hypertension in women of African descent (Sagnella et al., [29]), while, in the Framingham study (O'Donnell et al., [23]) and in the study conducted in Andhra Pradesh, India (Bhavani et al., [4]), the ACE I/D polymorphism was found to be associated with males rather than females. This might be because the gene regulation in renin--angiotensin system is significantly affected by the gonadal steroids, i.e. testosterone and oestrogen (Bachmann et al., [1]). Besides, both autosomal genes and genes involved in sexual dimorphism contribute to multi-factorial traits/diseases or are differentially expressed in tissues in males versus females (Rana et al., [27]). Various other genetic polymorphisms have also been studied that have influenced hypertension differentially in two genders. Nakayama et al., ([21]) identified the A allele of rs1394205 in the follicle stimulating hormone receptor in females of Tokyo to be linked to high blood pressure and Polimanti et al. ([25]) demonstrated the association of GSTT1 null phenotype of Glutathione S-transferase with hypertension in Italian females. Similarly the association of CC genotype of A1166C polymorphism of Angiotensin II Type 1 receptor gene in Siberian males (Stanković et al., [34]) and the a/a genotype of intron 4 variable number of tandem repeats (VNTR) polymorphism of endothelial nitric oxide synthase (eNOS) gene in Indian males with hypertension (Patkar et al., [24]) have been described.

In the case of the ACE2 rs2106809 polymorphism, hypertensives showed a higher frequency of polymorphism, but the results did not reach a statistically significant difference in univariate analysis. However, after adjustment for all the risk factors using logistic regression analysis the polymorphism (males: T versus C, females: TT versus CC/CT) was found to be associated with hypertension (p < 0.001) in the total population as well as in both males and females. Some studies have found a gender-specific effect of the gene, but it was not evident in our study. Fan et al. ([11]) found the ACE2 rs2106809 T allele to confer a 1.6-fold risk for hypertension in women but no association was found in men in a Chinese Han population. Chen et al. ([5]), in China, found the rs2106809 polymorphism to be associated with pulse pressure in women and men and with reduction in systolic blood pressure in women and also that it might act as an independent influencing factor of gene–gene interaction in BP reducing effects of benazepril. Fan et al. ([12]), however, did not find any significant association in either female or male Chinese patients with orthostatic hypertension. There are also some other studies on polymorphisms which did not show gender specification. The T1559C genotype of the E-selectin gene (Wang et al., [39]) and the 5′-uncoding region (UCR) -1248 A > G polymorphism of the Mitofusin2 gene (Wang et al., [40]) have been observed to be associated with hypertension in both males and females in China.

Because ACE and ACE2 act in counteracting ways (Crackower et al., [9]; Fan et al., [12]) we tested for any possible association, but, in our study no combined effect of ACE I/D and ACE2 rs2106809 polymorphisms was observed. Fan et al. ([11]), however, reported that ACE2 rs2106809 T allele when combined with the effect of the ACE DD genotype, increased the risk for hypertension by 2.34-fold in females.

Alcohol consumption was identified to be independently associated with hypertension. However, alcohol consumption was restricted to males and was the most significant independent risk factor in the male population and conferred ∼3.4-times higher risk in alcoholics compared to non-alcoholics. Alcohol is an important contributor to hypertension. Alcohol directly influences the heart or the vascular smooth muscle or stimulates the sympathetic nervous system or the renin--angiotensin--aldosterone system, causes severe dehydration and weight gain due to extra calories, acts as a diuretic, affects the calcium and magnesium absorption, diminishes the effects of antihypertensive medications and increases their side-effects. It also diminishes the effects of natural remedies. It may increase plasma cortisol levels through magnesium loss into the urine, by an increase in endothelin release or by a decrease in NO production in the arterial endothelium (Beilin et al., [3]). Alcohol as a positive risk for development of hypertension has been observed in various studies (Todkar et al., [38]). The HDL/LDL ratio was found to be lower in male controls in this study, which may be attributed to the higher levels of LDL in male normotensives. A notable feature is the prevalence of high triglyceride levels and BMI in our population, especially in males.

The main limitation of our study is the sample size. A larger sample size will be required for a more precise analysis. However, it can shed some light on mutations of two important candidate genes and their pattern in the Odisha population, where the ACE I/D polymorphism is associated with essential hypertension in females and the ACE2 rs2106809 polymorphism is associated in the case of both males and females. Further, in males consumption of alcohol can be considered as a significant confounding factor for expression of high blood pressure.

We acknowledge the Council of Scientific and Industrial Research, New Delhi, for providing a fellowship to Manisha Patnaik to carry out the research (Sanction number: 09/547/(0004)/2009-EMR-I). We also acknowledge the Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, for additional intramural financial help. The authors report no conflicts of interest.

We are grateful to the patients who participated in the study. We also thank the Director of Regional Medical Research Centre, Bhubaneswar, for providing necessary laboratory facilities for the study. Besides we also thank Mr. Subrat Barik for his help in statistical analysis.