Angiotensin-Converting Enzyme Genotype in Children and Coronary Events in Their Grandparents
Background It has been suggested that the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is an independent risk factor for coronary artery disease. The D/D genotype, which is associated with higher levels of circulating ACE than the I/D or I/I genotype, has been found significantly more frequently in patients with myocardial infarction and also in individuals with a parental history of myocardial infarction.
Methods and Results We explored the distribution of the ACE genotype in 404 school children, aged 6 to 13 years, and related the distribution to the number of their grandparents who had had vascular events. We found a significant association between the number of grandparents who had had coronary events and the ACE genotype (P=.01). In children with two or more grandparents who had had coronary events, there was an excess of both D/D (odds ratio=2.8 [95% confidence interval=1.16-6.56]) and I/D (odds ratio=1.4 [95% confidence interval=0.62-3.25]) genotypes compared with I/I genotypes. In addition, there was an association between the ACE genotype and lipoprotein(a) levels in children (P=.07). Both the ACE genotype and lipoprotein(a) were found to contribute significantly (P=.0042) and independently to family history of coronary artery disease, with the ACE genotype proving to be more predictive than lipoprotein(a) levels.
Conclusions We conclude that the I/D polymorphism of the ACE gene is an important independent risk factor for coronary artery disease and is more predictive that lipoprotein(a). The I/D polymorphism is not only associated with a parental history of myocardial infarction but also with coronary artery disease in second-degree relatives. A further study to explore the relation between the I/D polymorphism and circulating levels of lipoprotein(a) is indicated.
The established risk factors for coronary artery disease (CAD) only partly explain its occurrence and severity and the clustering of premature disease in families. In a recent study, we could explain only about 50% of the variance in CAD severity documented at coronary angiography in men and women.1 In those with a positive family history of premature CAD, lipoprotein(a) [Lp(a)], which is largely genetically regulated, was the most predictive variable.1 In a separate study, we found that levels of Lp(a) and apolipoprotein B (apoB) in children were correlated with the occurrence of coronary events in their grandparents.2 Recent evidence3 that a polymorphism of the angiotensin I–converting enzyme (ACE) gene might also be an independent risk factor for a genetically determined predisposition to CAD led us to explore the distribution of the polymorphism in a population of school children during a study aimed at identifying young high-risk families.
ACE mediates the conversion of the inactive angiotensin I to the potent vasoconstrictor angiotensin II. Circulating levels of ACE are strongly genetically determined. The insertion/deletion (I/D) polymorphism of the ACE gene (an I/D of a 287-bp alu repeat sequence in intron 16 of the ACE gene) acts as a polymorphic marker. This genetic variant accounts for up to 50% of the variance in enzyme level.4 It has been established that the D/D genotype of the I/D polymorphism is associated with higher levels of circulating ACE compared with the I/D and I/I genotypes.4 5 6 Cambien et al4 found the D/D genotype to be more frequent in patients with myocardial infarction, a finding that Bøhn et al7 could not confirm. However, case-control studies indicate that the D/D genotype is more frequent in patients with hypertrophic,8 ischemic, and idiopathic dilated cardiomyopathies9 and with left ventricular hypertrophy.10
In the present study we explored the distribution of the ACE genotype in school children in relation to Lp(a) and apoB levels. We related the findings to the occurrence of coronary events in their grandparents (because of the relatively young age of parents). Our primary aim was to identify young high-risk families and implement prevention. We reasoned that the finding of any association in second-degree relatives would indicate strong and important associations. In adult studies so far, the association of the ACE genotype with CAD, although significant, has not been strong and could be influenced by the confounding effect of early coronary death in those individuals homozygous for the D allele. This is avoided in a study of a population of school children, selected only on the basis of ethnicity, in that all were Caucasian.
We studied 404 white school children aged 5.8 to 13.3 years (mean, 9.9 years) from our family-based program for early prevention of CAD.2 There were approximately equal numbers of boys (53%) and girls (47%). We had complete family histories for the presence or absence of CAD in the children’s older family members. All information regarding coronary events in parents and grandparents was obtained by questionnaire from the parents as described previously.2 We classified parents and grandparents as having had a coronary event if there was a clear history of a myocardial infarction, death from CAD, or a history of coronary bypass surgery or angioplasty. We restricted the coronary event questionnaire to these major events to minimize error. We also checked by telephone the accuracy of reporting in 100 families randomly selected from among the 404 children studied. In 99 there was complete agreement with the previously completed and returned questionnaire. An event had been omitted in the remaining case.
Family History and Lipoprotein Measurements
The parents of the children were generally young (mean age of fathers, 41 years; range, 27 to 67 years; of mothers, 39 years; range, 24 to 57 years) and had had too few coronary events for a meaningful statistical analysis. We therefore confined the study to the occurrence of events in the children’s grandparents only. Family history was recorded as the number of grandparents who had had a coronary event. There were 189 children with no grandparent who had had a coronary event, 155 children with one grandparent and 60 children with two or more grandparents who had had coronary events (44 children with two affected grandparents, 14 with three affected grandparents, and 2 with all four grandparents affected). We measured apoA-I, apoB, the apoB/apoA-1 ratio, and Lp(a) in capillary blood samples obtained by finger prick in all children as previously described.2
We used finger-prick blood samples spotted onto filter paper as the source of DNA. The DNA (approximately 50 ng) was extracted from the filter paper by excising a 3-mm blood spot using a punch. The disks were placed into 0.5-μL microcentrifuge tubes, and two drops of pure methanol were added. After evaporation overnight, 30 μL of sterile water was added to the tube, overlaid with mineral oil, and heated to 100°C for 15 minutes. The tubes were then centrifuged at 10 000 rpm for 10 minutes. Approximately 2 μL of the supernatant was used as a template for the polymerase chain reaction (PCR) amplification of intron 16 of the ACE gene, which contains the insertion of a 287-bp alu repeat sequence. The primers and PCR conditions were from the protocol of Rigat et al11 using Taq polymerase (Boehringer Mannheim) and a Hybaid thermal cycler. The reaction included 5% dimethyl sulfoxide to ensure that the insertion allele was amplified in all heterozygotes.12 The PCR products were visualized on a 7% polyacrylamide gel with silver staining. The PCR product is a 190-bp fragment in the absence of the insertion sequence (D allele) and 490 bp in the presence of the insertion sequence (I allele).
Differences in genotype distribution in different family history, age, and sex groups were determined with χ2 tests. We used t tests to compare means [log-transformed for Lp(a)] of lipoproteins in the three genotype groups. We used multiple logistic regression analysis to assess the relation between family history of CAD, the ACE genotype, and lipoproteins. ACE genotype and lipoprotein levels were regarded as independent variables, and family history was regarded as the dependent variable. Statistical analyses were performed with the spss-x statistics software, version IV (SPSS Inc).
All blood samples were obtained with the informed consent of parents and agreement of the children themselves. The study was approved by the Ethics Committee of the University of New South Wales.
Genetic Characteristics of the Population
The ACE genotype was determined for all 404 children. The Figure⇓ shows a representative polyacrylamide gel showing the PCR products. In the total population, the frequency of the I allele was 0.48 and the D allele 0.52. The genotype distribution was I/I=0.21, I/D=0.54, and D/D=0.25. There were no significant differences in genotype distribution between different ages (P>.1) or sexes (P>.1).
Association Between ACE Genotype and Family History of CAD
There was a significant association between the number of grandparents with a history of CAD and the ACE genotype in the children (P=.01). Table 1⇓ shows the distribution of the ACE genotype in these children according to family history. There was no significant difference in the distributions of the ACE genotype in children who had only one grandparent with a coronary event and in children whose grandparents had had no coronary events. However, the frequency of both the D/D and I/D genotypes was increased in the children with two or more grandparents who had had a coronary event. There was a significant excess of the D/D genotype in these children (odds ratio=2.8, 95% confidence interval=1.16-6.56, P=.02) and a trend toward an increase in the I/D genotype (odds ratio=1.4, 95% confidence interval=0.62-3.25) of borderline significance (P=.07).
Association Between ACE Genotype and Lipoproteins
Table 2⇓ shows mean lipoprotein levels according to genotype. There was no difference in mean levels of either apoA-I, apoB, or the apoB/apoA-I ratio between the genotypes. However, there was an association between Lp(a) level and genotype, although this association fell short of the .05 significance level (P=.07). The highest Lp(a) level was in those children having the D/D genotype, the next in children with the I/D genotype, and the lowest was found in children with the I/I genotype.
Independent Contribution of ACE Genotype and Lp(a) Level to Family History of CAD
ACE genotype (χ2=6.5, P=.01) and Lp(a) level (χ2=5.5, P=.02) in the children were independent predictors of CAD in their grandparents. The ACE genotype showed a stronger correlation with coronary events in grandparents and was therefore more predictive than Lp(a) levels, the correlation coefficents being .92 (regression coefficent=.07, SEM=.03) and .19 (regression coefficent=.0015, SEM=.0007), respectively.
This study defines a significant association between the ACE genotype in children and the occurrence of coronary events in their grandparents. The study confirms our previously reported result that Lp(a) levels in children are associated with coronary events in grandparents.2 But our present results indicate that the ACE genotype is more predictive than Lp(a) level. In addition, they are suggestive of an association between the ACE genotype and levels of circulating Lp(a).
Significant differences in the genotype distribution were detected only in those children with two or more affected grandparents. This was also true for Lp(a) in our earlier study.2 These are not unreasonable findings in a disorder of multifactorial etiology if the ACE genotype and Lp(a) levels are indeed predictive of coronary risk. As children share only 25% of each of their grandparents’ genes, they would be more likely to inherit the relevant D allele with two or more affected grandparents than with only one affected grandparent.
Tiret et al13 assessed the ACE genotype in adults in relation to whether or not they had a parental history of fatal myocardial infarction. They identified an association between the D/D and I/D genotypes and a parental history of infarction and found an odds ratio of 2.6 between the D/D and I/I genotypes and of 1.9 between the I/D and the I/I genotypes. Bøhn et al14 obtained similar results. In the present study of second-degree relatives, the odds ratios for coronary events in grandparents was 2.8 between the D/D and I/I genotypes and 1.4 between the I/D and I/I genotypes. The odds ratios in the present study are large compared with the findings described above when the dilution of shared genes, due to the generation gap, is considered.
An important consideration in this study is the accuracy of the self-reported family history questionnaires. Førde and Thelle15 found 78% agreement between a self-reported history of myocardial infarction in first-degree relatives and the diagnosis from doctors’ records, hospital records, and death certificates. There was 86% agreement in a similar recent Australian study.16 Our evaluation of the questionnaires indicated that underreporting of coronary events was more likely than overreporting,2 as other researchers have found.17 In the present study we restricted our questionnaire to information on definitive coronary events (myocardial infarction, coronary bypass surgery, angioplasty, death from CAD) to minimize error. When we checked the data by telephone in a randomly selected 25% of the 404 families studied, there was an error in one family, an unreported event (see “Methods”). We concluded that the family history data obtained for our population was accurate and that any inaccuracies would tend to reduce rather than amplify our risk estimates.
An intriguing additional result in the present study is the possibility of an association between the ACE genotype and circulating Lp(a) levels. Although the association level at P=.07 is of borderline significance, the mean Lp(a) level increased with the number of D alleles present. Children with the I/D genotype had higher Lp(a) concentrations than those with the I/I genotype; the highest Lp(a) levels were in the children who had the D/D genotype (Table 2⇑). If further studies in larger populations confirm this association, the possibility of a regulatory effect, either direct or indirect, of the ACE genotype on Lp(a) expression would warrant investigation.
The ACE genotype and Lp(a) levels in children cannot of course be direct contributors to coronary events in their grandparents. Their significance as predictors of the occurrence of coronary events reflects the association between the ACE genotype and Lp(a) with coronary risk. In the present study the ACE genotype was more predictive than Lp(a) level. The multiple regression analysis showed that the ACE genotype was more strongly correlated with CAD in grandparents than Lp(a), consistent with the ACE genotype being a more important predictor of CAD in grandparents than Lp(a). The frequency of the D/D genotype in our population was 0.25. If we assume that relative risk is well approximated by the odds ratio, the percentage of children with two or more grandparents who had had a coronary event attributable to this genotype is 9%. This compares well with a figure of 8% found by Cambien et al3 for acute myocardial infarction, particularly when the present study assessed in second-degree relatives the occurrence of a disorder for which there are many other risk factors.
The mechanisms of the ACE gene–mediated effect remain speculative and the possibilities have been reviewed.3 18 They are likely to be related to the higher circulating ACE concentrations, and probably tissue ACE concentrations, associated with the D/D genotype as well as the coronary vasoconstrictor and increased vascular smooth muscle proliferative changes produced by locally released angiotensin II.
In summary, the present study shows that the D/D genotype is more prevalent in the children of families with CAD in older second-degree relatives and strongly supports an independent role for the ACE genotype as a predictor of increased coronary risk. There is evidence for an association between the D allele and circulating Lp(a) levels in high coronary risk families. The study further shows that the ACE genotype and Lp(a) levels may explain an important part of the contribution of a positive family history to increased coronary risk, one of greater magnitude than that of apoB.2 19 Genotyping of the I/D polymorphism of the ACE gene and measurements of Lp(a) and apoB are inexpensive and may facilitate the targeting of prevention in young families at increased coronary risk.
This study was supported by the National Heart Foundation of Australia. We would like to thank Judith Lynch and Michelle Marshall for collection of blood samples and questionnaire information from school children and A.S. Sim for laboratory assistance.
- Received September 26, 1994.
- Revision received October 24, 1994.
- Accepted November 1, 1994.
- Copyright © 1995 by American Heart Association
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