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Angiotensin-converting enzyme gene insertion/deletion polymorphism in Egyptian patients with
myocardial infarction
Ahmad Settin, Rizk ElBaz, Amr Abbas, Ayman Abd-Al-Samad and Ahmed Noaman
Journal of Renin-Angiotensin-Aldosterone System 2009 10: 96
DOI: 10.1177/1470320309105198
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Paper
Angiotensin-converting enzyme gene insertion/deletion
polymorphism in Egyptian patients with myocardial
infarction
Ahmad Settin,* Rizk ElBaz,* Amr Abbas,† Ayman Abd-Al-Samad,# Ahmed Noaman^
Key words:
angiotensinconverting
enzyme,
Egypt, gene
polymorphism,
ischaemic heart
disease,
myocardial
infarction
*
Department of
Genetics, Faculty of
Medicine, Mansoura
University, Mansoura,
Egypt.
†
Department of Medical
Physiology, Faculty of
Medicine, Mansoura
University, Mansoura,
Egypt.
#
Department of
Cardiology, Faculty of
Medicine, Mansoura
University, Mansoura,
Egypt.
^
Department of
Zoology, Faculty of
Science, Mansoura
University, Mansoura,
Egypt.
Correspondence to:
Prof Dr Ahmad Settin
Prof of Pediatrics and
Genetics,
Qassim University, PO
Box 6655,
Buraydah 51452,
Qassim, Saudi Arabia.
Tel: +966 6 380
0050/2523
Fax: +966 6 380 1228
Email: settin60@gmail.
com
Journal of
the ReninAngiotensinAldosterone
System
(Including other
Peptidergic systems)
Abstract
Introduction. This work aimed to test the
association of the angiotensin-converting enzyme
gene insertion/deletion (I/D) polymorphism with
myocardial infarction.
Subjects and methods. This study comprised 79
Egyptian myocardial infarction cases with a mean
age of 54.4±9.9 years including 60 males and 19
females, plus 238 healthy unrelated individuals of
nearly matched age and sex as a control group.
For all subjects, DNA testing for the angiotensinconverting enzyme gene I/D polymorphism was
done using PCR amplification for detection of
both the D and I alleles followed by a second run
PCR specific for the I allele for samples typed as
DD in the first run.
Results. Cases had a higher frequency of DD
(29.1%) and ID (62.0%) genotypes than II (8.9%)
genotype, with a higher frequency of D allele
than I allele (64.4% vs. 33.6%). Compared to
controls, cases had a significantly higher
frequency of ID genotype (62.0% vs. 47.5%,
p<0.05). This was more apparent among cases in
the low risk group (p=0.002) than in the high
risk group (p=0.041).
Conclusion. The angiotensin-converting
enzyme gene I/D polymorphism is
probably a risk factor for ischaemic
heart disease among Egyptian cases,
particularly if integrated with other
environmental and genetic risk factors.
Introduction
Coronary artery disease (CAD) is a multifactorial
disease influenced by environmental and genetic
factors. Family history of premature CAD in addition to other risk factors, such as smoking, obesity,
diabetes, and dyslipidaemia, are all interactive factors contributing to the occurrence of the disease.1
Although the role of these environmental factors
in the development of myocardial infarction (MI)
has been clearly established, the role of nonconventional risk factors remains undefined. In
the last few years, great interest has been focused
on genetic factors with the intention of finding
common markers that could identify a subgroup
of patients at higher risk of death or with a worse
prognosis in which new therapeutic timings and
interventions could be tested.2
In an enzymatic cascade, angiotensinogen is
cleaved by renin to produce angiotensin I,
which is further converted to the bioactive
octapeptide angiotensin II (Ang II) through
the action of angiotensin-converting enzyme
(ACE), a membrane- bound, zinc metalloendopeptidase involved in the metabolism of many
small peptides. ACE and angiotensinogen play
an important role in blood pressure and blood
volume homeostasis.3 Thus, it is not surprising
that the genes coding for this system are being
investigated in relation with MI.2
Concentrations of plasma and tissue ACE are
determined by the ACE gene located on chro­
mosome 17q23. This gene manifests a 287-bp
repeated Alu sequence insertion (I) or deletion
(D) polymorphism in intron 16.4 The homozygous DD genotype, which is associated with a
two- to threefold increase in levels of ACE, may
cause a variety of adverse cardiovascular effects.5
The increased risk of MI associated with the ACE
D allele is graded, with low risk for ACE II, intermediate risk for ACE ID, and high risk for ACE
DD genotypes, which suggests codominant
inheritance.6,7 It is hypothesised that in subjects
with the ACE DD genotype, higher levels of ACE
may contribute to coronary thrombogenesis.8
Recently, it has been reported that changes in
pulse pressure and cardiac remodelling were
detected among cases of MI having DD and ID
genotypes rather than the II genotype.9,10
Month
June 2009
2009
Volume X
10
Number X
2
SAGE Publications 2009 Los Angeles, London, New Delhi and Singapore
10.1177/1470320309105198
96
96
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Paper
Reports on the associations of the ACE gene I/D
polymorphism, in addition to being controversial
in various Arabic and Mediterranean countries,
are relatively lacking among Egyptian patients
with MI. Therefore, this work was planned to
investigate this association among cases from the
Nile Delta region of Egypt.
Subjects and methods
Subjects
This study comprised 79 cases with MI taken
randomly from those admitted in the Intensive
Care Unit of the Cardiology Department, Internal
Medicine Specialised Hospital, Mansoura University,
Mansoura, Egypt. Their mean age was 54.4±9.9
years with an age range of 25–75 years. There
were 60 (75.9%) males and 19 (24.1%) females.
Of them, 23 (29.1%) were smokers, 21 (26.6%)
had a positive family history of MI, 25 (31.6%)
were diabetic, and 16 (20.3%) were hyperlipidaemic. Regarding risk factors, cases were classified
into high risk group with two or more risk factors
and low risk group with no or only one risk factor. For comparison, 238 healthy individuals of
nearly matched age and sex and with no history
of any cardiac diseases or evident cardiovascular
risk factors were taken as a control group.
Informed consent was taken from all subjects in
addition to an approval from the University
Ethical and Scientific Committees.
Journal of
the ReninAngiotensinAldosterone
System
(Including other
Peptidergic systems)
June 2009
Volume 10
Number 2
Determination of the ACE gene I/D
polymorphism
From each subject, 3 ml venous blood was collected in a polyethylene tube containing EDTA
solution as an anticoagulant and kept frozen
until used for DNA extraction using the Generation
DNA Purification Capture Column Kit (Gentra
Systems Inc., MN, USA). PCR amplification was
done with the respective fragments from intron
16 of the ACE gene according to the method
described by Lindpaintner et al.11 Briefly, 20 µl of
a PCR master mix containing 1 mM primers, 200
mM deoxynucleotide triphosphates, 1.3 mM magnesium chloride, 50 mM potassium chloride, 10
mM TRIS-hydrochloric acid (pH 8.4 at 25ºC),
0.1% Triton X-100, and 0.35 unit of Taq polymerase was added. Optimised primer pair was
used to amplify the D and I alleles, resulting in
319-bp and 597-bp amplicons, respectively
(5’-GCC CTG CAG GTG TCT GCA GCA TGT-3’
and 5’-GGA TGG CTC TCC CCG CCT TGT
CTC-3’). The thermocycling profile (Genius;
Techne, UK) consisted of denaturation at 94ºC
for 30 seconds, annealing at 56ºC for 45 seconds,
and extension at 72ºC for 2 minutes, repeated for
Figure 1
Panel A (PCR products, first run) shows positive bands for both
the D (319 bp) and I (597 bp) alleles, i.e. ID genotype in lanes
3, 5, 7, and 8, and only one band for the D allele, i.e. DD
genotype in lanes 2, 4, and 6. Panel B (PCR products, second
run) shows a positive band for the I allele (335 bp) in lanes 2,
3, 5, 7, and 8. This denotes that the sample in lane 2 is actually
an ID genotype. D = deletion; I = insertion.
35 cycles, followed by a final extension at 72ºC
for 7 minutes. The amplification products of the
D and I alleles were identified by electrophoresis
on a 2% agarose gel and visualised on a 300-nm
ultraviolet transilluminator. Because the D allele
in heterozygous samples is preferentially amplified, each sample found to have the DD genotype was subjected to a second, independent
PCR amplification with a primer pair that recognises an insertion-specific sequence (5’-TGG
GAC CAC AGC GCC CGC CAC TAC-3’ and
5’-TCG CCA GCC CTC CCA TGC CCA TAA-3’),
with identical PCR conditions except for an
annealing temperature of 67ºC. The reaction
yields a 335-bp amplicon only in the presence of
an I allele, and no product in samples homozygous for DD (figure 1).
Statistical analyses
Data were processed and analysed using the
Statistical Package of Social Science (SPSS, version 10.0). The frequency of studied allelic polymorphisms among cases was compared to that of
controls and tested for positive association using
chi-square (c2), Fisher’s exact tests and odds ratio
(OR) with 95% confidence interval (95% CI).
A minimum level of significance was considered
if p was ≤ 0.05. Furthermore, the distribution
of alleles in studied groups was tested for fitting
to the Hardy-Weinberg equilibrium assuring no
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Paper
Table 1
Distribution of the ACE gene I/D polymorphism in a sample of patients with acute MI compared to controls.
Cases
(n=79)
Controls
(n=238)
Fisher p
OR (95% CI)
Genotypes
DD
23 (29.1%)
113 (47.5%)
0.0057**
ID
49 (62.0%)
113 (47.5%)
0.027*
II
7 (8.9%)
12 (5.0%)
0.27
Alleles
(n=158)
(n=476)
D
95 (64.36%)
339 (71.20%)
0.01*
I
63 (33.64%)
137 (28.78%)
0.01*
0.45 (0.26-0.79)
1.81 (1.07-3.04)
1.80 (0.69-4.83)
0.61 (0.42-0.88)
1.64 (1.13-2.39)
Key: * p<0.05 and ** p<0.01 (significance level testing each group versus controls). ACE = angiotensin-converting enzyme;
D = deletion; I = insertion; MI = myocardial infarction; OR (95% CI) = odds ratio (95% confidence interval).
Table 2
Distribution of ACE I/D genotypes among low risk and high risk subjects compared to controls using c2 test of trend.
Studied groups
Controls (n=238)
Total cases (n=79)
Low risk cases (n=30)#
High risk cases (n=44)
ACE genotypes
DD
n (%)
ID
n (%)
113 (47.5)
23 (29.1)
6 (20.0)
13 (29.5)
113 (47.5)
49 (62.0)
20 (66.7)
28 (63.6)
II
n (%)
c2
p
12 (5.0)
7 (8.9)
8.28 0.004**
4 (13.3)
9.58 0.002**
3 (6.8)
4.18
0.041*
Key: # Five cases dropped from the analysis with non-informative history to be assigned to particular risk group. * p<0.05 and
** p<0.01 (significance level testing each group versus controls). ACE = angiotensin-converting enzyme; D = deletion;
I = insertion.
significant difference between observed and
expected frequencies using the c2 test.
Results
The distribution of the ACE gene I/D poly­
morphism in studied cases of MI and controls
(table 1) showed that cases had a higher frequency of DD and ID genotypes than II genotype (29.1%, 62.0%, and 8.9%, respectively).
Compared to controls, cases had a significantly
lower frequency of the DD genotype (29.1% vs.
47.5%, p<0.05), but with a significantly higher
frequency of the ID genotype (62.0% vs. 47.5%,
p<0.05). On the other hand, cases showed a
higher frequency of the II genotype than controls, but this was statistically non-significant
(8.9% vs. 5.0%, p>0.05).
Journal of
the ReninAngiotensinAldosterone
System
(Including other
Peptidergic systems)
June 2009
Volume 10
Number 2
using c2 test of trend (table 2) showed that low
risk cases had a higher frequency of ID (66.7%)
than controls (47.5%), which was statistically significant (p=0.002). The same was observed in the
high risk group, but with a lower level of significance (63.6% vs. 47.5%, p=0.041).
On the other hand, comparing ACE I/D genotype
frequencies in cases-subgroups related to each
single risk factor, such as age of onset, sex,
hyperlipidaemia, smoking, positive family history, diabetes, and obesity, revealed no significant difference (data not shown).
Regarding allele frequency, the D allele was the
predominant allele in all cases (64.4% vs. 33.6%
for the I allele). However, when compared to
controls, the D allele frequency was found to be
significantly lower among cases than controls
(64.4% vs. 71.2%, p<0.05).
Discussion
CAD continues to be the main cause of death in
developed countries. During the last decade, there
has been a growing interest in the study of the
ACE gene I/D polymorphism as a potential risk
factor for conditions like MI.12 However, despite
the large number of studies with different designs
and populations, the role of the ACE gene I/D
polymorphism on MI is still controversial.13
Comparison of the ACE I/D genotypes among
low risk and high risk subjects with controls
This study of the ACE gene I/D polymorphism
among Egyptian controls showed an equal
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frequency of ID and DD genotypes that was
relatively high (47.4% each), with a lower frequency of the II genotype. The same finding has
also been reported in Saudi Arabia (47.1% for DD
and 41.0% for ID) and in Italy (44.5% and 42.8%,
respectively).14,15 However, almost all other studies showed a higher frequency of the ID genotype among their control groups (ID genotype
frequency was one and a half to two times as
much as the DD). Examples include studies in
Japan,16 South Asia,17 Chile,18 Australia,19 France,20
USA,11 and Germany.21
Regarding the ACE gene I/D polymorphism
among Egyptian MI cases, this study showed a
higher frequency of the ID than the DD genotype, with a lower frequency of the II genotype.
The frequency of the D allele was also higher
than the frequency of the I allele. This is in
agreement with most studies, e.g. the ones in
Colombia,22 India,23 Saudi Arabia,14 France,24
Poland,25 and Italy.2 However, compared to controls, this study showed a significantly higher
frequency of the ID genotype among MI cases,
but with a significantly lower frequency of the
DD genotype. The same finding was reported by
Dzimiri et al. in Saudi Arabia.14 Furthermore, in
Turkey, Acarturk et al. reported that the ID
genotype was the most frequent in all subjects
who underwent diagnostic coronary angiography, although in cases found to have CAD, the
DD genotype was higher compared to the
controls.26 In Poland, Zak et al. reported that the
D allele carriers (DD + ID genotypes) were more
frequent in the CAD patients compared to the
control group, whereas the familial CAD risk
group showed the highest frequency of the
ID genotype.25
Journal of
the ReninAngiotensinAldosterone
System
(Including other
Peptidergic systems)
June 2009
Volume 10
Number 2
The lower frequency of the DD genotype observed
among our cases may be due to the fact that our
sample was restricted to new MI cases in the
Intensive Care Unit rather than ambulant cases
under maintenance treatment. Here we can speculate that the patients with DD genotype can
manifest early with alarming signs of hypertension
or angina and receive treatment as ambulatory
cases. On the other hand, cases with ID genotype,
who may have an intermediate elevation of ACE,
may be neglected until coming under the effect of
other risk factors that lead together to the outcome of acute MI. This is manifested by having
63% of our cases with two or more risk factors. In
this respect, we recommend a bigger sample
including both acute cases with MI in addition to
ambulatory cases with ischaemic heart disease;
considering at the same time the various risk
factors pertaining to the disease.
Studies stressing the possible role of ACE DD
genotype as a risk factor in CAD related to
disease prognosis in terms of disease onset and
mortality include ones in France,6 New Zealand,27
UK,28 Colombia,22 Spain,29,30 and South Africa.4
However, in the Rotterdam Study (The
Netherlands), Sayed-Tabatabaei et al. reported
no association between the ACE gene I/D polymorphism and MI, although they observed an
increased risk of cardiovascular mortality for carriers of the D allele among smokers in younger
subjects and this diminished at later ages.31 On
the other hand, Agrawal et al. in India23 and
Franco et al. in Italy2 observed that the DD genotype was slightly more frequent in patients as
compared to controls; however, the differences
were not significant.
From this study, we conclude that the ACE gene
I/D polymorphism probably has some association with MI among Egyptian patients, particularly if integrated with other environmental and
genetic risk factors.
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Journal of
the ReninAngiotensinAldosterone
System
(Including other
Peptidergic systems)
June 2009
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Number 2
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