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2. disease

Egyptian Journal of Medical Microbiology, October 2009
Vol. 18, No. 4
The Association of Angiotensin-1 Converting Enzyme (ACE) Serum
Level and (I/D) Polymorphism in Egyptian Preeclamptic Women
Maha A. El Bassuoni MD1, Hanna Younes MD2, Randa M. Talaat MD3,
Hanaa Amer MD2. Enas S. Eissa MD1 and Riham G. Mahfouz MD4
1
Clinical Pathology Dept., Faculty of Medicine, Menoufiya University, 2 Obstetric and
Gynecology Dept., Faculty of Medicine, Al Azhar University, 3 Microbiology Dept.
Genetic Engineering and Biotechnology Sadat Institute, Menoufiya Universitym
4
Biochemistry Dept. Faculty of Medicine, Menoufiya University
ABSTRACT
Preeclampsia (PE) a major cause of maternal and neonatal mortality and morbidity worldwide in which
hypertensive disorders during pregnancy account for 25.7% of maternal deaths. Both maternal and fetal
genetic factors may predispose towards pre-eclamptic pregnancy, especially severe forms. However,
preeclampsia is thought to be the result of the interplay between important genetic components and
environmental influences; still, factors and mechanisms that lead to preeclampsia remain mysterious.
Insertion/deletion (I/D) polymorphism of of ACE gene has attracted significant attention and has been
extensively investigated with its serum activity in a spectrum of cardiovascular phenotypes. Aim to study
the potential association of I/D polymorphism of ACE gene in PE Egyptian women that gets us closer to
understanding the disease. Patients and Methods: One hundred hypertensive and age-matched
normotensive primigravidae were recruited from Menoufiya university Hospital. Routine investigations
were done for PE diagnosis. DNA was extracted from whole blood of patients and healthy controls. All
samples were genotyped for ACE I/D polymorphism according to Rigat et al. using amplification and
PCR of known allelic variants. ACE genotype was identified and followed by serum concentration of ACE
activity for both groups. Results: ACE DD genotype was found in 60% of PE patients while 34% of
normotensive subjects (P≤ 0.005). The odds ratio (95% CI) for developing hypertension in cases with DD
genotype was 2.91 (1.29-6.57), while with II genotype was 0.18 (0.02-1.63). DD genotype revealed
significantly more prevalent among cases than controls (P≤ 0.005), where it was 2.9 folds higher among
cases than controls. The incidence of D allele of ACE gene was higher in hypertensive (0.98) than in
normotensive (0.90, P > 0.05), although D allele was higher among cases than controls, but it did not
show significance (P > 0.05). High significance was revealed when comparing the mean total ACE
activity in the hypertensive patients (32.74 IU/l) and normotensive subjects (28.06 IU/l) (P <0.001). The
ACE activity in cases and controls carrying DD allele differed significantly (P<0.001). In contrast the
other ACE genotype ID and II did not show significance between cases and controls. Conclusion: These
findings might bear implications for precise management of pregnancy in high-risk DD genotype women.
Further large scale evaluation was required to provide added marker for risk assessment for PE patients.
Previous proposals found that there are both
maternal and fetal genetic factors that may
predispose towards pre-eclamptic pregnancy,
especially severe forms of preeclampsia.
Maternal family history has the greatest
influence on PE risk, but paternal family history
of the condition can also contribute (Skjærven
et al., 2005)
The causes of preeclampsia are not well
understood, but several factors are known to
contribute to the risk. Factors include diabetes,
high blood pressure prior to pregnancy, obesity,
and first pregnancy, also the possibility in part,
a genetic basis. The condition is more likely
among women whose relatives have also had it
(Norma, 2006).
INTRODUCTION
Preeclampsia (PE) is a maternal disease of
pregnancy associated with increased blood
pressure and proteinuria after 20 weeks of
gestation (Díaz, 2006). It is a major cause of
maternal and neonatal mortality and morbidity
worldwide and has account for 25.7% of
maternal deaths during pregnancy (WHO,
1988). The risk for an adverse pregnancy
outcome in women who have previously had
preeclampsia is markedly higher (from 20% to
40%) in comparison to women with history of
normal pregnancy, but the mechanisms involved
have not yet been identified (Mello, 2003).
89
Egyptian Journal of Medical Microbiology, October 2009
No definite genetic cause has yet been
confirmed. However, PE is thought to be the
result of the interplay between important genetic
components and environmental influences; still,
factors and mechanisms that lead to
preeclampsia remain elusive (Ray et al., 2005).
As a result, there is a lack of effective
preventive interventions (et al, 2002).
Angiotensin-1 Converting enzyme (ACE),
which codes for the ACE gene, has been linked
with preeclampsia in a number of different
studies. The protein encoded by ACE is
involved in controlling blood pressure and the
balance of fluid and salts in blood (Rigat et al.,
1990).
Over
the
past
decade,
the
insertion/deletion (I/D) polymorphism of a 287bp in intron 16 of (ACE) gene has attracted
significant attention and has been extensively
investigated in a spectrum of cardiovascular
phenotypes, because of its correlation with
serum ACE activity (Niu et al, 2002 ).
Inappropriate activation of the renin–
angiotensin system may play a part in the
development of many cardiovascular disorders,
including preeclampsia (Agerholm-Larsen et
al., 1990). A common insertion/deletion
polymorphism
within
the
angiotensin-I
converting enzyme gene (ACE-I/D) has been
reliably associated with substantial differences
in the plasma and tissue angiotensin-converting
enzyme (ACE) activity in a codominant
(additive) fashion not only in persons of
European descent, but also in other populations
such as Hispanics (Kammerer et al., 2005).
Previous experimental studies (Langer et
al., 1998) suggested that the "physiological
remodeling" of spiral arteries throughout
pregnancy is mediated by the renin-angiotensin
system (RAS), which is one of the main factors
regulating blood pressure, and fluid and
electrolyte balance (Ito.etal.,1992). Throughout
normal pregnancy, the RAS is stimulated;
plasma
renin
activity,
angiotensinogen,
angiotensin II, and aldosterone levels are all
increased (Ward.etal, 1993) At the same time,
pregnancy induces refractoriness to the pressor
effects of angiotensin II (Morgan et al., 1999)
Moreover, recent data have shown that
upregulation of angiotensin II type 1 (AT1)
receptor subtype in the syncytiotrophoblasts
could play a patho-physiological role patients
with PE (Jackson et al., 2000). Moreover, PE is
characterized by the loss of this physiological
refractoriness to angiotensin II (Morgan et al.,
1999 and Miller et al., 1988).
Vol. 18, No. 4
Plasma and tissue ACE activities are under
genetic control. Increased ACE activity due to
the deletion polymorphism of the ACE gene is
associated with diseases that exhibit endothelial
disturbance. Studies in various ethnic group
have shown contradictory evidence on the
association of ACE insertion/deletion (I/D)
polymorphism with preeclampsia PE (El
Shafei, 2007).
The researchers theorize that Ang II may
restrict the fetal vessels that lie within the
chorionic villi, which not only raises blood
pressure, but also lowers oxygen and nutrient
flow to the baby and may result in lower birth
weight and other complications of preeclampsia
(Anton, 2009).
ACE inhibitor drugs are currently used to
lower Ang II in non-pregnant women with
hypertension, but these drugs cannot be given to
pregnant women.
Other therapies aimed at regulating blood
pressure might be beneficial if they target the
chorionic villi rather than the system as a whole.
The current workis being done to determine if
growth factors that cause the placenta's blood
supply to develop may also be regulated by the
increase in Ang II (Anton, 2009).
Aim of the study
To investigate the hypothesis that
preeclampsia is associated both ACE gene
insertion-deletion polymorphism (I/D) and
serum ACE activity and analyzing the
relationship between preeclampsia and ACE
genotype.
PATIENTS & METHODS
One hundred hypertensive and age-matched
normotensive primigravidae were recruited
from Menoufiya university Hospital. These
subjects were followed up to delivery and
perinatal outcome noted. The Inclusion Criteria
were: Pregnant women with preeclampsia
between 20-32 weeks gestation. The criteria
used for the diagnosis of PE were in accordance
with the guidelines of the American College of
Obstetrics
and
Gynecology
(ACOG).
Preeclampsia is defined as a maternal blood
pressure of >140/90 mmHg on two readings at
least 6 hours apart with proteinuria of >300
mg/24 hours. The ACE genotype was identified
and serum ACE activity was measured.
Genotyping was performed for all the studied
cases taking into account some well-known
contributing factors in PE such as maternal age,
primiparity, gestational age and proteinuria. All
90
Egyptian Journal of Medical Microbiology, October 2009
these variables were significantly associated
with PE. All the pregnancies with known
maternal vascular diseases, e.g. essential
hypertension, diabetes and systemic lupus
erythematosis or congenital abnormalities were
excluded. None of the women had vaginal
bleeding or labor pains at the day of blood
sampling. The control groups were gestational
age-matched healthy pregnant women and had
uncomplicated pregnancies
2-Methods
A. Molecular Diagnosis for genotypes of ACE
gene
Peripheral venous blood samples were
collected from the antecubital vein in Vacutainer
tubes containing 0.129 mol/L sodium citrate, the
final blood/anticoagulant ratio being 9:1.
Genomic DNA was extracted from whole blood
leukocytes using a QIAmp Blood Kit
(QIAGEN, Hilden, Germany). All samples
were genotyped for ACE I/D polymorphisms
using amplification after PCR of known allelic
variants.I/D polymorphism in intron 16th of
ACE gene was determined according to the
Vol. 18, No. 4
method of Rigat et al. (Lindpaintner, et al.,
1995), DNA was amplified at an annealing
temperature of 60°C in the presence of 5%
dimethylsulfoxide (DMSO) to reduce the
incidence of mistyping ID as DD (Schimidt, et
al., 1995). Moreover, each DD genotype was
subjected to a second PCR amplification
without 5% DMSO at an annealing temperature
of 67°C and by using a primer pair that
recognizes the insertion-specific sequence.
These modifications were made to reduce
underestimation of heterozygotes (Forgarty et
al., 1996). Positive controls consisting of DNA
samples from subjects of known genotypes
(from previous work) and a negative control of
complete PCR reaction mix without DNA were
included in each PCR reaction. PCR
amplification yielded a fragment with (I allele)
or without the insertion allele (D allele) of ~ 490
and 190bp, respectively. All PCR products were
resolved
on a 2% agarose gel by
electrophoresis, these alleles were scored
accordingly.
Lane 1: Molecular weight marker (50-bp ladder);
lane 2: DD homozygote;
lane 3: ID heterozygote with presence of both 190-bp D fragment and 490-bp I fragment;
lane 4: II homozygote
Determination of ACE genotypes by PCR amplification on ethidium bromide-stained agarose gel. Three
patterns of PCR products (II, ID, and DD) for ACE gene I/D polymorphism are shown. Arrows on right
indicate molecular weights of PCR products (allele I and D correspond to 490- and 190-bp products,
respectively).
centrifuge at 4°C and 1000 x g, freezed
specimen at –20°C if not assayed within 5 days.
ACE activity in sterile serum is stable for up to
30 days at 2-8°C and 6 months at –20°C.
To avoid lipemic sera, blood samples
should be taken from fasting patients. Due to
interference with the photometric determination
B. Serum concentration of ACE activity
SPECIMEN
COLLECTION
AND
STORAGE
Since EDTA inhibits ACE activity, serum
specimen should be used for the determination
of ACE activity.Sufficient bloodwas collected
(at least 0.5 ml) by venipuncture into an
appropriate tube without anticoagulant.
91
Egyptian Journal of Medical Microbiology, October 2009
Vol. 18, No. 4
subjects with DD genotype was 2.91 (1.29-6.57)
, while the odds ratio for developing
hypertension in subjects with II genotype was
0.18 (0.02-1.63).This shows that DD was
significantly more prevalent among cases than
controls (P≤ 0.005), where it was 2.9 folds
higher among cases than controls. The incidence
of D allele of ACE gene was higher in
hypertensive
subjects
(0.98)
than
in
normotensive subjects (0.90, P > 0.05), and
although D allele was higher among cases than
controls, but there was no significant difference
betweenACE DD genotype in groups both (P >
0.05). However, the ACE genotype was similar
in different categories of PIH(Pre-eclampsia
induced hypertension) and did not affect the
perinatal outcome.
The mean total ACE activity in the
hypertensive subjects was (32.74 IU/l) and in
normotensive subjects (28.06 IU/l) were of high
significance (P <0.001). The ACE activity in
cases and controls carrying DD allele differed
significantly (P<0.001), in contrast of the other
ACE genotype ID and II which did not show
significant difference in between both cases and
controls groups(P> 0.05).
Icteric or hemolytic sera cannot be used for
ACE activity determination.
PRINCIPLE OF THE ASSAY
ACE catalyses the conversion of
angiotensin I to angiotensin II. The kinetic of
this cleavage reaction is measured by recording
the decrease in absorbance at 340 nm. The ACE
kinetic method is standardized with the
BÜHLMANN ACE colorimetric kit (order
code: 01-KK-ACE) according to the reference
method described (Ronca-Testoni, 1983 and
Bénéteau and Baudin et al. 1986).
CALCULATION
Calculate the corresponding enzyme activity
(Ex) of each unknown sample by dividing the
difference in absorbance of the individual
sample (∆Ax) through the mean of the
absorbance difference of the calibrator vial
(∆Ac) and multiply the results by the enzyme
activity (Ec).
RESULTS
ACE DD genotype was found in 60% of
subjects with PE but only in 34% of
normotensive subjects (P≤ 0.005). The odds
ratio (95% CI) for developing hypertension in
Table 1: The clinical data of PE patients and gestational age-matched controls
Variables
CASES
CONTROLS
t
(n.=50)
(n.=50)
Maternal age
28.731±5.759
31.5±3.5144
-1.26245
Systolic blood pressure
175.384±25.937
113.5±13.753
7.361628
Diastolic blood pressure
10.738±12.659
77.5±8.959
6.6338
Table 2: ACE Genotype Frequencies in Patients and Controls
ACE
CASES
CONTROLS
X2
P value
genotype
(n.=50)
(n.=50)
NO.
%
NO.
%
DD
30
60
17
34
6.78
<0.005
ID
19
38
28
36
3.25
>0.05
II
1
2
5
10
2.84
>0.05
Table 3: Incidence of D allele among cases and controls.
Group
Cases
Controls
(n.=50)
(n.=50)
NO.
%
NO.
%
D allele
49
98
45
90
92
P value
>0.05
<0.001
<0.001
Odds ratio (95% CI)
2.91 (1.29-6.57)
0.48 (0.22-1.06)
0.18 (0.02-1.63)
Z test
P value
1.26
>0.05
Egyptian Journal of Medical Microbiology, October 2009
Table 4: Total ACE activity Units/L
Patients and Controls
Studied
Mean Total ACE
Groups
activity Units/L
Hypertensive 32.74 ± 8.19
Controls
28.06 ± 5.34
P value`
<0.001
Vol. 18, No. 4
(mean value) in different ACE Genotype Frequencies of
Mean ACE activity IU / L
II
ID
21.3 ± 0.00
36.41 ± 7.58
19.61 ± 3.97
34.27 ± 6.4
>0.05
>0.05
DD
39.9 ± 3.14
23.46 ± 4.28
<0.001
(P<0.0001) between women with and those
without preeclampsia recurrence and fetal
growth restriction as well as an association
(P<0.0007) between DD genotype and risk of
recurrent preeclampsia or fetal growth
restriction. His study showed an association
between ACE DD genotype and a recurrent
pregnancy negative outcome after PE in a
previous pregnancy indicating that DD genotype
is not associated with risk of PE in the first
pregnancy. Finding an association between the
DD genotype and a negative outcome in the
successive pregnancy suggests that the DD
genotype can be related to PE and FGR(foetal
growth retardation only.
Nevertheless, Mando et al, 2009 set up that
the distribution of ACE genotypes was different
in PE. This was confirmed in mild PE, whereas
no significance was found in severe PE. This
could suggest that different factors may lead to
mild and severe PE, with ACE polymorphism
playing a more important role in the mild form.
Gürdöl et al., 2003 found an association
between the genotype II and low ACE activity
in preeclamptic women and an association
between D allele frequency and preeclampsia in
Turkish women. Pregnancy alone did not have
an effect on the ACE activity.
They explained by that uterine artery
resistance indexes were significantly lower in II,
higher in DD, and intermediate in ID genotype
carriers, whereas the
Umbilical artery pulsatility index values
were significantly higher in the DD group in
comparison to ID and II genotypes. their study
shows that the ACE I/D polymorphism affects
uteroplacental and umbilical flows and the
recurrence of an adverse pregnancy outcome in
women with history of preeclampsia.
Choi et al. 2004 established that the
frequency of DD genotype was significantly
greater in preeclampsia (0.36) than in controls
(0.14) (p<0.05). The frequency of D allele was
0.55 in preeclampsia and 0.40 in controls
(p<0.05). However, there were no differences in
Statistical Analyses
All data are presented as mean ± SD or
percent. Mean 2 tests were used for categoric
variables. Gene polymorphisms ACE DD vs.
ID/II; in addition, two-way interactions of these
latter variables were also tested. All statistical
analyses were performed using statistical
packages (Instat v.2; Graph Software for
Science, San Diego, CA USA).
DISCUSSION
Plasma and tissue ACE (angiotensin
converting enzyme) activities are under genetic
control. Increased ACE activity due to the
deletion polymorphism of the ACE gene is
associated with diseases that exhibit endothelial
disturbance. Studies in various ethnic group
have shown contradictory evidence on the
association of ACE
Insertion/deletion (I/D) polymorphism with
preeclampsia (PE) (El Shafei, 2007).
In the present study, ACE DD genotype
was found in 60% of subjects with PE but only
in 34% of normotensive subjects (P≤0.005). The
odds ratio for developing hypertension in
subjects with DD genotype was 2.91 (1.29-6.57
- 95% CI ) , while the odds ratio for developing
hypertension in subjects with II genotype was
0.18 (0.02-1.63).This shows that DD was
significantly more prevalent among cases than
controls (P≤0.005), where it was 2.9 folds
higher among cases than controls.
Carrying the DD genotype may have some
influence on the pathogenesis of preeclampsia,
perhaps through effects on placental hypoxia or
the interaction of hypertensive disease and
atherosclerosis, although this influence may not
be strong. Additional studies using a larger
number of patients and analyses that include
other genetic and environmental factors will be
necessary to confirm these results (Kobashi et
al., 2005).
In line with these results were Mello et al,
2003 who found a difference in genotype
distribution (P<0.0002) and allele frequency
93
Egyptian Journal of Medical Microbiology, October 2009
Vol. 18, No. 4
was 1.14 (0.56-2.32) he concluded that his study
has found no evidence that the insertiondeletion polymorphism in the angiotensinconverting enzyme gene is associated with preeclampsia.
In an Egyptian study, El Shafei et al., 2007
showed that the ACE II genotype may have a
predisposing effects on preeclampsia especially
in younger women and/or in women early
pregnancy. The ACE DD genotype didn’t show
any association with preeclampsia. They
concluded that the genetic susceptibility in
preeclampsia needs more studies about the role
of other candidate genes in addition to the ACE
gene.
CONCLUSIONS
ACE gene polymorphism in PE women
included: insertion homozygote II, deletion
homozygote (DD) and insertion/deletion
heterozygote (ID) was higher than in
controls.These findings suggested that ACE I/D
polymorphism maybe involved in maternal
uteroplacental modulation and fetal umbilical
flows which might bear implications for more
accurate management of pregnancy in high-risk
DD genotype women. The clinical relevance of
these results deserves to be further evaluated in
larger studies to provide an additional marker
for risk assessment of patients with PE history.
the onset of preeclampsia and pregnancy
outcomes according to the ACE genotypes.
Previous studies have documented that the
D/D polymorphism of the ACE gene is
associated with increased plasma ACE
concentrations, cardiac diseases such as
myocardial infarction and left ventricular
hypertrophy, and progression of diabetic
nephropathy. These associations, however, have
not been consistently replicated.
Norma et al., 2006 found that in an
additive model (per-D-allele) revealed a null
association between the ACE-I/D variant and
preeclampsia risk (crude OR = 0.95 [95% CI,
0.81-1.10]) in a case-control study. They
concluded that It is highly likely that the
observed small nominal increase in risk of
preeclampsia associated with the ACE D-allele
is due to small-study bias, similar to that
observed in cardiovascular disease. Reliable
assessment of the origins of preeclampsia using
a genetic approach may require the
establishment of a collaborating consortium to
generate a dataset of adequate size.
In addition, Zhang et al, 2006 study, did
not detect any association between the ACE
D/D polymorphism and renal functional
deterioration in univariate analysis.Several
studies have now examined the association of
the ACE D/D polymorphism with PE. We
believe that the discrepancies in the above
studies and the present work univariate and
multivariate analyses may be in part related to
sample size. Furthermore, Li.etal., 2007 found
no association of the ACE gene polymorphisms
with preeclampsia. However, ACE gene I/D
polymorphisms were associated with the severe
proteinuria and the increase in serum uric acid,
urea, and creatinine and renal dysfunction seen
in
preeclampsia.
They
inferred
that
Preeclampsia patients carrying the D allele may
be susceptible to renal dysfunction.
In contrast to the present finding,
Young.etal, 2004 indicated that the ACE
polymorphism play no significant role in
preeclampsia observed in Korean women. In
addition, neither the I-D genotype distributions
nor the allele frequencies differed significantly
between pre-eclamptic and normotensive
pregnancies in maternal or fetal samples in a
study conducted by Morgan et al., 1999. In his
study, the odds ratio for pre-eclampsia in
women with the DD genotype, compared with
the ID and II genotype, was 1.09 (95%
confidence interval 0.55-2.16). The odds ratio
associated with the DD genotype in the fetus
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