Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:304-309
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:304-309.)
© 1996 American Heart Association, Inc.
Deletion Polymorphism in the Angiotensin-Converting Enzyme Gene in Patients With a History of Ischemic Stroke
Maurizio Margaglione;
Egidio Celentano;
Elvira Grandone;
Gennaro Vecchione;
Giuseppe Cappucci;
Nicola Giuliani;
Donatella Colaizzo;
Salvatore Panico;
Francesco P. Mancini;
Giovanni Di Minno
From Clinica Medica, Istituto di Medicina Interna e Malattie
Dismetaboliche, Universita' di Napoli, and Unita' di Trombosi e
Aterosclerosi, IRCCS "Casa Sollievo della Sofferenza," S Giovanni
Rotondo, Italy.
 |
Abstract
|
|---|
Abstract We evaluated the genotypes of the
angiotensin-converting
enzyme (ACE) gene in 101
subjects with and 109 subjects without
a history of ischemic
stroke. All were attending a metabolic
ward. The two groups
were compared for major risk factors for
ischemic events.
Genotypes were determined by polymerase chain
reaction with
oligonucleotide primers flanking the polymorphic
region
in intron 16 of the ACE gene. Deletion polymorphism of the
ACE
gene (DD genotype) was shown to be more common in subjects
with
a history of stroke than in those without (relative risk, 1.76;
confidence
intervals, 1.02 to 3.05). A positive family history for
ischemic
complications of atherosclerosis was
also more common in subjects
with documented events (relative risk,
1.99; confidence intervals,
1.10 to 3.59). DD genotype and a
positive family history were
strong independent discriminators of
cerebral ischemia. Plasma
levels of tissue-type
plasminogen activator (TPA) and
plasminogen
activator inhibitor-1
help identify subjects with a history
of cerebral ischemic
episodes. When such fibrinolytic variables
were included in the
analysis, the DD genotype still strongly
and
independently discriminated subjects with a stroke history
and
significantly interacted with TPA levels >10 ng/mL in
such
identification. We conclude that in subjects attending
a
metabolic ward, homozygosity for a deletion
polymorphism of
the ACE gene consistently discriminates
subjects with a stroke
history. Interaction with TPA improves such
identification.
Key Words: ACE genotype • risk factor interaction • family history • fibrinolytic variables
|
Introduction
|
|---|
Stroke is a major
complication of atherosclerotic cardiovascular
disease
and a leading cause of morbidity and mortality in Western
countries.
Individuals who smoke, have high blood pressure or
high plasma levels
of cholesterol or glucose, or are obese are
at risk for
this
event.
1 2 3 4 5
However, such risk factors
account for only
about one third of the future ischemic
episodes.
6 7 8 The regulation of the
renin-angiotensin system is a central
event in
cardiovascular pathophysiology.
9 Family
and population
studies have reported that an insertion/deletion
polymorphism
in the ACE gene is associated with marked differences
in serum
10 and cellular
11 ACE levels, with
higher levels being associated
with homozygosity for the ACE D
allele (DD genotype).
10 11 The DD
genotype has been shown to be a risk factor for myocardial
infarction
in low-risk populations.
12 Moreover,
increased frequency in
the deletion has been associated with a parental
history of
myocardial infarction
13 14 and with
increased
coronary risk
in non–insulin-dependent diabetic
patients.
15 We have
evaluated the genotypes of the
ACE gene in subjects with a history
of ischemic stroke
(stroke-positive) and those without such
history
(stroke-negative) attending a metabolic ward.
A large-scale prospective study has shown that plasma levels of TPA
have a predictive power with respect to ischemic
stroke.16 In the population sample analyzed in
this report, we have previously documented abnormally high circulating
levels of TPA and its inhibitor, PAI-1.17
Infusion of angiotensin II results in a substantial
increase in the circulating levels of PAI-1.18 Since we
have found that the DD genotype consistently helps
discriminate subjects with a history of stroke, we evaluated whether
interactions between the molecular variation and TPA and PAI-1 levels
could help identify stroke-positive subjects in this setting.
|
Methods
|
|---|
Subjects
From February to December 1992, 210 subjects (108
men, 102
women;
mean age, 63.6 years; range, 31 to 86) were enlisted for the
study.
They were chosen among subjects who had been attending the
metabolic
ward of the outpatient clinic of our institution.
From 8 to
12 months before being enlisted, 101 of them (51 men, 50
women)
had survived an ischemic stroke. Demographic
characteristics
of the subjects, the manner in which they were enlisted
(inclusion/exclusion
criteria), and similarities and differences among
cases (stroke-positive)
and control subjects (stroke-negative)
have been reported elsewhere.
17 None of the 210 subjects
had clinical evidence of cancer or
acute or chronic inflammatory
disease. All had been repeatedly
instructed to stop smoking and
drinking alcohol and to control
food intake, and all were highly
motivated to follow the advice.
All had been on an isocaloric
Mediterranean diet for at least
6 months. A complete clinical summary
with emphasis on personal
and family history for angina pectoris,
myocardial infarction,
ischemic stroke, peripheral
arterial disease, and vascular risk
factors was obtained
from all subjects. Positive family history
was defined as the
occurrence of stroke or myocardial infarction
before the age of 55
years in male and 60 years in female parents
and
siblings.
19 The 109 subjects without any documented
episode
of ischemic stroke were comparable to those with a
history of
stroke with respect to sex, height, occupation, social
class,
cardiovascular risk factors, and use of drugs.
In particular,
no difference between stroke-positive and
stroke-negative individuals
was found with respect to mean plasma
concentrations of total,
HDL, and LDL cholesterol,
triglycerides, and Lp(a); nor were
differences found
between cases and control subjects with respect
to the number of
subjects with high blood pressure or diabetes
mellitus (most of type
II). After approval of the local ethical
committee, the studies were
carried out according to the Principles
of the Declaration of Helsinki.
Informed consent was obtained
from all subjects.
Materials
dNTP, KCl, MgCl2, gelatin, agarose, and
mineral oil were from Perkin Elmer-Cetus; proteinase K was from USB
Corp; Lymphoprep (d=1.077), from Nyegaard Oslo; HEPES,
Tris-HCl, EDTA, ethidium bromide, and SDS were from Sigma Chemical Co.
From each subject, after 12 to 15 hours of overnight fasting, 18 mL of
blood was collected at 9 to 9:30 AM without venous stasis
from the antecubital vein via a 19-gauge scalp vein needle into a
sterile tube containing 2 mL of sterile 3.8% trisodium citrate.
Samples were processed immediately. Concentrations of total
cholesterol, HDL cholesterol,
triglycerides, and plasma glucose were detected
enzymatically17 19 with the use of commercially
available
reagents (Roche). Plasma fibrinogen was assayed by the Clauss clotting
method,19 using reagents from Boehringer-Mannheim.
Lp(a) was assayed by ELISA methods using kits from Biopool-Menarini.
Imulyse for PAI-1 and TPA antigens were from Biopool-Menarini as well.
Based on our previous data and in agreement with the manufacturer's
recommendations, normal values of TPA in our laboratory are 3 to 10
ng/mL and those of PAI-1, 4 to 42 ng/mL. Reference pooled normal plasma
from apparently healthy drug-free volunteers was prepared and
stored under the same conditions as those from the subject samples of
the study. In both stroke-positive and stroke-negative
individuals, the intra-assay and interassay coefficients of
variation of PAI-1 and TPA never exceeded 4.5%.
Isolation of DNA and Genotype
Analysis
Eighteen milliliters of blood was drawn from each patient
into 2
mL of 3.8% sodium citrate. Peripheral blood
leukocytes19 were incubated overnight at 37°C in a
digestion buffer (100 mmol/L NaCl, 10 mmol/L Tris-HCl, 25 mmol/L EDTA,
1% SDS, and 0.1 mg/mL of proteinase K). DNA was isolated by
phenol/chloroform extraction and ethanol
precipitation.19 20 PCR was used to detect the I/D
polymorphism of the ACE gene. The primers and the PCR conditions
used were the ones suggested by Rigat et al.21 Briefly,
the amplification19 22 was carried out on 50
µL-volume
samples in a Perkin Elmer-Cetus thermal cycler. Each sample contained
0.1 µg of genomic DNA, 15 pmol of each primer, 100 µmol/L of dNTPs,
10 mmol/L Tris HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 0.001% (wt/vol) gelatin, and 1 U of
Taq polymerase. The solution was overlaid with 50 µL of
mineral oil. The 30 cycles were at 93°C for 1 minute, at 60°C for 1
minute, and at 72°C for 2 minutes. The amplification products
were electrophoretically resolved in a 2% agarose gel by a 40 mmol/L
TRIS-acetate buffer, pH 7.7, containing 1 mmol/L EDTA, stained with 0.5
µg/mL of ethidium bromide,23 and visualized by UV
light.
Statistical Analysis
All the analyses were performed
according to the SPSS/PC
V2.0 statistical package and following the recommended
procedures.24 The Kolmogorov-Smirnov test, a
nonparametric method, was used to compare the distributions
of the continuous variables in stroke-positive and
stroke-negative subjects. Pearson's 
2
statistics were used to evaluate the independent nature of the clinical
condition with respect to categorical variables. Odds ratios were
calculated to evaluate the interaction between the variables, and
the Mantel-Haenszel 2 was used to evaluate
confidence intervals. Appropriate models were also set up to evaluate
in a logistic analysis the independent contribution of each
variable to the ischemic event. An enter method was used to
set up the system; log likelihood and Wald 2
statistics are presented. For all the tests, significance was
established at a value of P<.05.
|
Results
|
|---|
In the stroke-negative subjects the frequencies observed for
the
D and I alleles were 62% and 38%, respectively. These
frequencies
were compared with those predicted from the Hardy-Weinberg
equilibrium,
and no significant differences were found
(
2 test). In the stroke-positive
group, the
frequencies of the D and I alleles were 72% and 28%,
respectively.
Relative risk of stroke positivity associated to the D/I
alleles
was 1.53, with confidence intervals ranging from 1.27 to
1.86.
When the sample was stratified according to DD and II/ID
genotypes,
homozygosity for the D allele was significantly
associated with
a stroke history (relative risk, 1.76; confidence
intervals,
1.02 to 3.05) (Table 1
). Regardless of such
history, no difference
in the ACE genotypes was found in men
compared with women. In
addition to the ACE DD genotype, the
stroke history was also
significantly associated with a positive family
history (Table
1
). Among the whole population analyzed, no
difference in the
genotype distribution was found with respect
to the age being
above or below 60 or 70 years, cigarette smoking,
hypertension,
diabetes mellitus, positive family history, TPA >10
ng/dL,
or PAI-1 levels >43 ng/dL. Likewise, no difference was found
with
respect to LDL cholesterol, triglycerides,
glucose, fibrinogen,
PAI-1 antigen, TPA, or Lp(a) (Table 2
). In
contrast, a statistically
significant difference
was found with respect to total cholesterol
(
P=.03)
and HDL cholesterol (
P=.05).
Moreover, in alcohol consumers
as well as in subjects with LDL
cholesterol >1.35 g/L, the
DD genotype was more
common than the II/ID genotype (Table 3
).
On the
other hand, association studies indicated that the DD
genotype
correlated with the levels of LDL cholesterol being
above
or below 1.35 g/L (relative risk, 2.18; confidence intervals,
1.15 to
4.16). When a positive family history was the variable
taken into
consideration, it appeared to be less common in the
DD genotype
than in the DI/II genotype (28.9% versus 34.5%).
Furthermore,
a logistic regression model in which several variables
were
included revealed the strong, independent nature of the
DD
genotype as a discriminator of subjects with a stroke history
(Table
4
). Stratification of the variables according
to stroke history
(Table 5
) confirmed that a positive
family history was actually
more common in the non-DD genotype
in this setting and revealed
a trend in stroke-positive individuals
for a higher frequency
of hypertension and raised TPA and PAI-1 levels
in the DD genotype
compared with the non-DD one. Finally, in
its homozygous state,
the D allele appeared to interact with
hypertension, with alcohol
consumption, or with the lack of familial
history in identifying
subjects with a stroke history (Table
5
).
View this table:
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[in a new window]
|
Table 5. Interaction Between D/I Genotypes of the ACE
Gene and Certain Vascular Risk Factors in Patients With History of
Stroke
|
|
We have recently reported that plasma TPA concentrations >10 ng/mL and
PAI-1 concentrations >43 ng/mL consistently help identify
subjects with a history of cerebral ischemic
episodes.17 When the plasma levels of these fibrinolytic
variables were taken into consideration in this setting, TPA
antigen was confirmed to be a strong discriminator of a stroke history
(relative risk, 4.23; confidence interval, 2.28 to 7.82;
2, 22.28; P<.0001). PAI-1
antigen behaved similarly (relative risk, 2.78; confidence interval,
1.39 to 5.53; 2, 8.79;
P<.005). When these fibrinolytic variables were
included in the logistic regression model depicted in Table 4
,
TPA
appeared to be the strongest discriminator of a stroke history
(B=1.305, Wald 2=14.68,
P=.001). Under
these conditions, familial history (B=0.808, Wald
2=5.98, P=.014) and the DD
genotype (B=0.602, Wald 2=3.67,
P=.055) strongly and independently discriminated between
stroke-positive and stroke-negative individuals. Moreover, TPA
improved the ability of the DD genotype to identify
stroke-positive subjects (Table 5).
|
Discussion
|
|---|
ACE is predominantly located on capillary
endothelial cells
of vascular beds, on cells of
absorptive epithelia such as those
of the renal proximal tubule, and on
other epithelia including
those of the brain.
12 ACE
activity is detectable in plasma
and, despite large interindividual
differences, its levels are
very stable within an
individual.
12 The latter phenomenon is
largely related to
the D/I polymorphism.
9 ACE converts
angiotensin
I into the antinatriuretic
vasoactive angiotensin II, an octapeptide
involved in
vasoconstriction, aldosterone production, and
norepinephrine
release from sympathetic nerve
endings.
11 On the other hand,
infusion of
angiotensin II results in an in vivo substantial
increase
in the circulating levels of PAI-1.
18 ACE also
inactivates
bradykinin, a vasodilator and
natriuretic substance.
11 ACE
is encoded by a
21-kb 26 exon gene
25 located on chromosome
17 at
q23.
26 A deletion polymorphism in the ACE gene,
consisting
of the absence of a 287-bp Alu repetitive sequence in
reverse
orientation near the 3' end of intron 16,
12 has a
frequency
of 0.6 and is likely to be a neutral marker in tight linkage
disequilibrium
with a functional variant of the sequence yet to be
identified.
In case-control and cross-sectional studies this
deletion has
been associated consistently with coronary
risk.
10 11 12 13 14 15
Stroke is a major ischemic complication
of atherosclerosis.
However, thus far, the involvement
of the ACE gene in this event
is not understood. In the present
report, we document that the
frequency of the DD genotype is
abnormally high in subjects
with a history of ischemic stroke
just as in those with a history
of myocardial infarction. The
"multiple risk factor" theory
implies that vascular risk factors
interact cumulatively to
create high-risk
individuals.
19 Tiret et al
27 have reported
a
synergistic effect for the interaction of the DD genotype
of
the ACE gene and the C allele of the angiotensin type 1
receptor
gene in identifying subjects with a history of myocardial
infarction.
Among potential predictors of cerebral ischemia, a
large-scale
prospective study has stressed the power of TPA
antigen.
16 In our logistic model, TPA was the strongest
discriminator of
stroke-positive individuals. Our interaction
analysis (Table
5
) suggests that certain variables greatly
enhance the ability
of the DD genotype in identifying
stroke-positive subjects.
It also implies that the impact of the DD
genotype on such identification
varies considerably, depending
on demographic characteristics
of the sample analyzed. A stroke
history was more common in
individuals with the DD genotype and
TPA plasma levels >10
ng/dL than in those with a comparable
genotype and lower concentrations
of the fibrinolytic
variable. Infusion of angiotensin II results
in a
substantial increase in vivo in the circulating levels
of
PAI-1.
18 Raised concentrations of TPA antigen are
present
in subjects with high plasma PAI-1 levels, and high levels
of
both are currently thought to reflect a hypofibrinolytic
state.
28 In our setting of stroke-positive
individuals, there was a
trend for a higher frequency of raised TPA and
raised PAI-1
in the DD genotype compared with the non-DD
genotype. TPA and
PAI-1 are released from perturbed
endothelial cells.
17 TPA
has been
suggested as a marker of preclinical atherosclerosis
in
apparently healthy individuals.
29 The extent to which
raised
levels of fibrinolytic indices reflect a vascular perturbation
in
this setting cannot be evaluated. However, the abnormally high
levels
of fibrin associated with a hypofibrinolytic state may well
play
a role in the ischemic risk associated with abnormalities
of
the renin-angiotensin system.
It is of interest to relate our findings to some recent data on the ACE
genotype in high-risk individuals. Morris et
al30 reported a marked selective decrease in the frequency
of the DD genotype in subgroups of hypertensive patients of
increasing age who were not selected for cardiac pathology and had two
hypertensive parents and suggested that the DD genotype
increases the risk of premature death. In our stroke-positive
setting, the frequency of the DD genotype was higher in
subjects who experienced more than one vascular event than in those
with only one ischemic episode (58.3% versus 46.3%, Table 5).
Moreover, in the whole sample, we find a trend to an increased
frequency of the DD genotype with age (Table 6).
Our patients differ in several instances from the hypertensive subjects
evaluated by Morris et al. The majority of our subjects had other risk
factors besides hypertension. Most of the persons described here are
older than the ones described in that study. Elevated ACE DD frequency
has been reported in centenarians with molecular variations of the
apolipoprotein E gene.31 ACE is involved in a wide range
of cellular functions including tissue repair and resistance to
inflammatory and proliferative events.32 There may be
beneficial effects related to ACE activity in some high-risk
individuals that would be relevant to avoid mortality due a vascular
accident. In survivors of myocardial infarction, the ACE DD
genotype modulates the relative risk conferred by high-risk
conditions (such as positive family history or type II diabetes
mellitus)13 14 15 and helps identify
previous
ischemic events among patients regarded as low-risk
individuals (such as those with low LDL
cholesterol).12 In our stroke-positive
individuals, the DD genotype was more common in subjects with
LDL cholesterol >1.35 g/L than in those with lower LDL
cholesterol values (67% [16 of 24] versus 49% [38 of
77]). The same differences in proportion are seen in
stroke-negative subjects (56% [15 of 27] versus 34% [28 of
82]) (Table 5). In keeping with our data, in their work on
697
subjects with angiographically defined coronary heart disease,
Ludwig et al33 found that the risk for myocardial
infarction associated with the DD genotype was independent of
apoprotein B values. On the other hand, at variance with diabetes
mellitus, a positive family history was actually less common in the DD
genotype than in the DI/II genotype in our setting. The
fact that it is not the DD genotype that has the greater
frequency of a positive family history in our individuals is further
supported by the observation that in the logistic regression, ACE
genotype and positive family history were strong independent
predictors for the cerebral ischemic event (Table 4). Thus,
despite the unambiguous association between ACE DD genotype and
stroke, the positive family history appears to be mediated little by
the DD genotype in our stroke-positive individuals. Table 5
implies that the impact of the DD genotype on the
identification of subjects at risk for ischemic events varies
considerably, depending on the criteria used to select the patients and
control subjects. A large-scale prospective study in middle-age
apparently healthy "low-risk" individuals34 has
disputed the predictive power of the ACE DD genotype with
respect to ischemic heart disease. Studies on linkage
disequilibrium (association) are known to be highly sensitive to the
selection of a genetically appropriate control sample. The differences
in genetic background in the samples examined may well provide likely
explanations of the differences between these data and earlier studies
on myocardial infarction. The frequency of the DD genotype in
our stroke-negative individuals was 43%. In the study by
Lindpaintner et al34 and in that by Ludwig et
al,33 the frequency of the DD genotype was less
than 31%. The latter figures were obtained in North American
individuals and are in agreement with the originally published data
reports on this polymorphism.10 However, our estimate
is in agreement with the data obtained in another population of
Southern Italy35 and is comparable to the value (39%)
reported by Bonh et al36 in their studies on the
association between this genotype and myocardial infarction.
Our figure was independently verified by the genotyping of an
additional cross-population sample consisting of 619 apparently
healthy people 25 to 60 years old. In the latter population, 263
individuals (42.5% of the total) were homozygous for the ACE D
allele. This provides a sample for estimates of D and I allelic
frequencies in our normal population that exceeds those of other
analyses and implies that with respect to the ACE locus, our
sample is likely to be genetically representative of
the regional population. These data show that in a group of subjects
attending a metabolic ward, the ACE DD genotype,
whether alone or in combination with TPA, identifies subjects with a
history of cerebral ischemic episodes. As of now, it is not
clear whether an index combining measurements of established risk
factors and DD genotype would be a better marker of
arterial risk than the genotype or TPA evaluated
singly. On the other hand, we believe that the similarities and
differences of the results between these data and previous reports on
the ACE genotype in coronary heart disease patients
provide the rationale for longitudinal analyses in cohorts of
healthy young people followed up over many years. Although this may
raise ethical concerns in view of the effectiveness of drugs that may
offset the deleterious consequences of carrying a DD
genotype,37 38 prospective studies would be
crucial to discriminate selective advantages or disadvantages carried
out by molecular variations at this genetic locus to provide
information on relative risk estimates39 and to identify
new strategies in vascular medicine.
|
Selected Abbreviations and Acronyms
|
|---|
| ACE |
= |
angiotensin-converting enzyme |
| Lp(a) |
= |
lipoprotein(a) |
| PAI-1 |
= |
plasminogen activator inhibitor-1 |
| PCR |
= |
polymerase chain reaction |
| TPA |
= |
tissue-type plasminogen activator |
|
|
Acknowledgments
|
|---|
The authors wish to thank Prof Massimo Volpe for helpful
suggestions
and Drs M. Grilli and P. Simone for the patient
selection.
|
Footnotes
|
|---|
Reprint requests to Giovanni Di Minno, MD, Clinica Medica, Istituto
di
Medicina Interna e Malattie, Dismetaboliche, Via S Pansini,
5, 80131,
Napoli, Italy.
Received January 11, 1995;
accepted December 1, 1995.
|
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Abstract
Introduction