Genotype Distribution of Angiotensin-Converting Enzyme Polymorphism in Australian Healthy and Coronary Populations and Relevance to Myocardial Infarction and Coronary Artery Disease
Abstract Angiotensin-converting enzyme is a key component of the renin-angiotensin system that plays an important role in cardiovascular regulation. An association between the angiotensin-converting enzyme insertion/deletion (I/D) polymorphism and increased coronary risk has been found in some studies but not in others. To explore this further in an Australian white population, we compared the ACE genotype distribution in 550 patients aged 37 to 65 years with coronary artery disease documented by angiography with the genotype distribution in 404 healthy school children aged 6 to 13 years. We also explored associations in the patients between the angiotensin-converting enzyme I/D polymorphism and a history of myocardial infarction and coronary artery disease severity assessed by the number of major coronary arteries with more than 50% luminal obstructions and by the Green Lane coronary score. The frequencies of the angiotensin-converting enzyme genotype in the coronary artery disease patients were 0.236 for I/I, 0.395 for I/D, and 0.369 for D/D genotypes. This distribution with an excess of the D/D genotype was significantly different (χ2=23.69, P<.0001) from that in the school children, in whom the genotype distribution was in Hardy-Weinberg equilibrium (I/I, 0.21; I/D, 0.54; D/D, 0.25). There was also a significant excess of D/D genotype among patients with a history of myocardial infarction (χ2=9.42, P=.009), and there was the same D/D excess in the subgroup of children (n=60) with two or more grandparents who had had coronary artery disease. We found no associations between the angiotensin-converting enzyme polymorphism and the number of significantly stenosed coronary arteries (χ2=2.069, P=.91). We conclude that the D/D genotype is a significant predictor for coronary artery disease events in the Australian white population but is not a marker for angiographically assessed coronary artery disease severity. The angiotensin-converting enzyme genotype–associated increased risk for coronary events may be mediated more by angiotensin II–induced coronary vasoconstriction than by an increase in injury-related smooth muscle cell proliferation in the coronary vasculature.
- Received August 22, 1995.
- Accepted October 30, 1995.
The renin-angiotensin system is involved in many aspects of cardiovascular regulation, including sodium homeostasis, cardiovascular remodeling, and maintenance of vascular tone,1 2 and ACE is a key component of it. The inactive angiotensin I is converted in vivo by ACE into the potent vasoconstrictor angiotensin II. An I/D polymorphism at intron 16 of the ACE gene has been identified and shown to explain up to 50% of the variance in circulating ACE activity, the D/D genotype being associated with higher circulating levels.3
In 1992, Cambien et al4 first reported an association of the ACE D/D genotype with MI survivors and particularly among those at ‘low‘ risk according to conventional risk factors. Several other groups have explored a possible relationship with CAD since then. Most retrospective studies supported the initial findings that the D/D genotype was more frequent in MI survivors and in cases of fatal MI and sudden cardiac death.5 6 7 8 Our own studies9 and those of Tiret et al10 and Bohn et al11 also identified an excess of the D allele in individuals with a positive family history of CAD in either first- or second-degree relatives. However, the recent large prospective American physician’s study showed no association between ACE I/D polymorphism and the prevalence of MI or ischemic heart disease.12 Furthermore, Ludwig et al13 have reported an association with MI but observed no relationship between the polymorphism and the development of coronary stenosis in the same patient population. Sanmani et al14 also found no association between the ACE polymorphism and restenosis after coronary angioplasty.
In view of these conflicting findings obtained in different patient population groups and the possibility of a population-specific effect in relation to the ACE I/D polymorphism, we sought to explore two questions relevant to an Australian white population in the present study. The first is whether the genotype distribution of the ACE I/D polymorphism is different in healthy and coronary white Australian subjects. The second is whether there is an association between the polymorphism and the occurrence of MI and severity of CAD.
The healthy population was a previously reported group of 404 school children of approximately equal numbers of boys and girls aged 6 to 13 years who were part of a family heart health education program.9 15 All children were considered to be healthy by parents and teachers. After prior parental consent they were selected solely on the basis of ethnicity, in that all were white.9 Capillary blood samples were obtained by finger prick and spotted onto filter paper and stored at −70°C as previously described.9
The patients were whites aged 65 years or younger, both men and women, consecutively referred to the Eastern Heart Clinic at Prince Henry Hospital for coronary angiography over a 16-month period in 1994 and 1995. These patients were referred by cardiologists from outpatient clinics of local hospitals and from medical centers for assessment because of a suspected or confirmed clinical diagnosis of ischemic heart disease for determination of subsequent management. We excluded from analysis only patients shown to have significant left main disease (>50% luminal obstruction), because it was difficult to categorize this small proportion of the total (5%) within the classification system we used (see below).
Written consent was obtained from all parents of the children in the healthy population and from every patient after a full explanation of the study, which was approved by the Ethics Committee of the University of New South Wales.
A 4-mL venous blood sample was drawn into an EDTA sample tube in the patients before the angiogram after at least a 6-hour fast. The blood sample was centrifuged within 2 hours, and plasma and cellular components were stored separately at −70°C until analysis.
DNA Analysis for the Detection of ACE I/D Polymorphism
DNA was extracted from the frozen cellular blood component by a salting-out method adapted from that described by Miller et al16 for whole frozen blood. The extracted DNA was stored at 4°C until analysis. ACE I/D polymorphism was determined from the protocol of Badenhop et al9 and Rigat et al17 with Taq polymerase (Boehringer Mannheim) and a thermal cycler (Hybaid). The reaction included 5% dimethyl sulfoxide to ensure that the I allele was amplified in all heterozygotes.18 Any one person is either an I/I homozygote, an I/D compound heterozygote, or a D/D homozygote for this polymorphism.
TC, HDL-C, and triglyceride levels were measured in the patients by the hospital’s clinical chemistry department with standard enzymatic methods. LDL-C levels were calculated by the Friedewald formula.
Documentation of CAD Severity
The severity of coronary stenosis was determined by the number of significantly stenosed coronary arteries as follows. Angiograms were assessed by two cardiologists who were unaware that the patients were to be included in the study. Each angiogram was classified as revealing either no coronary lesion with more than 50% luminal stenosis or one, two, or three major epicardial coronary arteries with more than 50% luminal obstructions. We also used the Green Lane coronary scoring system, which provides a numerical value for lesion severity and takes account of the amount of myocardium supplied by an affected vessel; the maximum score is 15.
Documentation of Other Relevant Variables
The relevant history was obtained for each patient with a questionnaire, with standardized choices of answers to be ticked during the interview. We recorded the presence or absence (yes/no) of a history of MI, hypertension requiring treatment, diabetes, and angina pectoris. Where these disorders were answered as positive, the patient’s medical records were consulted to confirm the diagnosis of MI based on standard criteria (clinical manifestation, abnormal enzymology, and electrocardiographic changes at the time of diagnosis). A “don’t know” box was also included for those patients not clear about aspects of their medical history that were also unavailable from the patient’s file. Current medications were recorded, in particular the use of lipid-lowering drugs. The presence or absence of CAD among first-degree relatives (parents and siblings) and the age of first onset were recorded for a quantitative assessment of family history of premature CAD. We recorded the presence and severity of angina according to whether each patient was experiencing no angina, stable angina, or unstable angina before and during the current hospitalization. All those classified as having unstable angina had an increase in pain frequency as well as rest pain. The lifetime smoking dose in pack-years was recorded as described previously.19
We determined whether the distribution of genotypes was in Hardy-Weinberg equilibrium by χ2 analysis as described by Emery.20 The frequencies of the alleles and genotypes among different subgroups were compared by χ2 test. We used a conditional logistic regression analysis in which either a history of MI or the number of significantly diseased vessels was the dependent variable and assessed the independent effect of ACE genotype while other measured variables were entered as independent variables. The categorical variables in the logistic analysis were coded so that the presence of diabetes, hypertension, family history of CAD, and use of lipid-lowering drugs were coded 1 and the absence of these disorders and nonlipid drug users as 0. Since there were only 2 patients with “don’t know” answers about family history, 2 about diabetes and hypertension, and 1 about history of MI, the “don’t know” was simply coded as a missing value and excluded from the analysis.
To compare our findings with those reported by others,4 5 6 7 8 we also conducted our χ2 analysis in low-risk subgroups. These were identified with reference to age (≤55 years for men and ≤60 for women), TC/HDL-C ratio (<5.0), BMI (<27 kg/m2), and smoking history (nonsmokers). We used the TC/HDL-C ratio instead of other lipid variables because it was the strongest lipid predictor for CAD severity in this patient population.19 The median BMI value in the patient population was 27 kg/m2. To stratify patients according to age, we used quartiles of the patient population for the analysis. The 25th, 50th, and 75th percentiles were 53, 57, and 60 years.
The demographic information for the 550 patients is shown in Table 1⇓.
Genotypes in the Patients and Healthy School Children
Although the distribution of the ACE genotype in the school children was in Hardy-Weinberg equilibrium, as we showed previously,9 in the 550 patients it was not (χ2=23.69, P<.0001). In the patients the expected and observed numbers of cases for the I/I genotype were 104.4 and 130 (23.6%), for the I/D genotype 272.2 and 217 (39.5%), and for the D/D genotype 177.5 and 203 (36.9%). The distribution pattern was virtually the same after patients with angiographically normal coronary arteries (n=103) were excluded (I/I, 106 [23.7%]; I/D, 179 [40.0%]; and D/D, 162 [36.2%]). The frequency distribution of the genotypes was not different between men and women, although the D allele frequency was slightly higher among women (0.588) than men (0.557). The D allele frequency for all patients was 0.566.
The frequency distribution of D/D genotypes among all patients who had reported having had an MI tended to be higher (χ2=3.00, P=.22). After patients with angiographically normal coronary arteries were excluded, there was a significant excess of the D/D genotype among patients with a history of MI (n=84, 40.4%) compared with those without (n=78, 32.6%; χ2=6.73, P=.027). This relationship was even stronger among patients with one or more significantly diseased (>50% luminal stenosis) vessels and a history of MI (χ2=9.42, P=.009; Table 2⇓). This significant association between the D/D genotype and MI remained statistically significant (r=.09, P=.02) in a logistic linear regression analysis after controlling for all other categorical variables (sex, number of significantly diseased vessels, diabetes, hypertension requiring treatment, family history of CAD) and quantitative variables (age, TC, HDL-C, LDL-C, triglyceride, lifetime smoking dose, lipoprotein[a]).
There was a significant excess of the D/D genotype among the patients compared with the distribution documented in the 404 school children (Table 3⇓). This difference was also true when the comparison was only among those with one or more significantly diseased (>50% luminal stenosis) vessels. However, as also shown in Table 3⇓, the frequency of the D/D genotype among those children who had two or more grandparents with a positive CAD history was the same as that in the CAD patients.
ACE I/D Polymorphism and CAD Severity
We found no statistically significant association between the ACE I/D polymorphism and the number of significantly diseased vessels in the patients, either in all patients or in men and women separately (Table 4⇓). The D allele frequencies in patients with zero, one, two, or three significantly diseased vessels were 0.578 (0.556 for men and 0.598 for women), 0.569 (0.561 for men and 0.592 for women), 0.553 (0.565 for men and 0.636 for women), and 0.563 (0.576 for men and 0.500 for women), respectively (χ2=2.069, P=.85). This lack of significant association was further confirmed in a logistic regression analysis in which age, sex, presence of diabetes, hypertension requiring treatment, angina, history of MI, usage of lipid-lowering drugs and adrenergic β-blockers, TC/HDL-C ratio, BMI, and lifetime smoking dose were controlled. The coronary scores for patients with the I/I (5.44±0.4), I/D (4.92±0.3), and D/D (5.46±0.3) genotypes were also not different (F=0.876, P=.42).
In the low-risk subgroup analysis, we cannot reject the null hypothesis that the ACE polymorphism and severity of coronary stenosis are independent. As shown in Table 5⇓, the distribution of ACE genotypes among men aged 55 years or younger and women aged 60 years or younger with different numbers of significantly diseased vessels was not different. The same result was observed in the age categories stratified by the population quartiles. There was also no statistically significant association among low-risk groups identified according to the TC/HDL-C ratio (lower than 5.0), absence of smoking, or BMI less than 27 kg/m2 (the 50th percentile of the patient population) or among those with no hypertension, no diabetes, no history of MI, or no angina. In fact, the frequency of the D/D genotype remained virtually constant among patients with different numbers of diseased vessels.
We believe that the two populations we studied are genetically representative, first of the normal white population of Sydney, Australia, of school children, and second of coronary patients in that subgroup of the Sydney white population aged 65 years and younger who are judged by cardiologists to require coronary angiography for the diagnosis and management of CAD. Thus, the key finding of our study—that the distribution of the ACE I/D polymorphism is very different in the healthy and coronary populations, with a highly significant increase in the D/D genotype in the latter—constitutes further evidence for the relevance of the D/D genotype to CAD in white Australians. The second finding of a strong association between the presence of the D/D genotype and a history of MI in patients with angiographically demonstrated significant CAD adds further support to the original observation made by Cambien and colleagues.4 Our further finding of no statistically significant association between ACE genotype and the severity of CAD is in accordance with the recent observations of Ludwig and associates13 ; they found no association with development of coronary stenosis but a definite one with occurrence of MI.
While all these observations raise interesting questions about mechanisms, the association between genotype and CAD emerges clearly in our population. This is further supported by our finding of a D/D genotype excess in that subset of children with two or more grandparents who had coronary events. This excess was the same as the one we identified in the coronary patients. The fact that these associations have not been found in some other populations, particularly in the recent study of American physicians,12 who probably represent a low-risk group, may indicate that D/D genotype–associated coronary risk is population specific.
Despite the associations with coronary risk that we demonstrated, the D/D genotype distribution was virtually the same in the groups of patients with coronary syndromes and no or mild CAD and in those with single, double, and triple vessel disease (Table 4⇑). Nor was there an association with the coronary score. A lack of this statistically significant association was also seen among low-risk patients identified by age, TC/HDL-C ratio, BMI, or smoking status. The older patients tended to have more significantly diseased vessels, but the ACE genotype was still not associated with CAD severity after controlling for age as a quantitative or stratification variable. Furthermore, the frequency of the D allele was not different in different age quartiles or in men younger than 55 or women younger than 60 years. Although these findings are supported by those of Ludwig and colleagues,13 it is also relevant that Sanmani et al14 found no association between ACE I/D polymorphism and the development of restenosis after angioplasty in 233 patients.
All these findings would be consistent with ACE genotype–associated cardiovascular risk being more relevant to angiotensin II–induced coronary vasoconstriction than to any enhancing effects on smooth muscle cell proliferation. This could contribute to the association of the genotype with the occurrence of fatal and nonfatal MI, sudden cardiac death, and positive family history of coronary events5 21 and to associations with dilated cardiomyopathy7 and sudden death in families with hypertrophic cardiomyopathy.22 It could also be consistent with beneficial effects of ACE inhibitors in reducing the incidence and death from MI documented in drug trials.23
In conclusion, our study shows that there is a highly significant excess of the D/D genotype in Australian white patients with established CAD and that this is also associated with an increased risk of MI. We found no evidence for a relationship between the D/D genotype and the severity of CAD documented by angiography.
Selected Abbreviations and Acronyms
|BMI||=||body mass index|
|CAD||=||coronary artery disease|
This work was supported by grants from the National Health and Medical Research Council of Australia. We wish to thank Dr Bridget Wilcken for reviewing the manuscript; Lily Fenech, Shelly Brown, Steven Brouwer, Dr Greg Cranny, and all nurses in the Eastern Heart Clinic for their assistance in clinical data collection; A.S. Sim and Dr Jun Wang for their laboratory assistance; and J. Kessey for the data entry. We are also most grateful to the cardiologists in the department for allowing us to study their patients.
Dzau VJ. Cell biology and genetics of angiotensin in cardiovascular disease. J Hypertens. 1994;12(suppl 4):S3-S10.
Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990;86:1343-1346.
Cambien F, Poirier O, Lecerf L, Evans A, Cambou J-P, Arveiler D, Luc G, Bard J-M, Bara L, Ricard S, Tiret L, Amouyel P, Alhenc-Gelas F, Soubrier F. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature. 1992;359:641-644.
Evans AE, Poirier O, Kee F, Lecerf L, McCrum E, Falconer T, Crane J, O’Rourke DE, Cambien F. Polymorphisms of the angiotensin-converting-enzyme gene in subjects who die from coronary heart disease. Q J Med. 1994;87:211-214.
Mattu RK, Needham EWA, Galton DJ, Frangos E, Clark AJL, Caulfield M. A DNA variant at the angiotensin-converting enzyme gene locus associates with coronary artery disease in the Caerphilly Heart Study. Circulation. 1995;91:270-274.
Badenhop RF, Wang XL, Wilcken DEL. Angiotensin-converting enzyme genotype in children and coronary events in their grandparents. Circulation. 1995;91:1655-1658.
Ludwig E, Corneli PS, Anderson JL, Marshall HW, Lalouel JM, Ward RH. Angiotensin-converting enzyme gene polymorphism is associated with myocardial infarction but not with development of coronary stenosis. Circulation. 1995;91:2120-2124.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.
Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCPI) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res. 1992;20:1433.
Wang XL, Tam C, McCredie RM, Wilcken DEL. Determinants of severity of coronary artery disease in Australian men and women. Circulation. 1994;89:1974-1981.
Emery AEH. Hardy-Weinberg equilibrium: the estimation of gene frequencies. In: Emery AEH, ed. Methodology in Medical Genetics: An Introduction to Statistical Methods. Edinburgh, UK: Churchill Livingstone; 1976:3-9.
Morgan K. Diverse factors influencing angiotensin metabolism during ACE inhibition: insights from molecular biology and genetic studies. Br Heart J. 1994;72:3-10.