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Association of insertion/deletion polymorphism of angiotensin-converting

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inhibitors

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 Journal of Renin-Angiotensin-Aldosterone
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Association of insertion/deletion polymorphism of angiotensin-converting enzyme gene among Malay

male hypertensive subjects in response to ACE inhibitors
Farzad Heidari, Ramachandran Vasudevan, Siti Zubaidah Mohd Ali, Patimah Ismail, Ali Etemad, Seyyed Reza Pishva,
Fauziah Othman and Suhaili Abu Bakar
Journal of Renin-Angiotensin-Aldosterone System published online 7 July 2014
DOI: 10.1177/1470320314538878

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538878
research-article2014
JRA0010.1177/1470320314538878Journal of the Renin-Angiotensin-Aldosterone SystemHeidari et al.

Article

Journal of the Renin-Angiotensin-

Association of insertion/deletion Aldosterone System

1­–8

polymorphism of angiotensin-converting
© The Author(s) 2014
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enzyme gene among Malay male DOI: 10.1177/1470320314538878
jra.sagepub.com

hypertensive subjects in response

to ACE inhibitors

Farzad Heidari1, Ramachandran Vasudevan2, Siti Zubaidah Mohd

Ali3, Patimah Ismail1, Ali Etemad1, Seyyed Reza Pishva1, Fauziah
Othman4 and Suhaili Abu Bakar1

Abstract
Introduction: Several studies show that the insertion/deletion (I/D) polymorphism of the angiotensin-converting
enzyme (ACE) gene has been associated with hypertension in various populations. The present study sought to determine
the association of the I/D gene polymorphism among Malay male essential hypertensive subjects in response to ACE
inhibitors (enalapril and lisinopril).
Materials and methods: A total of 72 patients with newly diagnosed hypertension and 72 healthy subjects were
recruited in this study. Blood pressure was recorded from 0 to 24 weeks of treatment with enalapril or lisinopril.
Genotyping of the I/D polymorphism was carried out using a standard PCR method.
Results: Statistically significant association of the D allele of the ACE gene was observed between the case and control
subjects (p < 0.01). There was a decrease in blood pressure in the patients carrying the DD genotype (SBP=18.5±8.1
mmHg, DBP=15.29±7.1 mmHg) rather than the ID (SBP=4.1±3.3 mmHg, DBP=9.1±3.5 mmHg) and II genotypes (SBP=
3.0±0.2 mmHg, DBP 0.11±6.1 mmHg) of the ACE gene.
Conclusion: Patients carrying the DD genotype had higher blood pressure-lowering response when treated with ACE
inhibitors enalapril or lisinopril than those carrying ID and II genotypes, suggesting that the D allele may be a possible
genetic marker for essential hypertension among Malay male subjects.

Keywords
Hypertension, I/D, ACE, polymorphism, RAAS, lisinopril, enalapril

Introduction
High blood pressure (BP) or hypertension is the most com-
mon chronic condition, affecting 20%–30% of the adult
population,1 and it is a growing concern in Malaysia.2
Most (90%–95%) hypertension is idiopathic and appar-
ently primary or essential hypertension (EHT), and the
remainder are said to be secondary hypertension that leads
to renal and adrenal diseases.3 Evidence suggests that
genes may contribute to 30% of the variation of BP; how-
ever, the gene-gene and gene-environment interactions are
still remains unknown.4,5 The renin-angiotensin system
(RAS) regulates BP and fluid homeostasis.6 Angiotensin-
converting enzyme (ACE) converts angiotensin I to the
vasoactive angiotensin II and inactivates bradykinin. An
insertion/deletion (I/D) polymorphism of the 187-bp Alu
element in intron 16 of the ACE gene predicts approxi-
mately half of the inter-individual variability in serum
ACE levels.7 Predicting the effect of a particular antihyper-
tensive agent in an individual is a difficult task. To over-
come this problem, researchers are currently considering
which genes influence the response to various antihyper-
tensive drugs.8 Some studies have investigated the effect
1Genetic Research Group, Department of Biomedical Science, Faculty
of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia
2Institute of Gerontology, Universiti Putra Malaysia, Malaysia
3Clinic Kesihatan Senawang, Malaysia
4Department of Human Anatomy, Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, Malaysia
Corresponding author:
Ramachandran Vasudevan, Institute of Gerontology, Universiti Putra
Malaysia, Serdang, Selangor 43400, Malaysia.
Email: vasuphd@gmail.com

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2 Journal of the Renin-Angiotensin-Aldosterone System
of the ACE I/D polymorphism on BP response in patients
with cardiovascular disease treated with ACE inhibitors
(ACEIs).9–14 Several studies have indicated that the D
allele had a stronger BP-lowering effect,15–17 while a few
studies failed to show the association of I/D polymorphism
in lowering the blood pressure.18,19 The conflicting results
made unclear to ability to predict whether the insertion or
deletion allele of the I/D polymorphism of the ACE gene
influences the response to ACEIs. Taking this into an
account, we have evaluated the relationship between the
I/D polymorphism of the ACE gene on the mean difference
in systolic blood pressure (SBP) and diastolic blood pres-
sure (DBP) among ACEIs in Malay male hypertensive
subjects.
Material and methods
Study population
The study was conducted in conformity with the Universiti
Putra Malaysia (UPM) declaration of ethical clearance
from the National Medical Research Register, Malaysia
(reference: NMRR-12-1062-12650). An additional
approval letter was also obtained from Clinic Kesihatan
Senawang, Seremaban, prior to recruiting subjects. This
study was conducted in 72 EHT Malay men with a mean
age of 47±11.3 years while visiting the clinic of non-
communicable disease, Department of Hypertension,
Seremban, Malaysia, with SBP more than 140 mmHg and/
or DBP more than 90 mmHg. Subjects with a history of
diabetes mellitus, renal failure, secondary form of hyper-
tension and major infectious disease were excluded. A sim-
ple questionnaire was prepared both in Malay and English
to obtain their socio-demographic and risk factors such as
age and history of family disease. Informed consent was
obtained from all the subjects. Each hypertensive patient
received either enalapril or lisinopril (20 mg, once daily)
for 24 weeks. During the follow-up period, BP was moni-
tored once every four weeks. Following a 24-week period,
10 patients were withdrawn because they did not meet
entry criteria or were no longer receiving either enalapril or
lisinopril. Lipid profiles such as low-density lipoprotein
(LDL), high-density lipoprotein (HDL), triglycerides, total
cholesterol (T. chl), fasting glucose (FBG) and body mass
index (BMI) were measured from both groups of subjects.
Seventy-two patients were assigned enalapril or lisinopril
20 mg once daily (20 mg/d). Prior to treatment initiation, all
patients received advice with emphasis on lifestyle changes
according to the clinical protocol including body weight
control, alcohol moderation, sodium reduction, and
increased consumption of fresh fruits and vegetable and
low-fat dairy products.20 The study also included 72 healthy
volunteer controls recruited from the residents of Seremban
and surrounding areas. BP was measured in all the study
subjects using a sphygmomanometer at least three times at
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an interval of five minutes in accordance with the proce-
dures recommended by the seventh Joint National
Committee on Prevention, Detection, Evaluation, and
Treatment of High Blood Pressure (JNC VII) guidelines.21
Sample collection
Prior to receiving the ACEIs, 4–5 ml of blood samples of
hypertensive subjects were collected from the peripheral
blood leukocytes into an ethylenediaminetetraacetic acid
(EDTA) tube (Becton Dickinson, NJ, USA) by a qualified
phlebotomist. Plasma was separated from the blood by
centrifugation method and stored at –20ºC for further
analysis.
Genotyping of I/D polymorphism
Genomic DNA from peripheral blood was isolated with
the QIAamp Blood DNA Mini Kit (QIAGEN, Germany).
The purity of extracted DNA was checked using
Biophotometer (Eppendorf, Germany). To determine the
I/D polymorphism of the ACE gene, a flanking primer
pair18 5’ CTGGAGACCACTCCCATCCTTTCT-3’ and
5-GATGTGGCCATCACATTCGTC ACGAT-3’ (synthe-
sized by Research Biolabs, Malaysia) was used to amplify
the genotypes of the I/D polymorphism of the ACE gene
by conventional polymerase chain reaction (PCR) tech-
nique. PCR amplification was performed with a 25 μl reac-
tion mixture that contained 20 pmol of each primer, 0.4
mmol/l each deoxynucleotide triphosphate (dNTP), 2
mmol/l MgCl2, 1XTaq buffer and one unit of NEB Taq
DNA polymerase (New England Biolabs, Beverly, MA,
USA). The PCR cycling conditions were carried out on an
iCycler machine (BioRad Laboratories, Hercules, CA,
USA) with an initial denaturation step of five minutes at
94°C, followed by 30 cycles of denaturation at 94°C for 30
seconds, annealing at 58°C for one minute and extension
at 72°C for two minutes, followed by a final extension for
five minutes at 72°C before the storage of the samples at
4°C. PCR products were separated by agarose gel electro-
phoresis (Promega, Madison, WI, USA). DNA fragments
were stained in ethidium bromide and visualized by Alpha
Imager (Alpha Innotech, San Leandro, CA, USA) under
ultraviolet (UV) light. The PCR fragments showed three
genotypes: a 490 bp band (II), a 190 bp band (DD), and
both a 490 and a 190 bp band (ID). Mistyping of ID
heterozygotes as D homozygotes may occur. To
increase the specificity of DD genotyping, PCR ampli-
fications were also performed with an insertion-specific
primer pair 5’-TGGGACCACAGCGCCCGCCACTAC-3’
and 5’-TCGCCAGCCCTCCCATGC CC-ATAA-3’ used
in each sample that had the DD genotype.22 PCR condi-
tions were performed with one minute of initial denatura-
tion at 94°C, followed by 30 cycles of denaturation at 94°C
for 30 seconds, annealing at 67°C for 45 seconds and
Heidari et al. 3

Table 1.  Distribution of ACE I/D polymorphism in Asians and other populations.

Ethnicity Allele frequency References

  I D p value  
Malaysian pooled 0.65 0.35 0.03a  
Malays 0.21 0.71 0.01a  
Chinese 0.63 0.37 0.04a Jayapalan et al., 200825
Indians 0.58 0.42 0.04a  
Malays 0.23 0.77 0.00a Current study
Asian Indians 0.36 0.64 0.04a Movvaa et al., 200726
Han Chinese 0.56 0.44 0.08b Ji et al., 201027
Southern China 0.32 0.68 0.03b Chang et al., 200728
Singaporeans 0.41 0.69 0.03a Jiang et al., 200929
Japanese 0.61 0.39 0.01a Mondry et al., 200530
Asian Indians 0.44 0.56 0.03a Das et al., 200931
Indonesians 0.36 0.64 0.80b Rasyid et al., 201232
Europeans 0.47–0.58 0.37–0.48 0.09b Bleumink et al., 200533
Australians 0.42 0.58 0.000a Jones et al., 200834
Americans 0.51 0.49 0.07a Arnett et al., 200514
Arabs 0.21–0.42 0.66–0.77 0.03a Bayoumi et al., 200635
Koreans 0.31 0.69 0.01a Bae et al., 200736
Mexican 0.65–0.79 0.35–0.45 0.07b Vargas-Alarcón et al., 200337
Indians 0.33 0.67 0.07b Das et al., 200831
Bahraini 0.44 0.66 0.00a Al-Harbi et al., 201238
South Turkey 066 0.44 0.08b Acarturk et al., 200539

ACE: angiotensin-converting enzyme; I: insertion; D: deletion. ap < 0.05, bp > 0.05.

extension at 72°C for two minutes. Under these condi-

tions, the reaction showed the presence of an I allele at the
335 bp amplicon and no products in samples that were
homozygous for the DD genotype. Identical results were
obtained when genotyping was performed on two separate
occasions.
Statistical analysis
All computations were carried out with the SPSS program,
version 20. Results are expressed as mean±SD (standard
deviation). Sample size was adequate for the present study
determined by using standard statistical method at the ratio
of 1:1 for case and control groups, at the significance level
of 0.05 at power 80% on the basis of prevalence of minor
alleles referred from previous studies.23,24 Chi square (X2)
goodness of fit was used to verify the agreement of
observed genotype frequencies with those expected
(Hardy-Weinberg equilibrium). Differences between gen-
otype groups were tested with analysis of variance
(ANOVA) using Bonferroni’s method for multiple com-
parisons between genotype classes. The multilinear step-
wise regression was made to assess the factors responsible
for the reduction of BP. P < 0.05 was considered statisti-
cally significant. Calculation of mean arterial pressure
(MAP) is determined by the formula given below:
MAP = ( 2 × diastolic ) + systolic  / 3
Where diastole counts twice as much as systole because
two-thirds of the cardiac cycle is spent in diastole. The
usual range of MAP is 70–110, and a MAP of about 60 is
necessary to perfuse coronary arteries, brain and kidneys.
Results
A total of 72 hypertensive patients and 72 controls were
recruited in our study. The D allele of the ACE I/D poly-
morphism was significantly associated with Malaysian
Malay male hypertensive subjects. Our study results are
well in accordance with other populations and differed
from few ethnicities. Table 1 shows the list of populations
with conflicting results in relation to the ACE I/D gene
polymorphism. The mean age of hypertensives was
47.22±11.3 and that of normotensives was 46.92±12.7
years. Mean BMI, heart rate, different biochemical param-
eters and SBP and DBP of the study subjects are shown in
Table 2.
Genotypes of I/D polymorphism were determined by
incubating the PCR product. The fragments were resolved
on 2% agarose gel. Figure 1 shows the PCR product and
gene polymorphisms. The absence/presence of a 287-bp

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4 Journal of the Renin-Angiotensin-Aldosterone System

Table 2.  Demographic and clinical characteristics of the study

population.

Hypertensives Normotensives p value

Total 72 72 –
Age (years) 47.22±11.3 46.92±12.7 0.81
BMI (km/m2) 27.42 ± 2.7 21.60 ± 3.1 0.0001a
HR (beats/min) 76.7 ± 8.1 75.8 ± 7.7 0.79
FBG (mg/dl) 92.54 ± 17.5 91.9 ± 14.3 0.75
LDL-C (mg/dl) 87.3±21.6 92.3±27.3 0.11
HDL-C (mg/dl) 42.4±6.1 38.3±6.2 0.08 Figure 2.  Detection of mistyping of ID heterozygotes in 2%
TG (mg/dl) 166.1±30.3 169.9±20 0.78 agarose gel electrophoresis. Lanes 1, 2, 4 and 5 show mistyping
TC (mg/dl) 149.8±31.9 150.2±28.4 0.81 of ID heterozygotes as D homozygotes, which show the
SBP (mmHg) 152.0±13.0 120±13.3 0.0001a presence of the I allele (335 bp), whereas lanes 3, 6 and 7 show
DBP (mmHg) 94.6±8.7 80.6±2.8 0.0001a no products, which means there are confirmed homozygous
DD genotypes of I/D polymorphism. M represents a 100-bp
Values in parenthesis indicate percentages. Mean ± SD for age, body DNA ladder. I: insertion; D: deletion; ACE: angiotensin-
mass index (BMI), systolic, heart rate (HR), fasting glucose (FBG), blood converting enzyme.
pressure (SBP), diastolic blood pressure (DBP), total cholesterol (TC),
low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein
cholesterol (HDL-C) and triglycerides (TG). aSignificant difference at p associations of ACE genotypes (ID+DD) with EHT, as the
< 0.05 from controls.
observed difference in genotypes between the controls and
patients was statistically significant: X2 = 29.415, degree
of freedom (df) = 1, p < 0.0001, odds ratio (OR) = 3.13
(1.9150–5.2868) at 95% confidence interval (CI). A statis-
tically significant association of the D allele was observed
between the case and control subjects: X2 = 14.67, p =
0.00013 (Table 3). OR and relative risk adjusted for age in
both genotypes and alleles. The observed changes in SBP
and DBP were analyzed to determine their association
with genotypes at the ACE gene locus. There was a statisti-
cally significant reduction in SBP in patients with II, ID
and DD genotypes, 3±0.2 mmHg, 4.1±3.3 mmHg,
Figure 1.  Detection of I/D polymorphism of the ACE gene 18.5±8.1 mmHg, respectively (p < 0.05) when treated with
in 2% agarose gel electrophoresis. Lanes 3, 7, 8 and 9 show ACEI enalapril or lisinopril for 24 weeks. Similarly, there
homozygous II genotypes; lanes 1, 2, 4, 6 and 10 show was a significant reduction in DBP genotypes, i.e. 0.11±6.1
heterozygous ID genotypes; lane 5 shows homozygous DD mmHg, 9.1±3.5 mmHg and 15.29±7.1 mmHg, respec-
genotypes of I/D polymorphism. M represents a 100-bp DNA
ladder. I: insertion; D: deletion; ACE: angiotensin-converting
tively (p = 0.06) when treated with ACEI enalapril or lisin-
enzyme. opril. The decrease in SBP and DBP after 24 weeks of
treatment of the patients carrying the DD genotype (SBP =
18.5±14.1 mmHg, DBP = 15.29±7.1 mmHg) was greater
Alu repeated sequence of I/D polymorphism represents the than the groups carrying ID (SBP = 4.1±3.3 mmHg, DBP
DD genotype (190 bp), whereas II indicates the homozy- = 9.1±3.5) and II genotypes (3± 0.2 mmHg, DBP =
gous (490 bp) and ID heterozygote genotypes (490 and 0.11±6.1 mmHg. MAP before ACEI treatment in patients
190 bp). Figure 2 shows the mistyping of ID heterozygotes with three genotypes, i.e. DD, ID and II, were comparable
of I/D polymorphism of the ACE gene. (p < 0.05). In our study, the MAP in patients with DD
The distribution of genotypes DD, ID and II in the con- genotypes (117.9±13.5 mmHg) was higher than the nor-
trols and patients did differ significantly from that expected mal range of 70–110 mmHg and there was significantly
under Hardy-Weinberg equilibrium. In the total of 72 greater reduction in MAP after treatment with enalapril or
patients, the DD genotype was observed in 43 (59.42%), lisinopril as compared to patients with ID (p = 0.03) and
the ID genotype was observed in 25 (35.33%) and the II II genotypes (p = 0.001) (Table 4). Allele frequencies of
genotype in four (5.25%) of the case subjects. The observed each polymorphism in the study population were in the
genotypes in controls were 19 (26.41%) DD, 35 (49.96%) Hardy-Weinberg equilibrium. Table 5 shows the changes
ID and 18 (23.65%) II genotypes. The allele frequencies in of BP in response to lisinopril and enalapril drugs among
patients were found to be 0.77 and 0.27 for the D and I I/D genotypes. Although two given BP-lowering drugs
alleles, respectively, whereas 0.51 and 0.49 for D and I was reduced MAP significantly, enalapril has a greater reduc-
observed in control subjects. We found significant tion level (II: 1.5±4.3, ID: 8.4 ± 3.7, DD: 16.4 ± 7.8)

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Heidari et al. 5

Table 3.  Genotypic and allelic frequencies of I/D polymorphism of the ACE gene among hypertensives and normotensives.

Genotypes Hypertensives (HT) (%) Normotensives (NT) (%)

DD (%) 43 (59.42%) 19 (26.41%)
ID (%) 25 (35.33%) 35 (49.96%)
II (%) 4 (5.25%) 18 (23.65%)
X2  
p value 0.0003a  
Alleles  
D 111 (77.08%) 74 (51.3%)
I 33 (22.92%) 70 (48.61%)
X2  
p value 0.000a  
OR (95% CI) HT versus NT 3.18 (1.9150–5.2868)  

Genotype frequencies are indicated as absolute values and percentages. Allele frequencies are indicated as fractions. I: insertion; D: deletion; ACE:
angiotensin-converting enzyme; OR: odds ratio; CI: confidence interval. ap < 0.05 is considered to be significant.

Table 4.  Intergenotypic (I/D variant of the ACE gene) variations in systolic and diastolic blood pressure response when treated
with ACE inhibitors (enalapril, lisinopril) in patients with essential hypertension.

BP (mmHg) ACE I/D genotypes p value

  II ID DD  
SBPB 148.2±3.1 149.4±13.40 158.6±22 0.043a
SBPA 145.2± 2.9 145.3±10.1 140.1±13.9 0.0002a
SBPΔ 3± 0.2 4.1±3.3 18.5±8.1 0.0001a
DBPB 96.21±13.2 94.7±10.9 97.6±9.2 0.05
DBPA 94.10±7.1 88.3±7.4 82.31±2.1 0.0001a
DBPΔ 0.11±6.1 9.1±3.5 15.29±7.1 0.0001a
MAPB 113.5±9.8 112.5±11.7 117.9±13.5 0.06
MAPA 111.1±5.7 107.3±8.3 101.6±6.0 0.0001a
MAPΔ 2.4± 4.1 5.2± 3.4 16.3±7.5 0.0001a
  p value
  II vs. DD DD vs. ID ID vs. II
SBPB 1.0 0.059 0.092
SBPA 0.019 0.0001 0.016
DBPB 0.22 1.00 0.11
DBPA 0.22 1.00 0.10
MAPB 0.012 0.72 1.0
MAPA 0.0001 0.001 0.03

I: insertion; D: deletion; ACE: angiotensin-converting enzyme; SBPA: systolic blood pressure after treatment; SBPB: systolic blood pressure before
treatment; SBPΔ: average systolic blood pressure; DBPA: diastolic blood pressure after treatment; DBPB: diastolic blood pressure before treatment;
DBPΔ: average diastolic blood pressure; MAPA: mean arterial pressure after treatment; MAPB: mean arterial pressure before treatment; MAPΔ:
average mean arterial pressure. SBP, DBP, and MAP were compared with respect to genotypes with t-test of significance test at one degree of free-
dom adjusted for age. p < 0.05 is considered to be significant. aAnalysis of variance (ANOVA) using Bonferroni’s method for multiple comparisons
between genotype classes.

compared with lisinopril (II: 0.8± 4.3, ID: 7.5±3.5, DD:
14.2 ±13.1) (Table 5).
Discussion
Researchers are still investigating whether the ACE gene
polymorphism helps in predicting the success of ACE
inhibition among hypertensives and other complications.
Taking that into an account and to the best of our knowl-
edge, the current study is the first comprehensive report on
the I/D polymorphism of the ACE gene in response to
ACEIs among Malay male hypertensive subjects. Our
results show that there was a significant association in car-
riers of genotype DD compared with the individuals with

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6 Journal of the Renin-Angiotensin-Aldosterone System
Table 5.  I/D polymorphism and BP response to enalapril and lisinopril among hypertensive patients.
Enalapril
BP (mmHg) ACE I/D genotypes
  II ID DD
SBPΔ 4.2± 0.3 7.1±4.2 19.7±9.2
DBPΔ 0.11±6.1 9.1±3.5 15.29±7.1
MAPΔ 1.5± 4.3 8.4± 3.7 16.4±7.8
I: insertion; D: deletion; BP: blood pressure; ACE: angiotensin-converting enzyme; SBPΔ: average systolic blood pressure; DBPΔ: average diastolic
blood pressure; MAPΔ: average mean arterial pressure. ap < 0.01.
ID and II genotypes in lowering BP (p < 0.0001). Several
studies have reported associations between essential
hypertension and ACE (I/D) gene polymorphism such as in
Taiwan,15 Turkey,40 Malaysia41 and Chinese populations.42
However, studies reported the lack of association of the
I/D gene polymorphism with EHT.43,44 We have conducted
this study in Malay male subjects to find an association
between the I/D gene polymorphism and EHT. In our
study, a co-dominant pattern of inheritance was seen in the
study subjects as the genotype frequencies in both groups
were all in accordance with the Hardy-Weinberg equilib-
rium. Results from our study have shown the distribution
of I/D genotypes among patients in the order of DD>ID>II,
which was similar to Malaysian41 and China45 but quite
different from the Tunisian.46 The frequency of the D allele
among controls in our study, which was 0.51, is lower than
the study in the Malaysian population41 and approximately
lower than in the Turkish population.47 Multivariate logis-
tic regression analysis (regressed for age) revealed that
individuals with the D allele were at 1.8 times higher odds
(3.18 (1.9150–5.2868) at 95% CI, X2 = 24.415, p =
0.00013) of developing EHT. Our study showed a higher
OR of 3.18 (95% CI, 1.9150–5.2868) compared to a recent
meta-analysis indicated that D allele was associated with a
20% increase risk of hypertension (OR = 1.22, 95% CI,
1.10–1.29).11 The relative risks for hypertension are 1.3 for
subjects carrying the DD genotype and 1.28 for those hav-
ing the D allele of the ACE (I/D) gene polymorphism. This
study provides the normal distribution of the genotypes
and alleles in I/D polymorphism among Malay male in
Malaysian population and suggests that genetic factors
play an important role in the etiology of hypertension
among Malay male hypertensive subjects. Our study
results are well in accordance with other studies.16,17,48
ACEIs are recommended for managing cardiovascular dis-
eases and renal diseases.49–51 However, there is substantial
variability in individual responses to these agents. For
example, fewer than 50% of hypertensive patients achieve
adequate BP control with ACEI monotherapy.52 An
increasing number of studies have indicated that patients
from different ethnic groups have different responses to
ACEIs.53 Heterogeneity in the individual BP response to
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Lisinopril
ACE I/D genotypes
p value II ID DD p value
0.0001a 2.21±1.1 6.10±2.9 17.90±11.1 0.0001a
0.0001a 0.13±5.92 8.13±3.8 12.94±4.6 0.0003a
0.0001a 0.8±4.3 7.5±3.5 14.2±13.1 0.0001a
ACEI therapy still represents an obstacle in the treatment
of hypertension. Comparison of genotypes has shown a
significant difference in the frequencies of ACE (I/D) gene
polymorphisms that vary significantly by race.15,41,47 Thus,
it is possible that the ethnic diversity in genotypes contrib-
utes to the observed variability in ACEI responses among
races. Arnett et al.14 reported the association of the D allele
was associated with greater BP response in women patients
with EHT treated with hydrochlorothiazide for four weeks.
Milionis et al. found no relationship between the renin-
angiotensin-aldosterone system (RAAS) gene polymor-
phisms and BP response to ACEIs among patients with
hypertension and metabolic syndrome.54 The inconsist-
ency in results might be explained by the genetic and envi-
ronmental heterogeneity among different ethnic groups,
differences in ACE inhibitors55 used and study sample
size. The conflicting results among the various populations
led us to determine the relationship between the ACE I/D
gene polymorphism and BP response to an ACEIs enal-
april and lisinopril in an appropriate sample size of 142
Malay male hypertensive subjects. Our study reported that
the association of the D allele was associated with greater
BP response in 72 newly diagnosed Malay male subjects
and then treated with ACEI enalapril or lisinopril for 24
weeks. In our study, the MAP in patients with the DD gen-
otype was higher than the normal range of 70–110 mmHg
and there was significantly greater reduction in MAP after
treatment with enalapril or lisinopril as compared to
patients with the ID (p = 0.03) and II genotypes (p =
0.001). The present study has some limitations but,
although our study sample was relatively small, the results
show that the D allele has a strong association with EHT in
response to ACEIs. However, further studies with larger
sample sizes are recommended to confirm the association
of the I/D polymorphism of the ACE gene.
Conclusion
Our study shows that individuals with the D allele of I/D
polymorphism of the ACE gene were strongly associated
with Malay male hypertensive subjects in blunting the BP
response to ACEIs.
Heidari et al. 7
Acknowledgements
The authors gratefully like to extend their gratitude to all the vol-
unteers participated in this study.
Conflict of interest
None declared.
Funding
This research received no specific grant from any funding agency
in the public, commercial, or not-for-profit sectors.
References
1. Hansen TW, Li Y, Boggia J, et al. Predictive role of the
nighttime blood pressure. Hypertension 2011; 57: 3–10.
2. Rampal S, Rampal L, Rahmat R, et al. Variation in the prev-
alence, awareness, and control of diabetes in a multiethnic
population: A nationwide population study in Malaysia.
Asia Pac J Public Health 2010; 22: 194–202.
3. Carretero OA and Oparil S. Essential hypertension. Part I:
Definition and etiology. Circulation 2000; 101: 329–335.
4. Zhu X, Chang YP, Yan D, et al. Associations between
hypertension and genes in the renin-angiotensin system.
Hypertension 2003; 41: 1027–1034.
5. Ehret GB. Genome-wide association studies: Contribution
of genomics to understanding blood pressure and essential
hypertension. Curr Hypertens Rep 2010; 12: 17–25.
6. Konoshita T. Do genetic variants of the renin-angiotensin
system predict blood pressure response to renin-angiotensin
system-blocking drugs? A systematic review of pharmacog-
enomics in the renin-angiotensin system. Curr Hypertens
Rep 2011; 13: 356–361.
7. Rigat B, Hubert C, Alhenc-Gelas F, et al. An insertion/dele-
tion polymorphism in the angiotensin I-converting enzyme
gene accounting for half the variance of serum enzyme lev-
els. J Clin Invest 1990; 86: 1343–1346.
8. Taverne K, de Groot M, de Boer A, et al. Genetic polymor-
phisms related to the renin-angiotensin-aldosterone system
and response to antihypertensive drugs. Expert Opin Drug
Metab Toxicol 2010; 6: 439–460.
9. Hermida RC, Ayala DE, Fernández JR, et al. Circadian
rhythms in blood pressure regulation and optimization of
hypertension treatment with ACE inhibitor and ARB medi-
cations. Am J Hypertens 2011; 24: 383–391.
10. Brugts JJ, Isaacs A, Boersma E, et al. Genetic determinants
of treatment benefit of the angiotensin-converting enzyme-
inhibitor perindopril in patients with stable coronary artery
disease. Eur Heart J 2010; 31: 1854–1864.
11. Johnson AD, Newton-Cheh C, Chasman DI, et al. Association
of hypertension drug target genes with blood pressure and
hypertension in 86,588 individuals. Hypertension 2011; 57:
903–910.
12. Nordestgaard BG, Kontula K, Benn M, et al. Effect of ACE
insertion/deletion and 12 other polymorphisms on clini-
cal outcomes and response to treatment in the LIFE study.
Pharmacogenet Genomics 2010; 20: 77–85.
13. Baudin B. Angiotensin I-converting enzyme gene polymor-
phism and drug response. Clin Chem Lab Med 2000; 38:
853–856.
Downloaded from jra.sagepub.com at Universiti Putra Malaysia on July 7, 2014
14. Arnett DK, Davis BR, Ford CE, et al. Pharmacogenetic
association of the angiotensin-converting enzyme inser-
tion/deletion polymorphism on blood pressure and cardio-
vascular risk in relation to antihypertensive treatment: The
Genetics of Hypertension-Associated Treatment (GenHAT)
study. Circulation 2005; 111: 3374–3383.
15. Chung CM, Wang RY, Chen JW, et al. A genome-wide
association study identifies new loci for ACE activ-
ity: Potential implications for response to ACE inhibitor.
Pharmacogenomics J 2010; 10: 537–544.
16. Taverne K, de Groot M, de Boer A, et al. Genetic polymor-
phisms related to the renin-angiotensin-aldosterone system
and response to antihypertensive drugs. Expert Opin Drug
Metab Toxicol 2010; 6: 439–460.
17. Schelleman H, Klungel OH, van Duijn CM, et al. Drug-gene
interaction between the insertion/deletion polymorphism of
the angiotensin-converting enzyme gene and antihyperten-
sive therapy. Ann Pharmacother 2006; 40: 212–218.
18. Schelleman H, Klungel OH, van Duijn CM, et al. Insertion/
deletion polymorphism of the ACE gene and adherence to
ACE inhibitors. Br J Clin Pharmacol 2005; 59: 483–485.
19. Yu H, Zhang Y and Liu G. Relationship between polymor-
phism of the angiotensin-converting enzyme gene and the
response to angiotensin-converting enzyme inhibition in
hypertensive patients. Hypertens Res 2003; 26: 881–886.
20. Ahmad N, Hassan Y, Tangiisuran B, et al. Guidelines
adherence and hypertension control at a tertiary hospital in
Malaysia. J Eval Clin Pract 2013; 19: 798–804.
21. Bajaj JK. Prescription patterns of antihypertensive drugs
and adherence to JNC VII guidelines in a tertiary care hos-
pital in north India. International Journal of Medical and
Clinical Research 2012; 3: 118–120.
22. Shanmugam V, Sell KW and Saha BK. Mistyping ACE het-
erozygotes. PCR Methods Appl 1993; 3: 120–121.
23. French B, Joo J, Geller NL, et al. Statistical design of per-
sonalized medicine interventions: The Clarification of
Optimal Anticoagulation through Genetics (COAG) trial.
Trials 2010; 11: 108.
24. Schork NJ and Topol EJ. Genotype-based risk and pharma-
cogenetic sampling in clinical trials. J Biopharm Stat 2010;
20: 315–333.
25. Jayapalan JJ, Muniandy S and Chan SP. Angiotensin-1
converting enzyme I/D gene polymorphism: Scenario in
Malaysia. Southeast Asian J Trop Med Public Health 2008;
39: 917–921.
26. Movvaa S, Alluric RV, Komandur S, et al. Relationship of
angiotensin-converting enzyme gene polymorphism with
nephropathy associated with Type 2 diabetes mellitus in
Asian Indians. J Diabetes Complications 2007; 21: 237–241.
27. Ji L, Zang L, Shen P, et al. Association of angiotensino-
gen gene M235T and angiotensin-converting enzyme gene
I/D polymorphisms with essential hypertension in Han
Chinese population: A meta-analysis. J Hypertens 2010;
28: 419–428.
28. Chang YC, Wu WM, Chen CH, et al. Association between
the insertion/deletion polymorphism of the angiotensin
converting enzyme gene and risk for psoriasis in a Chinese
population in Taiwan. Br J Dermatol 2007; 156: 642–645.
29. Jiang X, Sheng H, Li J, et al. Association between renin-
angiotensin system gene polymorphism and essential
8 Journal of the Renin-Angiotensin-Aldosterone System
hypertension: A community-based study. J Hum Hypertens
2009; 23: 176–181.
30. Mondry A, Loh M, Liu P, et al. Polymorphisms of the inser-
tion/deletion ACE and M235T AGT genes and hypertension:
Surprising new findings and meta-analysis of data. BMC
Nephrol 2005; 6: 1.
31. Das M, Pal S, Ghosh A, et al. Angiotensin converting
enzyme gene polymorphism (insertion/deletion) and hyper-
tension in adult Asian Indians: A population-based study
from Calcutta, India. Hum Biol 2008; 80: 303–312.
32. Rasyid H, Bakri S and Yusuf I. Angiotensin-converting
enzyme gene polymorphisms, blood pressure and pulse
pressure in subjects with essential hypertension in a South
Sulawesi Indonesian population. Acta Med Indones 2012;
44: 280–283.
33. Bleumink GS, Schut A, Sturkenboom MC, et al. Mortality
in patients with hypertension on angiotensin-I converting
enzyme (ACE)-inhibitor treatment is influenced by the
ACE insertion/deletion polymorphism. Pharmacogenet
Genomics 2005; 15: 75–81.
34. Jones GT, Thompson AR, van Bockxmeer FM, et al.
Angiotensin II type 1 receptor 1166C polymorphism is
associated with abdominal aortic aneurysm in three inde-
pendent cohorts. Arterioscler Thromb Vasc Biol 2008; 28:
764–770.
35. Bayoumi RA, Simsek M, Yahya TM, et al. Insertion dele-
tion polymorphism in the angiotensin-converting enzyme
(ACE) gene among Sudanese, Somalis, Emiratis, and
Omanis. Hum Biol 2006; 78: 103–108.
36. Bae Y, Park C, Han J, et al. Interaction between GNB3
C825T and ACE I/D polymorphisms in essential hyperten-
sion in Koreans. J Hum Hypertens 2007; 21: 159–166.
37. Vargas-Alarcón G, Hernández-Pacheco G, Rodríguez-Pérez
JM, et al. Angiotensin-converting enzyme gene (ACE)
insertion/deletion polymorphism in Mexican population.
Hum Biol 2003; 75: 889–896.
38. Al-Harbi EM, Farid EM, Gumaa KA, et al. Genotypes and
allele frequencies of angiotensin-converting enzyme (ACE)
insertion/deletion polymorphism among Bahraini popula-
tion with type 2 diabetes mellitus and related diseases Mol
Cell Biochem 2012; 2: 219–223.
39. Acarturk E, Attila G, Bozkurt A, et al. Insertion/deletion
polymorphism of the angiotensin converting enzyme gene
in coronary artery disease in southern Turkey. J Biochem
Mol Biol 2005; 14: 887–990.
40. Bayram B, Sayin E, Gunes HV, et al. DD genotype of ace
gene I/D polymorphism is associated in a Turkish study
population with osteoarthritis. Mol Biol Rep 2011; 38:
1713–1716.
41. Ramachandran V, Ismail P, Stanslas J, et al. Association
of insertion/deletion polymorphism of angiotensin-
converting enzyme gene with essential hypertension and
Downloaded from jra.sagepub.com at Universiti Putra Malaysia on July 7, 2014
View publication stats
type 2 diabetes mellitus in Malaysian subjects. J Renin
Angiotensin Aldosterone Syst 2008; 9: 208–214.
42. Bai Y, Wang L, Hu S, et al. Association of angiotensin-
converting enzyme I/D polymorphism with heart failure: A
meta-analysis. Mol Cell Biochem 2012; 361: 297–304.
43. Chiang FT, Lai ZP, Chern TH, et al. Lack of associa-
tion of the angiotensin converting enzyme polymorphism
with essential hypertension in a Chinese population. Am J
Hypertens 1997; 10: 197–201.
44. Gu XX, Spaepen M, Guo C, et al. Lack of association
between the I/D polymorphism of the angiotensin-converting
enzyme gene and essential hypertension in a Belgian popu-
lation. J Hum Hypertens 1994; 8: 683–685.
45. Liu T, Korantzopoulos P, Xu G, et al. Association between
angiotensin-converting enzyme insertion/deletion gene
polymorphism and atrial fibrillation: A meta-analysis.
Europace 2011; 13: 346–354.
46. Arfa I, Nouira S, Abid A, et al. Lack of association between
renin-angiotensin system (RAS) polymorphisms and hyper-
tension in Tunisian type 2 diabetics. Tunis Med 2010; 88:
38–41.
47. Nacak M, Nacak I, Sanli M, et al. Association of angioten-
sin converting enzyme gene insertion/deletion polymor-
phism with lung cancer in Turkey. Cancer Genet Cytogenet
2010; 198: 22–26.
48. Srivastava K, Sundriyal R, Meena PC, et al. Association
of angiotensin converting enzyme (insertion/deletion)
gene polymorphism with essential hypertension in north-
ern Indian subjects. Genet Test Mol Biomarkers 2012; 16:
174–177.
49. Januszko-Giergielewicz B, Kubiak M and Gromadziński
L. The role of combination therapy in the management of
hypertension in patients with chronic kidney disease [article
in Polish]. Przegl Lek 2013; 70: 199–204.
50. Neutel JM. The role of combination therapy in the manage-
ment of hypertension. Nephrol Dial Transplant 2006; 21:
1469–1473.
51. Plat F and Saini R. Management of hypertension: The
role of combination therapy. Am J Hypertens 1997; 10:
262–271.
52. Nelson M. Drug treatment of elevated blood pressure. Aust
Prescr 2010; 33: 108–112.
53. Arnett DK, Claas SA and Glasser SP. Pharmacogenetics of
antihypertensive treatment. Vascul Pharmacol 2006; 44:
107–118.
54. Milionis HJ, Kostapanos MS, Vakalis K, et al. Impact of
renin-angiotensin-aldosterone system genes on the treat-
ment response of patients with hypertension and metabolic
syndrome. J Renin Angiotensin Aldosterone Syst 2007; 8:
181–189.
55. Gradman AH, Basile JN, Carter BL, et al. Combination ther-
apy in hypertension. J Am Soc Hypertens 2010; 4: 90–98.