© 1996 American Heart Association, Inc.
Articles |
Deletion Polymorphism in the Angiotensin-Converting Enzyme Gene in Patients With a History of Ischemic Stroke
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.
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Abstract |
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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
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Introduction |
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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 serum10 and cellular11 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 infarction13 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.
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Methods |
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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.
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Results |
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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).
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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).
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Discussion |
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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 gene25 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 al27 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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The authors wish to thank Prof Massimo Volpe for helpful suggestions and Drs M. Grilli and P. Simone for the patient selection.
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Footnotes |
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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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