Deletion Polymorphism of the Angiotensin I-Converting Enzyme Gene Is Associated with Increased Plasma Angiotensin-Converting Enzyme Activity but Not with Increased Risk for Myocardial Infarction and Coronary Artery Disease

  1. Bernhard R. Winkelmann, MD;
  2. Markus Nauck, MD;
  3. Barbel Klein, MD;
  4. Andreas P. Russ, MD;
  5. Bernhard O. Bohm, MD;
  6. Rudiger Siekmeier, MD;
  7. Kai Ihnken, MD;
  8. Matti Verho, MD;
  9. Werner Grobeta, MD; and
  10. Winfried Marz, MD
  1. From the Johann Wolfgang Goethe-University, Frankfurt, Germany. Grant Support: By a research grant from Bristol-Myers Squibb GmbH, Munich, Germany, and a research grant from Forschungsschwerpunkt Land Baden-Wurttemberg. Requests for Reprints: Bernhard R. Winkelmann, MD, Hoechst AG, Klinische Forschung H840, 65926 Frankfurt, Germany. Current Author Addresses: Drs. Winkelmann and Verho: Hoechst AG, Klinische Forschung H840, 65926 Frankfurt, Germany.
     
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    Abstract

    Background: Previous research has shown that the insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene is a major determinant of plasma ACE activity. It has been suggested that persons with the DD genotype (those who express, on average, the highest levels of circulating ACE) have an increased risk for myocardial infarction and coronary artery disease, particularly if they are otherwise at low risk. Subsequent studies, however, have not confirmed that ACE I/D gene polymorphism is a risk factor for coronary artery disease and myocardial infarction.

    Objective: To investigate the association between the I/D polymorphism of the ACE gene and the risk for coronary artery disease and myocardial infarction in patients in whom coronary artery disease status was documented by angiography.

    Design: Cross-sectional study.

    Setting: University medical center.

    Patients: 209 male case-patients with coronary artery disease and 92 male controls without coronary artery disease, as documented by coronary angiography.

    Measurements: Assessment of the cardiac risk profile by questionnaire; classification of patients by the degree of coronary artery stenosis; levels of lipoproteins, apolipoproteins, and fibrinogen; and ACE I/D gene polymorphism assessed by polymerase chain reaction amplification.

    Results: Plasma ACE activity was significantly associated with ACE I/D gene polymorphism. The ACE genotype was not associated with the presence of coronary artery disease or myocardial infarction. If a recessive effect of the D allele was assumed (DD compared with DI and II), the relative risk was 1.00 (95% CI, 0.76 to 1.30) for coronary artery disease and 1.03 (CI, 0.77 to 1.38) for myocardial infarction. Results of analyses were also negative when a dominant effect of the D allele was assumed and when low-risk subgroups were examined. The established risk factors age and apolipoprotein B level emerged as the most important risk predictors in multivariate analyses, followed by diastolic blood pressure and fasting glucose levels.

    Conclusions: In an angiographically defined study sample, ACE I/D gene polymorphism was not associated with an increased risk for coronary artery disease or myocardial infarction, despite its effects on plasma ACE activity.

    The angiotensin I-converting enzyme (ACE) gene (encoding kininase II, EC 3.4.15.1) is involved in the regulation of vascular tone [1]. Angiotensin I-converting enzymes convert angiotensin I to the vasoactive angiotensin II and are involved in the degradation of bradykinin [2]. The ACE gene contains a polymorphism characterized by either insertion (I) or deletion (D) of a 287-base pair alu repetitive sequence in intron 16. This polymorphism has been shown to account for as much as half of the interperson variability of the ACE level in the circulating blood [3, 4]. The polymorphism itself is thought to be a marker for a closely linked but unidentified sequence variant that modulates the expression of the ACE gene, such that the deletion allele at this gene site is associated with increased plasma ACE activity [4].

    Cambien and coworkers [5] first reported an association between an insertion/deletion (I/D) polymorphism of the ACE gene and myocardial infarction in the ECTIM (Etude Cas-Temoin de l'Infarctus du Myocarde) study. The DD genotype occurred significantly more frequently in 610 patients who had had myocardial infarction than in 733 controls, especially in patients considered to be at low risk according to plasma apolipoprotein B levels and body mass index [5].

    Other studies [6-14] have found conflicting evidence on the relation between ACE I/D gene polymorphism and the risk for myocardial infarction or coronary artery disease. Analysis of 1250 case-patients and 2340 matched controls in the Physician's Health Study [9] did not confirm any association between the ACE genotype and the risk for myocardial infarction or coronary artery disease. Completely negative results were also reported in studies from Germany [11], Austria [12], and New Zealand [14]. Mattu and coworkers [10] failed to find a significant association of the DD genotype with coronary artery disease in their entire cohort, but they reported a significantly increased prevalence of the ACE DD genotype in patients with coronary artery disease who were at low cardiovascular risk (as defined by classic risk factors). However, the risk was quantitatively small and lost its significance once the low-risk definition proposed by Cambien and colleagues [5] was used. It is not clear whether the original observation of a weak risk for myocardial infarction (odds ratio, 1.34) conferred by the ACE genotype in Cambien and coworkers' entire study cohort was a chance finding.

    In this context, ruling out significant, possibly asymptomatic disease in controls is at least as important as confirming the diagnosis. To study ACE I/D polymorphism, we prospectively enrolled persons (both case-patients and controls) in whom coronary vasculature was documented by angiography.

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    Methods

    Study Design and Study Participants

    Eligible participants were men who were born in 1927 or later, had recently (within the previous 4 months) had coronary angiography, and were scheduled for coronary bypass surgery or open-heart surgery not related to the coronary arteries at the Department of Cardiothoracic Surgery, Frankfurt University. The study protocol was approved by the ethics committee of the Johann Wolfgang Goethe-University and conformed to the declaration of Helsinki. To enroll participants on a continuous daily basis and to recruit enough controls, we amended the enrollment criteria within 2 months after the onset of the study. The amendment allowed us to enroll participants from the Department of Cardiology when no surgical candidate could be recruited. Fewer then 5% of the persons we contacted refused to participate in the study.

    Risk factors and patient history were assessed by a questionnaire; data were verified by reviewing medical records or by contacting the patient's family physician. Fasting blood samples were obtained early in the morning for measurement of routine clinical chemical variables and lipoprotein and apolipoprotein levels and for isolation of DNA; an oral glucose load was done, and hourly glucose levels were measured during a 3-hour period. Diabetes mellitus was diagnosed according to World Health Organization criteria [15] or by a history of diabetes. All angiograms were reviewed by two cardiologists. Coronary artery disease was defined as the presence of one or more stenoses greater than 50% in at least one major artery or a left main stem stenosis greater than 30%. Sixty patients (20%) had had coronary angioplasty. In these patients, the luminal narrowing before treatment was substituted for the severity of the treated segment or segments. The classification was based on the visual assessment of 15 coronary segments, as defined by an ad hoc committee of the American Heart Association [16].

    After informed consent was obtained, 301 consecutive white men living in Germany (age range, 20 to 78 years) were enrolled between July 1992 and September 1993; 11 participants were older than the upper age limit of 66 years. The 209 case-patients were recruited as follows: 137 (66%) before bypass surgery, 60 (29%) before coronary angiography, and 12 (6%) during an outpatient visit. Of the 92 controls, 56 (61%) were recruited before valve replacement surgery, 2 (2%) were recruited before other heart surgery, 11 (12%) were recruited before catheterization to rule out coronary artery disease, 8 (9%) were recruited before diagnostic catheterization not related to coronary artery disease, and 15 (16%) were recruited at an outpatient visit.

    Laboratory Analyses

    Plasma ACE activity was measured by radioimmunoassay using 3H-hippuryl-glycyl-glycine as a substrate (ACE activity test, HYCOR, Inc., Irvine, California).

    Genomic DNA was prepared from leukocytes with a commercial spin-column method (blood polymerase chain reaction kit, Diagen GmbH, Hilden, Germany). The ACE ID genotype was determined by polymerase chain reaction as described previously [17] but was then subjected to a second, independent polymerase chain reaction amplification so that mistyping was avoided [18]. Polymerase chain reaction products were subjected to electrophoresis in 1.2% agarose gels, and DNA was visualized by ethidium bromide. The genotypes were assigned independently by two investigators who were unaware of the patients' clinical condition.

    Total cholesterol and triglyceride levels were measured with standard enzymatic methods (CHOD-PAP and GPO-PAP, respectively, Boehringer Mannheim, Mannheim, Germany). High-density lipoprotein cholesterol levels were determined in the supernate after plasma was subjected to precipitation with magnesium chloride and phosphotungstic acid (Boehringer Mannheim) [19]. Apolipoprotein B levels were measured by automated rate nephelometry using an Array Protein System (Beckman Instruments, Brea, California). Fibrinogen levels were measured using the thrombin time method [20].

    Statistical Analysis

    We compared categorical variables using the chi-square test with the Yates continuity correction. We analyzed between-group differences in continuous variables with the Wilcoxon rank-sum test. Allele frequencies were estimated by the gene counting method, and Hardy-Weinberg equilibrium was tested by the chi-square test. We did univariate and multivariate logistic regression analyses using the maximum-likelihood method to study the association of the ACE genotype with coronary artery disease and myocardial infarction (Proc Logistic, Proc Catmod, SAS Institute, Cary, North Carolina). In a model in which a codominant (additive) effect of the D allele was assumed, the genotypes II, DI, and DD were coded as 0, 1, and 2, respectively; when a dominant effect was assumed, genotype II was coded as 0, and DI and DD combined were coded as 1. Accordingly, scores of 0 for II and DI combined and of 1 for DD were used in a model that assumed a recessive effect.

    All participants were categorized as case-patients (who had coronary artery disease) or controls (who did not) according to the degree of stenosis: cutoffs for controls were 49%, 24%, and 10% stenosis. Among the case-patients, a low-risk subgroup was defined by the presence of no more than one of the following risk factors: hypercholesterolemia (cholesterol level more than equals to 6.20 mmol/L), hypertriglyceridemia (triglyceride level more than equals to 2.26 mmol/L), body mass index greater than 27 kg/m2, history of hypertension, current smoker or history of smoking, and diabetes mellitus (a history of diabetes mellitus or disease diagnosed on the basis of results of oral glucose tolerance testing during the study). We also stratified our study sample by plasma apolipoprotein B levels and body mass index, using the medians of both as cutoffs (1.26 g/L and 26 kg/m2, respectively). All statistical analyses but one were done using the SAS statistical package (versions 6.04 and 6.10, SAS Institute). The exception was chi-square testing with the Yates continuity correction, for which we used Instat software (version 2.0, Graph PAD Software, San Diego, California).

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    Results

    Study Participants

    Case-patients were older than controls (mean age ±SD, 57.8 ± 6.9 years and 50.7 ± 12.6, respectively; P < 0.001), and their risk factor profile differed significantly from that of controls (Table 1). Although more case-patients than controls had smoked in the past (75.6% compared with 68.5%; P = 0.20), significantly more controls than case-patients currently smoked (25.0% compared with 8.1%; P < 0.001). This difference is primarily due to the fact that most case-patients had stopped smoking after they had had their first myocardial infarction. The risk profile of the subgroup of case-patients who had had myocardial infarction did not differ from that of case-patients who had not had myocardial infarction; the risk profile of the low-risk coronary artery disease group was similar to that of the control group (Table 1). The group at low risk for coronary artery disease comprised 47 patients, 8 of whom had no risk factors and 39 of whom had only one risk factor (27 smokers, 6 hypertensive patients, 4 overweight patients, and 2 patients with isolated hypercholesterolemia).

    View this table:
    Table 1. Baseline Characteristics of Case-Patients and Controls*

    Frequencies of Angiotensin I-Converting Enzyme Genotypes and Activity

    The ACE genotype distributions in case-patients and controls were in Hardy-Weinberg equilibrium. The relative frequencies of the ACE DD genotype or the D and I alleles in controls did not differ from the frequencies in all case-patients, case-patients who had had myocardial infarction, and low risk case-patients (Table 2). We also investigated the frequencies of ACE genotypes in different age groups (older and younger than ages 50, 55, and 60 years; stepwise increases by 5- and 10-year intervals from age 40 years). No significant differences in the ACE genotype distribution were seen in any of these comparisons. In particular, the prevalence of the DD genotype in elderly case-patients did not decrease.

    View this table:
    Table 2. Frequencies of the ACE Genotypes in All Case-Patients, the Subgroup with Myocardial Infarction, the Low-Risk Subgroups, and Controls*

    To rule out ACE treatment effects, we confined analysis of the association between plasma ACE activity and ACE genotypes to persons who had not received ACE inhibitor treatment (n = 237). Among all study participants, mean plasma ACE activity was lowest in persons homozygous for allele II (86.0 ± 26.2 U/mL; n = 48), intermediate in persons heterozygous for the ID allele (97.4 ± 23.0 U/mL; n = 117), and highest in persons homozygous for the DD allele (114.4 ± 32.2 U/mL; n = 72) (P < 0.001). The association between ACE genotype and plasma ACE activity was preserved when the case-patients and controls were analyzed separately (Figure 1).

    Figure 1. In each patient group, ACE activity differed significantly depending on ACE genotype (p less than 0.001). Values are expressed as means with 95% CIs.
    View larger version:
      Figure 1. In each patient group, ACE activity differed significantly depending on ACE genotype (p less than 0.001). Values are expressed as means with 95% CIs. Plasma angiotensin I—converting enzyme (ACE) activity in patients with coronary artery disease (squares) and controls (circles) not treated with an ACE inhibitor.

      No significant association was seen between the ACE genotype and any of the clinical and biochemical characteristics reported in Table 1, either in the entire study sample or in any of the subsets (such as case-patients who had had myocardial infarction and low-risk case-patients).

      Odds ratios were calculated as measures of the association of the ACE genotype with coronary artery disease or with myocardial infarction. The odds ratios indicated no significant increase in risk for coronary artery disease, either when all case-patients or when the coronary artery disease subgroups were compared with controls, regardless of whether the D allele was assumed to have a dominant, a codominant, or a recessive effect (Table 3). Similar nonsignificant results were obtained for the relative risk estimates in the myocardial infarction subgroup and in the low-risk subgroup (Table 3). By stratifying the data according to the low-risk criteria of Cambien and coworkers [5], which were almost identical to the respective medians in our control group (body mass index, 26 kg/m2 and apolipoprotein B level, 1.26 g/L), 23 of 209 case-patients and 31 controls were selected. No increased risk for cardiac disease was seen in any comparisons analyzing these low-risk subsets.

      View this table:
      Table 3. Risk for Coronary Artery Disease according to Genotype When Comparing Controls with All Case-Patients, the Subgroup with Myocardial Infarction, and the Low-Risk Subgroup

      Multivariate analyses adjusting for age, apolipoprotein B levels, diastolic blood pressure, fasting glucose levels, and cigarette smoking showed no effect of the ACE genotype on disease; with the exception of smoking, however, the relative risk estimates of the well-established risk factors indicated significant increases in cardiac risk (Table 4). Except for age, these risk factors were not significant in the low-risk subgroup. None of the logistic regression analyses comparing low-risk patients with low-risk controls showed an association between the D allele and the risk for coronary artery disease (data not shown).

      View this table:
      Table 4. Odds Ratios for Coronary Artery Disease according to ACE Genotype and Other Risk Factors by Multivariate Logistic Regression Analysis*

      Further analyses, in which participants were defined as case-patients if at least one stenosis of the coronary arteries exceeded 24% or as controls if maximum stenosis was 10% or less (including an analysis in which stenoses between 11% and 24% were excluded), also showed no significant overrepresentation of the D allele in case-patients.

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      Discussion

      In a study of four groups of patients from different parts of Europe, Cambien and coworkers [5] found that the frequency of the ACE DD genotype increased in survivors of myocardial infarction compared with controls. The association was strongest in patients who lacked identifiable risk factors for coronary artery disease [5]. Further evidence that ACE I/D gene polymorphism might be associated with coronary atherosclerosis came from studies in French patients with diabetes [21], studies in Italian patients [22], and studies in U.S. patients [23]. These reports, however, have been challenged by several studies that found no association in their entire study samples [8-14] and in subgroups defined to have a low risk for the development of coronary artery disease [8, 9, 12]. Many other studies had conflicting results [24-31]. The inconsistency of these results may have several explanations: 1) differences in the study samples themselves, 2) failure to adequately separate case-patients from controls, and 3) the possibility of premature deaths in persons carrying the ACE DD genotype.

      The use of patient-based controls rather than population controls is one of our study's strengths rather than a limitation. In contrast to other investigators, we were able to rule out relevant coronary artery disease in each control. Asymptomatic coronary artery disease is almost impossible to rule out clinically. Our control group included persons who primarily had aortic or mitral valve disease. We are convinced, however, that this did not introduce any relevant selection bias. The frequencies of the ACE genotypes in our control group were similar to those found in population-based studies [9, 10]. Beyond this, the control group had a low prevalence of atherogenic risk factors. In addition, patient characteristics in the control and coronary artery disease groups were almost identical to those in a large angiography-based European case–control study [32] and a German population cohort study [33], respectively.

      We could not, however, show an increased frequency of the ACE DD genotype in case-patients, regardless of whether we examined case-patients who had or had not had myocardial infarction. In four other studies [11-13, 22], angiography was used to characterize the entire study sample, including controls. All but one of these studies [22] reported negative results for an association of ACE I/D gene polymorphism with coronary artery disease or with myocardial infarction. One study [13] did report that the occurrence of myocardial infarction was significantly associated with the D allele in a comparison of patients with coronary artery disease who had had myocardial infarction with patients who had not [13]. This finding was not reproduced in other studies [9, 12, 21]. Furthermore, we and others [9, 12, 13] could not detect an increase in cardiac risk in low-risk subsets.

      We confirmed that the ACE D allele is associated with elevated ACE activity in plasma. However, no apparent correlation was seen between plasma ACE activity and the risk for coronary artery disease or myocardial infarction.

      Although experimental evidence implicates angiotensin II in the development of atherosclerotic lesions because it modulates the growth of smooth-muscle cells [34], there are reasons for cautioning against a direct link between the level of ACE activity and the risk for coronary atherosclerosis. First, although ACE gene polymorphism is associated with plasma ACE activity, no such association exists with angiotensin II [35, 36]. It is well known that renin, not ACE, is the rate-limiting step in the production of angiotensin II [2, 35]. Second, experimental studies indicate that pulmonary expression of ACE is subject to negative feedback by angiotensin II [37, 38]. Third, experimental evidence suggests that other enzymes, such as chymase [39] or chymostatin-sensitive angiotensin II-generating enzyme [40], bypass ACE in vivo.

      Although convincing evidence suggests that ACE inhibition represents a major advance in the treatment of hypertension, congestive heart failure, and acute myocardial infarction, it is doubtful whether the ACE D allele will be confirmed as a risk factor for early death, as has been suggested [41] or shown [42] by others. In a recent study [43], the DD genotype was found in 39.6% of 338 centenarians and in 26.6% of 164 healthy controls who were 20 to 70 years of age. The finding is difficult to reconcile with the concept that the DD genotype is a cardiovascular risk factor.

      Our study did not provide evidence of a significant increase in the risk for coronary artery disease or myocardial infarction. We believe that our study was strengthened by the use of angiographic evaluation to discriminate between case-patients and controls, which prevented the inclusion of persons with early coronary artery disease in the control group. This approach was considered an important prerequisite in a study on candidate gene polymorphisms in lipid metabolism [44]. In conclusion, ACE I/D gene polymorphism seems to be of no use as a predictor of risk for coronary arteriosclerosis and thrombosis.

      Acknowledgments: The authors thank Bettina Donnerhak, Ulrike Stein, and Daniela Wittmann for technical assistance in lipid and DNA analyses; Konstanze Schmitt-van Baal and Maria Czempik for help in literature research and secretarial assistance; Norbert Bender, MD, Heinz Metzger, PhD, and Ute Maier for the ACE activity measurements; and Kurt Loffler, PhD, and Birgit Keller, MS, for statistical advice and help in programming the logistic regression models.

      Dr. Russ: Wellcome/CRC Institute, Tennis Court Road, Cambridge CB2 IQR, United Kingdom.

      Drs. Gro β and Klein: Gustav-Embden Center of Biological Chemistry, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.

      Drs. Nauck and Marz: Division of Clinical Chemistry, Albert Ludwigs-University, Hugstetter Strasse 55, 79106 Freiburg, Germany.

      Dr. Siekmeier: Institute of Clinical Chemistry, Building 53, Carl Gustav Carus-Technical University, Fetscherstrasse 74, 01307 Dresden, Germany.

      Dr. Ihnken: 54 Park Terrace, Mill Valley, CA 94941.

      Dr. Boehm: Division of Endocrinology, Universitatsklinikum, Robert-Koch-Strasse 8, 89081 Ulm, Germany.

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      1. Ann Intern Med July 1, 1996 vol. 125 no. 1 19-25
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