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Gene Polymorphisms Are Associated with the Angiographic Extent of Coronary Artery Disease
Department of Medicine, Hadassah University Hospital on Mount Scopus (A.R.), Jerusalem, 91240 Israel; Departments of Cardiology (A.P.) and Ophthalmology (A.B.), and Endocrinology and Metabolism Service, Department of Internal Medicine (L.Re., R.D.-P.), Hadassah-Hebrew University Medical Center, Jerusalem, 91120 Israel; Authority for Computation and Information Medical Area Branch, Hebrew University (L.Ro.), Jerusalem, 91120 Israel; and Unit of Epidemiology, Hebrew University-Hadassah School of Public Health (Y.F.), Jerusalem, 91120 Israel
Address all correspondence and requests for reprints to: Rivka Dresner-Pollak, M.D., Endocrinology and Metabolism Service, Hadassah Hebrew-University Medical Center, P.O. Box 12000, Jerusalem, 91120 Israel. E-mail: rivkap{at}md.huji.ac.il.
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Abstract |
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gene (ESR1) may alter the atheroprotective effects of estrogens, and be associated with the severity of coronary artery disease (CAD). Objective: This study seeks to investigate the association between the ESR1 haplotype created by the c.454-397 T>C and c.454-351 A>G polymorphisms, the length of the (TA)n repeats, and the angiographic extent of CAD.
Design: Consecutive subjects with age younger than or equal to 55 yr who had undergone coronary angiography between November 2003 and January 2004 were included in the study.
Setting: The study was conducted in a referral center.
Patients: One hundred five subjects with age younger than or equal to 55 yr (87 males, 18 females) participated in the study.
Main Outcome Measures: The angiographic extent of CAD was graded by number of: 1) major coronary vessels with more than 50% narrowing (NMCV); 2) narrowed major coronary vessels and/or their second-order branch (NCV); and 3) coronary segments with any narrowing (NN). Analysis of covariance was used to test the effect of haplotype and (TA)n length on the angiographic extent of CAD with gender and number of CAD risk factors (hyperlipidemia, diabetes, hypertension, obesity, smoking, and family history of CAD) as covariates.
Results: The ESR1 haplotype c.454-397C and c.454-351G was associated with NCV and NN (P = 0.008 and 0.02, respectively). Carriers of two copies of haplotype C-G had a higher number of NCV compared with subjects with one or no copies combined (3.5 ± 2.2 vs. 2.3 ± 1.9, P = 0.012, respectively). A longer (TA)n repeat was associated with NCV (P = 0.04).
Conclusions: The ESR1 c.454-397C and c.454-351G haplotype and longer (TA)n repeats are associated with the extent of CAD in young subjects, independent of the known CAD risk factors.
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Introduction |
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It is also plausible that genetic variation in estrogen-mediated pathways can modulate the individual’s response to exogenous or endogenous estrogens, and contribute to the variation in cardiovascular risk. The actions of estrogens are mediated by two distinct receptors, estrogen receptor type (ER-) and estrogen receptor type ß (ER-ß), which are expressed in various cell types in the cardiovascular system. ER- is expressed in endothelial, smooth muscle, and myocardial cells, and was shown to play an important role in the development of coronary atherosclerosis in both females and males (5, 6).
Few studies suggested an association between common polymorphisms in the ER- gene (ESR1) and cardiovascular disease susceptibility (7, 8, 9, 10, 11, 12, 13, 14, 15).
Two large prospective, population-based cohort studies, the Rotterdam study and the Framingham study, examined the association between the T/C polymorphism in the first intron of the ESR1 gene (c.454-397 T>C), defined by the PvuII restriction enzyme, and the risk of cardiovascular disease (10, 11). In both studies, carriage of a specific genotype was associated with a 2- to 3-fold increase in myocardial infarction (MI) risk. However, the results of these two studies seemed to contradict each other. While in the Framingham study, male carriers of the C/C genotype had a 3-fold increased MI risk, in the Rotterdam study, female carriers of the T/T or T/C genotype had a 2-fold increased risk of MI. It has been speculated that there may be a gender difference in the role of ESR1 in the cardiovascular system (16). Furthermore, it is unknown whether increased MI risk associated with ESR1 c.454-397 T>C genotype, observed in these studies, is related to acute thrombosis or atherosclerosis.
The aim of this study was to determine whether the c.454-397 T>C and the c.454-351 A>G polymorphisms and the length of the (TA)n repeats in the ESR1 gene are associated with the extent of coronary atherosclerosis, as indicated by coronary angiography conducted in an unselected population of young subjects undergoing cardiac catheterization.
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Subjects and Methods |
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One hundred fourteen consecutive subjects with age younger than or equal to 55 yr who had undergone coronary angiography at Hadassah-Hebrew University Medical Center between November 2003 and January 2004, and agreed to participate in the study, were included. Clinical data and cardiovascular risk factors were obtained from medical charts and included: sex, ethnic origin, age, smoking habits, usage of medications, premature CAD in first-degree relatives, hypertension, hyperlipidemia, obesity, and diabetes mellitus. Ethnic origin was defined based on place of birth of four grandparents. The following definitions were used for coronary risk factors: hypertension, blood pressure greater than or equal to 135/85 mm Hg or antihypertensive therapy; dyslipidemia, low-density lipoprotein more than 130 mg/dl, high-density lipoprotein less than 45 mg/dl, triglycerides more than 200 mg/dl or lipid lowering treatment; diabetes, fasting plasma glucose more than 126 mg/dl or glucose lowering treatment; obesity, body mass index greater than or equal to 30 kg/m2, smoking (>10 cigarettes per day) current or recent (<2 yr before CAD onset).
To determine ethnic variability in the distribution of the ESR-1 c.454-397 T>C and c.454-351 A>G genotypes and alleles in Jewish Israeli ethnic groups, 256 randomly selected healthy blood donors from three main ethnic groups were genotyped: Ashkenazi Jews (n = 107), North African Jews (n = 80), and Asian Jews, mainly Iraqi Jews (n = 69). The study was approved by the institutional ethics committee, and informed consent was obtained from all participants
Angiographic findings
The angiographic extent of CAD was graded, as previously described by us (13). Briefly, CAD extent was assessed by three ways. 1) The number of major coronary vessels with greater than 50% occlusion (NMCV). Namely, single-, double-, or triple-vessel disease, classifying grades 0–3. A greater than 50% lesion in the left main coronary artery was regarded as two-vessel disease. 2) The number of narrowed major coronary vessels (left main, left anterior descending, left circumflex, and right coronary arteries) and/or their second-order branches (up to two large diagonals, two large marginals, and the posterior descending artery) with any narrowing, classifying grades 0–9 (NCV). 3) The number of coronary segments with any narrowing, using a 15-segment scheme, classifying grades 0–15 (NN). Coronary angiograms were interpreted by two independent cardiologists.
Determination of ESR1 genotypes and haplotypes
DNA was extracted from peripheral blood. The region containing the polymorphic c.454-397 T>C and the c.454-351 A>G sites was amplified by PCR with the following primers: forward primer 5'-CTCTACATGTTCCTAAAGAGG-3'; reverse primer 5'-CGATTATCTGAATTTGGCCTGG-3'. PCR was carried out in 35 cycles by the following steps: denaturation at 94 C for 30 sec, annealing at 61 C for 40 sec, and extension at 37 C for 90 sec. The 0.6-kb product was cleaved with the PvuII and XbaI restriction endonucleases. For PvuII, genotypes were termed CC, CT, or TT where the T allele codes for the presence of the PvuII site and the C allele codes its absence. For XbaI genotypes were termed AA, AG, or GG, where the A allele codes for the presence of the XbaI site and the G allele codes for its absence. Genotype data for each of the two polymorphisms was used to infer the haplotype alleles present in the population using the program PHASE version 2.0, which reconstructs haplotypes from population genotype data (17). Alleles were then defined as the following four haplotypes, T-A (representing a thymine 397 and adenosine 351 bp up-stream of exon 2, respectively), T-G, C-A, and C-G, and were coded as haplotype numbers 1–4 (1, T-A; 2, T-G; 3, C-A; 4, C-G).
The length of the (TA)n repeats 1174 bp upstream of exon 1 were determined as previously described by us (13).
Statistical methods
Observed and expected genotype frequencies were compared using 
2 test to determine whether they were in Hardy-Weinberg equilibrium. Genotype and allele frequencies among the three ethnic groups of healthy controls were compared by 2 test. Subjects were stratified by allele copy number (0, 1, or 2) of each haplotype. Four possible haplotype alleles were observed in our study population in the following frequencies: haplotype 1 (T-A) 47%, haplotype 2 (T-G) 3%, haplotype 3 (C-A) 2%, haplotype 4 (C-G) 48%. Based on our findings, we chose haplotype 4 (C-G) as the haplotype of interest. Comparisons of clinical variables among subjects with none, one, or two copies of haplotype 4 were performed using Pearson correlation coefficients and 2 analysis for continuous and categorical variables, respectively. Analysis of covariance was used to test the effect of ESR1 haplotype 4 and the mean length of the (TA)n on the angiographic extent of CAD with gender and the number of CAD risk factors as covariates. We initially examined the possible interaction between gender and haplotype 4 on measures of CAD. This interaction was not statistically significant; and we therefore deleted the interaction term from the final model. Because earlier studies suggested an opposite effect of ESR1 genotypes on ischemic heart disease outcomes in females and males, we performed an additional subanalysis stratified by gender. P < 0.05 was considered significant. Statistical analyses were performed using SAS version 8.2 (SAS Institute, Inc., Cary, NC).
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Results |
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There were no significant differences in the distribution of the c.454-397 T>C and c.454-351 A>G alleles and genotypes among healthy Jewish controls originated from three ethnic groups (Table 1
). Genotype distributions among controls were in Hardy-Weinberg equilibrium.
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The prevalence of the C and G alleles in the study population was 0.5 and 0.51, respectively, similar to those reported in other populations. Genotype distributions were in Hardy-Weinberg equilibrium. The prevalence of none, one, or two copies of haplotype 4 were 31.4, 41.9, and 26.7%, respectively. Clinical characteristics of the study population according to the number of copies of ESR1 haplotype 4 (none, one, or two) are presented in Table 2. There were no statistically significant differences among haplotype groups. A trend for increased prevalence of obesity with increased number of copies of haplotype 4 was noted (P = 0.052).
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The number of CAD risk factors significantly affected the extent of CAD for NMCV (P < 0.0001), NCV (P = 0.0009), and NN (P = 0.0002), indicating that our study population was representative of the population undergoing coronary catheterization.
The ESR1 haplotype 4 was associated with two out of the three measures of CAD extent after adjusting for gender and the number of CAD risk factors (P = 0.008 and 0.02 for NCV and NN, respectively) (Table 3).
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). There was also a trend for higher number of total narrowings (NN) in carriers of two copies of haplotype 4 compared with carriers of one and no copies combined (4.21 ± 2.8 vs. 3.0 ± 2.7, P = 0.07 in subjects with two vs. one and no copies combined).
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In an additional analysis, the model was run with the mean (TA)n repeats length, ESR1 haplotype 4, gender, and the number of risk factors. Higher mean (TA)n length was found to be significantly associated with NCV (P = 0.04), independently of the effect of ESR1 haplotype 4.
In a subanalysis, the models were rerun stratified by gender. The results showed similar effect in the same direction of haplotype 4 on CAD measures in both women and men (P = 0.26, 0.07, 0.11 for NMCV, NCV, and NN in women, respectively, and P = 0.29, 0.04, 0.09 for NMCV, NCV, and NN in men, respectively). Thus, the findings support using a model that includes both men and women.
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Discussion |
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gene was found to affect the angiographic extent of CAD, independent of the known CAD risk factors. Carriers of the two copies of haplotype 4 had a significantly higher number of narrowed coronary vessels compared with carriers of one and no copies of haplotype 4. These findings are in agreement with an earlier autopsy study in which Finnish males with the c.454-397 C/C genotype had significantly larger areas of coronary artery wall atherosclerosis compared with those with the c.454-397 C/T or c.454-397 T/T genotypes. Moreover, theses results are consistent with the data from the Framingham Heart study in which carriers of the c.454-397 C/C genotype had a 3-fold greater risk of MI, and a 2-fold increased risk of major atherosclerotic cardiovascular disease, defined as coronary insufficiency, CAD death, or atherothrombotic stroke (11). Our findings are driven by the results in men, but are also supported by the results that were observed in the small number of women who were included in our study. Interestingly, previous studies suggested apparently opposite effects of the ESR1 c.454-397 T>C polymorphism on CAD risk in females and males. In the Rotterdam study, Schuit et al. (10) found that female carriers of the T allele but not the C allele had a 2-fold increased risk of MI and ischemic heart disease. No increased risk was found in that study in males carrying the T allele (10). On the other hand, in the Framingham study, Shearman et al. (11) reported a 3-fold increase in MI risk among male carriers of two copies of the C allele. Because only five women in that study had an MI, the authors could not evaluate the role of this polymorphism in women. In addition, in another study, the T/T genotype but not the C/C genotype was found to be an independent predictor for in-stent restenosis in women but not in men (18). Finally, an association between the C/C genotype and systolic hypertension was observed in men but not in women (19). Future studies should include a significantly larger number of women undergoing coronary angiography to further investigate the question of gender differences in the effect of ESR1 variants on measures of CAD.
A borderline association between the ESR1 c.454-397C and c.454-351G haplotype and obesity was observed in our study. Although this finding was not a confounder of the effect of ESR1 genotype on CAD extent, it is in agreement with an earlier study, which also showed an association between the C/C genotype and an elevated body mass index (20). Deng et al. (20) reported that elderly women with the C/C genotype tend to gain weight with age, whereas those with the C/T or T/T genotypes tend to lose weight with age.
A known limitation to genetic association studies is the possible influence of ethnic heterogeneity of the studied population. Although the Israeli Jewish population is heterogeneous in its ethnic origin, we have shown that the frequencies of the ESR-1 c.454-397 T>C and c.454-351 A>G polymorphisms in alleles and genotypes were similar among the various Israeli ethnic groups, and also similar to those reported in other studies of white individuals. We were unable to genotype healthy Muslim controls. However, this ethnic group comprised a minority in our study population. The possibility of residual population stratification is an additional limitation of a genetic association study especially with a small sample size. Thus, our findings may be related to other yet unrevealed characteristics of these subjects, which may affect the risk of CAD. Family-based association studies or use of a panel of genetic markers in unrelated populations could be considered for larger studies in the future to overcome the limitation of population stratification.
The mechanism by which the ESR1 haplotype is associated with CAD is still unknown, and may be related to the contribution of yet undefined tightly linked locus to CAD. In addition, our results suggest that carriers of one copy of the C-G haplotype may have the lowest number of obstructed coronary vessels when compared with carriers of no or two copies of this haplotype. The possibility of some sort of advantage to the heterozygous state is remote. Although we used a haplotype approach rather than a single nucleotide polymorphism, the two sites used to create this haplotype are only 46 bp apart, and thus are showing high linkage disequilibrium. Therefore, a plausible explanation is that the group of carriers of one copy of the C-G haplotype is by chance comprised of subjects with a favorable genetic or other background factors with respect to CAD. At this stage, we have no other explanation for this apparently heterozygous advantage.
The ESR-1 c.454-397 T>C site was previously shown to be in linkage disequilibrium with the (TA)n repeats site in the promoter region of the ESR1 (21). In this study, we have demonstrated an association between the (TA)n repeats polymorphism and the extent of CAD, further confirming others’ and our previous data (12, 13). Thus, the plausibility that the ESR-1 c.454-397 T>C polymorphism affects promoter usage and influences the expression of the ESR1 should be further explored.
In conclusion, we reported an association between common polymorphisms in the ER- gene and the extent of coronary artery atherosclerosis. Our study further supports earlier studies, which suggested that sequence variants in the ER- gene might modify the effects of estrogens on the cardiovascular system, and be associated with cardiovascular disease susceptibility. Large studies in women could help resolve the issue of the possible differential response of ER- variants to exogenous estrogens.
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Footnotes |
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First Published Online September 13, 2005
Abbreviations: CAD, Coronary artery disease; ER, estrogen receptor; ET, estrogen therapy; MI, myocardial infarction; NCV, number of coronary vessels with any narrowing; NMCV, number of major coronary vessels with more than 50% narrowing; NN, number of narrowed coronary segments; WHI, Women’s Health Initiative.
Received February 4, 2005.
Accepted September 6, 2005.
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genotypes with body mass index in normal healthy postmenopausal Caucasian women. J Clin Endocrinol Metab 85:2748–2751 gene and their relationship to bone mass variation in postmenopausal Italian women. Hum Mol Genet 9:2043–2050This article has been cited by other articles:
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