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Angiotensin-Converting Enzyme Gene Polymorphisms and Obesity: An Examination of Three Black Populations Holly Kramer,* Xiaodong Wu,* Donghui Kan,* Amy Luke,* Xiaofeng Zhu,* Adebowale Adeyemo,† Colin McKenzie,‡ and Richard Cooper*

Abstract KRAMER, HOLLY, XIAODONG WU, DONGHUI KAN, AMY LUKE, XIAOFENG ZHU, ADEBOWALE ADEYEMO, COLIN MCKENZIE, AND RICHARD COOPER. Angiotensin-converting enzyme gene polymorphisms and obesity: an examination of three black populations. Obes Res. 2005;13:823– 828. We examined the association between obesity and 13 angiotensin-converting enzyme (ACE) gene polymorphisms, including the presence (I) or absence (D) of an Alu element in intron 16 (I/D polymorphism), and performed haplotype analysis using data collected from participants of a community survey of hypertension among blacks living in Ibadan, Nigeria; Spanish Town, Jamaica; and Chicago, IL. Transmission distortion of ACE gene polymorphisms and haplotypes from heterozygous parents to affected offspring was examined in each study population. To estimate haplotypes, polymorphisms were divided into three groups based on their position on the ACE gene. No ACE gene polymorphism was consistently overtransmitted from parents to obese offspring among the three populations. However, the haplotype ACE1-ACE5 TACAT, located in the promoter region, was significantly overtransmitted from parents to obese offspring in both the U.S. and Nigerian populations. No haplotype was significantly overtransmitted from parents to obese offspring among the Jamaicans. In conclusion,

Received for review July 26, 2004. Accepted in final form March 7, 2005. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. *Department of Preventive Medicine, Loyola Medical Center, Maywood, Illinois; †Department of Pediatrics/Institute of Child Health, College of Medicine, University of Ibadan, Ibadan, Nigeria; and ‡Tropical Medicine Research Unit, University of the West Indies, Mona, Jamaica. Address correspondence to Holly Kramer, Loyola Medical Center, Department of Preventive Medicine, 2160 First Avenue, Maywood, IL 60153. E-mail: [email protected] Copyright © 2005 NAASO

we noted the overtransmission of a particular ACE gene promoter region haplotype from parents to obese offspring in two separate black populations. These data suggest that ACE gene polymorphisms may influence the development of weight gain. Key words: angiotensin-converting enzyme gene, polymorphisms, haplotype The worldwide epidemic of obesity has resulted primarily from an imbalance of caloric intake and expenditure in societies becoming more affluent and sedentary. However, for any given individual, obesity is a multifactorial trait influenced by both genetic and environmental factors. The strong link between hypertension and obesity makes the genes composing the renin-angiotensin system interesting targets for study. Previous studies examining the association between angiotensin-converting enzyme (ACE)1 gene polymorphisms and obesity focused on the presence (I) or absence (D) of an Alu element in intron 16 (I/D polymorphism), and results have not been consistent (1–3). Differences in risk of obesity with the ACE I/D genotype have been attributed to sex and racial differences (1). However, many studies lack sufficient power to detect small contributions from a single polymorphism in a complex polygenic trait. In this study, we examined the association between BMI, percent body fat, overweight, and obesity and 13 ACE gene polymorphisms, including the I/D polymorphism, and performed haplotype analysis in three populations of black adults living in differing environments: Nigeria, Jamaica, and Maywood, IL.

1

Nonstandard abbreviations: ACE, angiotensin-converting enzyme; I/D polymorphism, the presence (I) or absence (D) of an Alu element in intron 16; TDT, transmission/disequilibrium test; PGI2, prostacyclin.

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ACE Gene and Obesity, Kramer et al.

Figure 1: Schematic representation of the human ACE gene with the 13 polymorphisms. Positions characterized by the use of ⫹1 represent the first nucleotide of the first codon in exon 1. UTR, untranslated region.

Participants included 1158 adults from 312 families in Nigeria; 546 adults from 136 Jamaican families; and 1080 adults from 312 African-American families. The total number of overweight and obese offspring in each of the three study populations was 187 and 113 in Nigeria, 117 and 65 in Jamaica, and 400 and 277 in the United States, respectively. Heritability for BMI was 0.36 for Nigeria, 0.49 for Jamaica, and 0.61 for the United States. A schematic representation of the ACE gene with the 13 polymorphisms genotyped across a 27-kb segment is shown in Figure 1. No

significant deviations from Hardy-Weinberg equilibrium were found except ACE2.1 and ACE6 (p ⫽ 0.05 and p ⫽ 0.03, respectively). The minor allele frequencies of each of the 13 ACE gene polymorphisms among the three populations are shown in Table 1. Approximately one-third of the three study populations carried the Alu segment in intron 16 (I allele). Linkage disequilibrium among the 13 polymorphisms has been previously described (4). In the Jamaican population, log-transformed BMI was significantly associated with ACE2 (A-5499C; p ⫽ 0.04)

Table 1. Minor allele frequencies of the 13 ACE polymorphisms

Marker

Minor allele

Location

Nigeria Frequency (%)

ACE1 ACE2 ACE3 ACE4 ACE5 ACEs2.1 ACEs1.1 ACE6 New ACE6* ACE7 I/D ACE8 ACE9

C(C/T) C(A/C) T(C/T) T(A/T) C(C/T) C(C/T) A(A/G) T(C/T) G(A/G) G(A/G) I(I/D) G(A/G) 2(2/3)

5’-UTR 5’-UTR 5’-UTR 5’-UTR 5’-UTR Intron 7 Intron 8 Exon 8 Intron 8 Exon 15 Intron 16 Exon 17 3’-UTR

34.46 34.22 5.91 33.23 5.98 8.15 3.88 9.48 0.61 36.61 35.74 21.24 43.71

Allele frequencies were estimated using the counting method from the extended founders. * Allele frequency ⬍1% in each population.

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Jamaica Frequency (%)

United States Frequency (%)

39.65 37.17 11.96 39.34 12.92 7.89 9.41 16.49 0.50 39.11 38.13 24.60 42.74

33.09 32.16 17.40 32.56 10.97 7.73 9.01 18.30 0.25 40.55 38.60 20.37 41.28


and ACE5 (C-115T; p ⫽ 0.03) after adjustment for age and sex. Log-transformed percent body fat was significantly associated with ACE5 (C-115T; p ⫽ 0.03) and ACE8 (A11860G; p ⫽ 0.0003) after adjustment for age and sex. In contrast, no allele was significantly associated with logtransformed BMI or percent body fat in the Nigerian or U.S. populations. No allele was significantly overtransmitted from parents to overweight offspring in the Nigerian or U.S. study populations, but ACE1 (T-5529C; p ⫽ 0.04) and ACE3 (T-3925C; p ⫽ 0.03) were both significantly overtransmitted among the Jamaicans. In Nigeria, ACE7 (A9596G; p ⫽ 0.02), I/D (p ⫽ 0.03), and ACE8 (A11860G; p ⫽ 0.05) were all significantly overtransmitted from parents to obese offspring. In the U.S. study sample, only ACE3 (T-3925C; p ⫽ 0.03) was significantly overtransmitted from parents to obese offspring. No polymorphism was significantly overtransmitted from parents to obese offspring in the Jamaican study population. Thus, no ACE gene polymorphism was consistently overtransmitted from parents to overweight or obese offspring across the three study populations. The transmission/disequilibrium test (TDT) results for Group 1 haplotypes [promoter region haplotypes (ACE1 to ACE5)] are shown in Table 2. (No significant association was noted between Groups 2 and 3 haplotypes and overweight or obesity in any of the three populations; data not shown.) The haplotype TACAT was significantly overtransmitted from parents to overweight offspring in the Nigerian population (p ⫽ 0.04) and reached borderline significance in the U.S. study population (p ⫽ 0.06). This same promoter region haplotype was significantly overtransmitted from parents to obese offspring in both the U.S. and Nigerian populations. The haplotype CCCTC was also significantly overtransmitted from parents to obese offspring in the U.S. population (p ⫽ 0.05). The global tests for ACE gene promoter region haplotypes reached statistical significance for both overweight and obesity in the U.S. (p ⫽ 0.03 and p ⫽ 0.02, respectively) and Nigerian populations (p ⫽ 0.04 and p ⫽ 0.04, respectively). No significant results were observed among the Jamaican participants. The ACE gene promoter region haplotype TACAT was in linkage disequilibrium with the D allele of the I/D. Among the Nigerian and Jamaican participants, 91% of the ACE gene promoter region TACAT haplotypes carried the D allele, and the percentage was 86% among U.S. participants. The characteristics of the three study populations are shown in Table 3. In this study of three black populations living in diverse environments, we examined the association between 13 ACE gene polymorphisms and BMI, overweight, and obesity. This study differs from previous studies that relied solely on the ACE gene I/D polymorphism (1,2). Although no single ACE gene polymorphism was consistently overtransmitted from parents to overweight or obese offspring in all three populations, we noted that a particular ACE gene

promoter region haplotype was overtransmitted from parents to overweight and obese offspring in both the Nigerian and U.S. participants. This ACE gene promoter region haplotype (ACE1ACE2-ACE3-ACE4-ACE5 TACAT) contains the ACE gene polymorphism ACE4, which is associated with increased serum ACE levels (5). In vitro studies suggest that higher ACE levels could increase the risk of obesity by increasing angiotensin II concentrations. Angiotensin II accelerates the conversion of preadipocytes to adipocytes and enhances lipid storage through stimulation of prostacyclin production (6 –9). In the presence of arachidonic acid, preadipocytes, adipocytes, and endothelial cells all produce prostacyclin (PGI2) through the metabolism of arachidonic acid by cyclooxygenase I or II. PGI2 promotes the terminal differentiation of preadipocytes into adipocytes through several intracellular signaling pathways (8,10). Darimont and colleagues (8,10) showed that differentiated adipocytes exposed to angiotensin II may also produce PGI2, and these effects seemed to be mediated through angiotensin II type 1 receptors. We noted no association between the ACE gene I/D polymorphism and the presence of overweight or obesity in each of the three populations. Strazullo et al. (1) reported that Italian men with the D/D allele had 82% higher odds of being overweight and were more likely to gain weight with age compared with men with the I/D or I/I allele. The prevalence of obesity was higher in the Italian men with the D/D allele compared with those with the I/D or the I/I allele, but the difference was not statistically significant (18.1% vs. 15.5%; p ⫽ 0.2) (1) perhaps because of the low number of obese study participants. The reported association between the ACE gene D/D allele and overweight in Italian men may reflect linkage disequilibrium between the D allele and the TACAT haplotype. In a previous report, we constructed a cladogram using the 13 ACE gene polymorphisms among the three study populations (4). The ACE gene promoter region haplotype TACAT is a member of a particular ACE genotype clade associated with hypertension. This cladogram, or tree diagram that estimates the evolutionary relationships between variants in the ACE gene, showed the presence of four common clades or groups of haplotypes, which have previously been described in detail (4). The haplotype groups comprising Clade 1 were significantly overtransmitted to offspring with hypertension, and all of the haplotypes that were overtransmitted from parents to overweight and obese offspring among the three study populations in this analysis also belong to Clade 1. The fact that this particular clade contains the ACE gene promoter haplotype TACAT and the D allele suggests that variation in ACE gene expression may influence both blood pressure and development of obesity. The lack of an association between ACE gene haplotypes and overweight and obesity in Jamaica may have been OBESITY RESEARCH Vol. 13 No. 5 May 2005

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Table 2. TDT results for association between overweight and obesity and ACE gene promoter region haplotypes (ACE1-ACE2-ACE3-ACE4-ACE5) Haplotype

Observed*

Expected†

TDT

p‡

TDT results for overweight and ACE gene promoter region haplotypes United States 11111 (TATAT) 11211 (TACAT) 22221 (CCCTT) 22222 (CCCTC) Global test Jamaica 11111 (TATAT) 11211 (TACAT) 12211 (TCCAT) 22221 (CCCTT) 22222 (CCCTC) Global test Nigeria 11211 (TACAT) 22221 (CCCTT) Global test

100.71 380.35 141.82 66.741

95.992 366.55 143.85 64.031

0.868 3.213 0.106 0.368

0.4 0.06 0.7 0.6 0.03

13.002 98.21 13.406 41.439 26.884

14.156 101.41 14.243 38.624 22.936

0.506 0.662 0.176 0.873 2.392

0.5 0.4 0.7 0.4 0.2 0.7

211.58 87.216

198.74 89.367

4.794 0.174

0.04 0.7 0.04

0.6 0.009 0.7 0.05 0.02

TDT results for obesity and ACE gene promoter region haplotypes United States 11111 (TATAT) 11211 (TACAT) 22221 (CCCTT) 22222 (CCCTC) Global test Jamaica 11111 (TATAT) 11211 (TACAT) 12211 (TCCAT) 22221 (CCCTT) 22222 (CCCTC) Global test Nigeria 11211 (TACAT) 22221 (CCCTT) Global test

59.255 272.35 98.994 50.233

61.724 255.28 101.15 47.034

0.355 7.168 0.171 7.168 13.923

10.473 48.503 7.409 22.999 14.131

10.388 52.623 8.355 21.434 13.525

0.004 2.108 0.347 0.495 0.087 3.678

0.9 0.2 0.6 0.5 0.7 0.6

137.51 45.057

125.15 49.657

6.979 1.486

0.01 0.3 0.04

* Observed number of transmitted haplotypes. † Expected number of transmitted haplotypes under the assumption of no linkage and no association (e.g., 50% chance of transmission of the specific haplotype). ‡ p value based on 10,000 permutations.

caused by the smaller number of participants or sampling variation. Environmental differences between the populations may also have accounted for the null findings in 826


Jamaica. We did not adjust the level of statistical significance for multiple comparisons and cannot rule out that these findings were simply caused by chance alone. How-


Table 3. Demographic characteristics of the study sample by environment

Male (%) Age (years) Percent body fat BMI (kg/m2) Overweight (%) Obese (%)

Nigeria (n ⴝ 1059)

Jamaica (n ⴝ 428)

United States (n ⴝ 956)

p

54 42.0 ⫾ 19.5 18.3 ⫾ 10.8 21.3 ⫾ 4.3 28 17

42 41.8 ⫾ 16.9 27.8 ⫾ 12.4 25.7 ⫾ 6.3 36 21

39 39.5 ⫾ 14.9 34.9 ⫾ 12.4 29.3 ⫾ 7.7 56 39

⬍0.0001 0.012 ⬍0.0001 ⬍0.0001 ⬍0.001 ⬍0.001

ever, the overtransmission of a particular ACE gene promoter region haplotype from parents to overweight and obese offspring was noted in two separate populations living in different environments, which makes the results less likely to be caused by chance. The association between the ACE gene promoter region haplotype TACAT and body size was more consistent for obesity than overweight. Associations between a genetic variant and a phenotype strongly influenced by environment such as BMI may be easier to detect at extreme values where environmental variance is lower. In conclusion, we noted the overtransmission of a particular ACE gene promoter region haplotype from parents to obese offspring in two separate black populations. Because this haplotype is in strong linkage disequilibrium with deletion of the Alu element in intron 16 (D allele), these results support the previously reported association between the ACE gene D allele and overweight and provide further evidence that ACE gene polymorphisms may influence the development of obesity.

Research Methods and Procedures Sampling Frame The sampling frame consisted of the participants of the International Collaborative Study of Hypertension in Blacks, a community survey of hypertension in Ibadan, Nigeria; Spanish Town, Jamaica; and Chicago, IL (11). Information on study protocol and recruitment procedures has been previously published (11). Study protocols were approved by the review boards of the participating institutions, and all participants provided written informed consent. Overweight and Obesity Weight and height were collected by trained research staff. Percent body fat was estimated using bioelectrical impedance analysis. These measurements were validated and cross-validated against deuterium dilution in each of the three populations (12,13). In Jamaica and the United States, overweight and obesity were defined as a BMI ⱖ27 and

ⱖ30 kg/m2, respectively, consistent with previous studies on ACE and obesity (1). In Nigeria, the distribution of BMI was markedly narrower compared with Jamaica and the United States, and the 75th percentile for BMI was 23.1 kg/m2. Because of marked differences in the distribution of BMI in Nigeria compared with Jamaica and the United States, overweight and obesity were defined as a BMI ⱖ23 and ⱖ25 kg/m2, respectively, in Nigeria (approximately the 75th and 80th BMI percentiles). Genotype and BMI data were available for 1059 Nigerians, 428 Jamaicans, and 956 U.S. blacks. Statistical Analysis Differences in continuous variables between the three populations were compared using ANOVA, and categorical variables were compared using the ␹2 test. Allele frequencies were estimated from the founders by the counting method, and the ␹2 test was used to assess Hardy-Weinberg equilibrium (14). Heritability was defined as the proportion of BMI variance caused by additive genetic variance and was calculated by variance component methods using the Statistical Analysis for Genetic Epidemiology package (SAGE v 4.4) (15). We examined the association between ACE gene polymorphisms and log-transformed BMI and log-transformed percentage body fat while controlling for age and sex, using the computer program ASSOC, available in the SAGE package. This program uses a mixed-effect model that treats the genetic marker and covariates (age and sex) as fixed effects, whereas the familial effect among relatives and individual environmental effects are treated as random effects. The familial effect was modeled on the assumption that the residual correlation between pairs of family members was entirely caused by polygenic effects. An additive effect model was assumed within each locus. To account for admixture in the Jamaican and U.S. samples, we used the generalized TDT (TRANSMIT program) proposed by Clayton (16) to determine transmission distortion of ACE polymorphisms from heterozygous parents to affected offspring. Haplotype analysis for overweight and obesity was performed using TRANSMIT. For multiple polymorphisms, OBESITY RESEARCH Vol. 13 No. 5 May 2005

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TRANSMIT reconstructs haplotypes through an E-M algorithm and tests individual haplotypes and overall transmission distortion (global test). To estimate haplotypes, we divided 12 polymorphisms into three groups based on their location on the ACE gene (Figure 1). Group 1 included ACE1 to ACE5 in the promoter region, whereas Group 2 included ACE2.1 and ACE1.1 (intron 7) and ACE6 (exon 6). New ACE6 was deleted because it had ⬍1% frequency. ACE7 (exon 15), I/D (intron 16), ACE8 (exon 17), and ACE9 (3⬘-untranslated region) comprised haplotype Group 3. The significance level was assessed by a bootstrap procedure based on 10,000 replicates. Multiple comparisons in each region were adjusted by the ␹2 statistic with the increased number of degree of freedoms based on the number of observed haplotypes.

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