Association between ESR2 genetic variants and risk of myocardial infarction.
Extensive studies of the role of the ER[alpha] and ER[beta] receptors in cardiovascular disease have shown that both receptors are expressed in humans in vascular endothelial, smooth muscle, and myocardial cells (2). ER[beta] is expressed more highly in vascular smooth muscle cells in women than men, whereas ER[alpha] is present in equal quantities (3 ). In the human heart, ER[beta] is up-regulated by pressure overload, and ER[beta] mRNA levels have been observed to be increased in hearts of patients with aortic stenosis, with the greatest increases seen in women (4 ). ER[alpha] has also been associated with the development of coronary artery disease in women (5).
Single nucleotide polymorphisms (SNPs) in both ERs have been associated with cardiovascular disease. ESR1  (estrogen receptor 1; formerly ESR) polymorphisms have been associated with coronary artery disease (6-8), myocardial infarction (9), and blood pressure (10). Polymorphisms in ESR2 [estrogen receptor 2 (ER[beta])] have been associated with cardiovascular disease (11) in a North American population and left ventricular mass and left ventricular wall thickness in women with hypertension (12), blood pressure (13, 14), and premature coronary artery disease (15).
In animal models, ER[beta] knockout mice are characterized by right and left ventricular hypertrophy (16) and systemic hypertension (17). In females, ER[beta] has been shown to attenuate the hypertrophy response to pressure overload (18), play a role in the protection against ischemia/reperfusion injury in the heart (19) and against left ventricular hypertrophy (20), attenuate clinical and biochemical manifestations of heart failure, and maintain normal ventricular repolarization and ventricular automaticity after MI (21 ). In males, ER[beta] mRNA expression is induced after vascular injury in blood vessels (22), and ER[beta] seems to regulate the small artery tone more strongly than in females (23 ).
ESR2 was discovered in 1996 by Mosselman et al. (24). ER[beta], the nuclear hormone receptor encoded by ESR2, is a 54.2-kDa protein comprising 485 amino ac ids. It has 86% structural homology with ER[alpha] in the DNA-binding domain and 58% in the ligand-binding domain (25). The gene is located on chromosome 14g22-24 and has 8 transcribed exons and a 5'-un-translated exon which exists in 2 isoforms (26). In the absence of hormone, ER[beta] is sequestered in a multiprotein inhibitory complex in the nuclei. The binding of a ligand causes activation and conformational change of the protein and homodimerization through phosphorylation. The dimers bind with high affinity to estrogen response elements (EREs), located in the regulatory regions of target genes, leading to regulatory functions. Various polymorphisms have been described in the nucleotide sequence of the ESR2 gene; one of them resulted in a nonsynonymous change from leucine to valine, but this polymorphism was not observed in our population (27, 28).
The aim of this study was to investigate the relationship between various SNPs in the human ESR2 gene and the risk of MI through a nested-case control study in a Spanish population.
Materials and Methods
We used a nested case-control design. We recruited 710 consecutive patients [566 men and 144 women, mean (SD) age 61.5 (11.0) years] with a first MI admitted to the only reference coronary unit in the catchment area between January 1996 and December 1998 in Gerona, Spain, as part of the REGICOR (Registre Gironi del Cor) study.
We randomly selected 2379 control subjects [640 men and 1739 women, age 46.2 (12.9) years] from a representative population of a cross-sectional study de signed to establish the prevalence of the major cardiovascular risk factors in the province of Gerona, Spain (29, 30). Control subjects were judged free of angina or MI by history, physical examination, electrocardiography, and routine laboratory data.
Written consent was obtained from all subjects, and the local ethics committee approved the study. Using a standardized questionnaire, we collected details on socioeconomic and demographic characteristics, in addition to information on smoking, hypertension, diabetes, and current medication use, from all subjects. Presence of a dyslipidemic state was determined by a self-reported history of increased lipid concentrations and/or use of measures controlling dyslipidemia (diet or lipid-lowering drug therapy). Presence of diabetes was determined by a self-reported history of diagnosis of diabetes and/or measures controlling diabetes (diet, oral antidiabetic medication, or injected insulin).
Hypertension was defined according to WHO criteria, and 542 participants were taking antihypertensive drug therapy. Blood pressure measurements were taken after a 5 min rest and 20 min later, and the value used was the mean of the 2 determinations. Standardized hypertension questionnaires were used as described (30).
Body mass index (BMI) was determined as weight divided by squared height (kg/[m.sup.2]). The mean BMI of the participants in the study was 27.2 kg/[m.sup.2].
SCREENING OF ESR2 GENE
The promoter region and 9 exons encoding the ESR2 RNA were amplified from the genomic DNA of 23 participants. The PCR products were analyzed by direct sequencing (dideoxynucleotide-sequencing method ABI Prism BigDye Terminator 3.0; Applied Biosystems) and confirmed by sequencing of the second strand. The DNA samples of the participants were then analyzed by TagMan assay (ABI Prism 7900HT; Applied Biosystems). Three interesting SNPs were identified in the ER[beta] gene (NCBI GeneID 2100):
* rs1256049, or RsaI, a synonymous SNP 1082A[right arrow]G transition in exon 6, using the forward and reverse primers 5'-TGGCAGCCAAGCATCAACAT-3' and 5'-TGCTGCTGCACCACAGATTA-3' and the FAM AAGTGCGGCTCTT-TAMRA and VIC-CCAAGT ACGGCTCTT-TAMRA probes.
* rs4986938, or AIuI, a 1730G[right arrow]A transition in the nontranslated region downstream of exon 9, using the forward and reverse primers 5'-TCGTCCTTGC CCTTGAGCCTAAAT-3' and 5'-ACGCTGCATTC AAATGTGCCCT-3' and the FAM-ACGCTTCA GCCTGTGA-TAMRA and VIC-CACGCTTCAGCT TGTGA-TAMRA probes.
* rs1271572, a G[right arrow]T transition in the promoter region, using the forward and reverse primers 5'-ATT TGCCAGCGACACACTCT-3' and 5'-AGGCCTT TCGCGTTAGATCA-3' and the FAM-ATTGTGA GACCCCCC-TAMRA and VIC-TTGTGAGAAC CCCC-TAMRA probes.
We assessed deviation from Hardy-Weinberg equilibrium using a [chi square] test with 1 degree of freedom to compare the observed and expected genotype frequencies among the subjects. We used a [chi square] or Fisher exact test, as appropriate, to compare categorical variables between groups, and Student t test to compare continuous variables between groups. We estimated odds ratios (ORs) and 95% CIs for the effect of ESR2 polymorphisms on MI risk using logistic regression analyses adjusted for the effects of other cardiovascular risk factors. Logistic regression analysis was also used to test for interactions. Bonferroni correction was used to adjust for multiple comparison testing.
We quantified pairwise linkage disequilibrium (LD) between the polymorphisms using the Shi standardized coefficient D' ([absolute value of D']). We examined haplotype frequencies and haplotype-based analysis for confounding variables using a Bayesian algorithm implemented in the R software, available at http://www. R-project.org. The dominant model (where heterozygotes and homozygotes for a particular haplotype are assumed to have equivalent effects) was fitted by the function "haplo.glm" in the R package "haplo.stats." To deal with haplotype phase ambiguity, this function uses a method that performs an iterative 2-step EM (expectation maximization), with the posterior probabilities of pairs of haplotypes per subject used as weights to update the regression coefficients, and the regression coefficients used to update the posterior probabilities (31 ). P values <0.05 were considered statistically significant. We used the SPSS statistical software package version 15.0 for statistical analysis.
ESR2 genotypes were obtained for 3089 subjects classified as having a first MI (710 subjects) or as controls (2379 subjects). Established risk factors, including age, BMI, sex (male), diabetes, hypertension, and cigarette smoking, were observed at higher frequencies in the MI group (as shown in Table 1). Furthermore, HDL cholesterol values were lower and triglyceride concentrations higher in the MI patients than in controls. Total cholesterol and LDL cholesterol values were higher in the control group, probably because these risk factors are well controlled in MI patients.
ESR2 GENOTYPES, HAPLOTYPES, AND ALLELE FREQUENCIES
To search for sequence variants of the ESR2 gene, we sequenced the promoter, exons, and intron--exon junctions of the gene in 23 unrelated individuals. Of the 7 SNPs identified, 3 were selected and genotyped in our study population because they seemed interesting for their specific frequencies in this group of patients or their putative biological function, or because they were previously described in the literature.
The first SNP, rs1271572, is located in the promoter region, 839 base pairs from the first exon, and corresponds to a G-to-T transition. The second SNP, rs1256049, is situated in exon 6 and corresponds to a synonymous G-to-A transition. The third SNP, rs4986938, is located 39 base pairs downstream from the last exon 9, and corresponds to a G-to-A transition. Genotype frequencies did not differ significantly from those predicted by Hardy-Weinberg equilibrium for rs1271572 (P = 0.160) or for rs1256049 (P = 0.480), but they differed in the case of rs4986938 (P = 0.028).
Genotype frequencies did not differ between controls and MI subjects for the rs1256049 and rs4986938 SNPs, whereas for rs1271572, a higher percentage of MI patients carried the T allele compared with controls (Table 2), indicating an association between this polymorphism and MI (P < 0.001). Frequencies of the rs1271572 SNP differed significantly between men and women (P = 0.004), but were not found to differ between controls and MI subjects (data not shown). The 3 SNPs were found to be in strong linkage disequilibrium. In particular, the rs1271572 polymorphism was in strong linkage disequilibrium with rs1256049 ([absolute value of D'] = 0.959) and rs4986938 ([absolute value of D'] = 0.828) and rs1256049 with rs4986938 ([absolute value of D'] = 0.926). Five common haplotypes (frequency >2%) and 3 uncommon haplotypes were detected (Table 3).
ASSOCIATION BETWEEN ESR2 rs1271572 AND MI
The association between the presence of T allele of the rs1271572 SNP and MI remained statistically significant in a multivariate analysis that adjusted for sex, age, BMI, diabetes, smoking, hypertension, and HDL cholesterol (P = 0.003) (Table 4). When the data were stratified by sex, this association persisted in men (P = 0.003 ) but not in women (P = 0.754). The comparison between the 3 genotypes revealed that the most relevant difference of frequencies was between the heterozygote carriers and the GG carriers. Assessment of the other 2 SNPs did not reveal an association with MI.
ASSOCIATION BETWEEN ESR2 HAPLOTYPE AND MI
The haplotype analysis revealed an association between MI and the very common haplotype TGG (defined as T allele for rs1271572, G allele for rs1256049, and G allele for rs4986938), which had an estimated frequency of 38.2% in the population. The association was statistically significant in both the overall population (P = 0.020) and in men considered separately (P = 0.009), with a relative risk of 1.41 (95% CI 1.06-1.87) and 1.57 (95% CI 1.12-2.21), respectively, for each copy of haplotype carried (Table 5). The most common haplotype, with a frequency of 38.8%, corresponds to GGA.
Although most studies have focused on the effect of estrogens on the cardiovascular system and the role of ER[alpha], there is accumulating evidence that ER[beta] may also play an important role in the development of cardiovascular disease. Recent studies have shown that ER[beta] knockout mice suffer from systemic hypertension and heart ventricular hypertrophy (32) but that 17-[beta]-estradiol, through ER[beta]-mediated mechanisms, protects the murine heart against left ventricular hypertrophy (33). There is also growing evidence suggesting a role for ERs in regulation of vascular healing and proliferation after injury as well as ER-mediated regulation of endothelial-dependent vasodilator reactivity responses (34).
In this study, we examined and observed an association between MI and ESR2 rs1271572 SNP in the overall population, an association that seems to involve men but not women. This association did not change after adjustment for clinically relevant cardiovascular risk factors, suggesting that rs1271572 SNP is an independent risk factor for MI risk.
This SNP is located in the promoter region of ESR2 but does not belong to any consensus regulatory sequence. It is possible that the translation to RNA might be affected if the SNP belongs to a site not yet described, such as transcription factor binding sequences. The observed association of the SNP with MI may also be related to a change in the structure of either the DNA or RNA molecules, e.g., a change in the ability of the DNA to wrap itself into a nucleosome around histones, thus affecting the access of regulatory proteins to their binding sites (35). A third possibility is that the SNP could be in linkage disequilibrium with other functional modifications.
The statistical significance of these results persisted after correction for multiple testing by the Bonferroni method. Therefore, it is extremely unlikely that these results are false positives, although the possibility cannot be totally excluded. The use of the Bonferroni method to adjust for multiple testing is controversial in genetics, because it assumes that there is total independence between different polymorphisms. Many polymorphisms are known not to be independent because their variation in the population can be structured into haplotypes, transmitted as blocks of alleles in linkage disequilibrium with each other. Haplotype analysis reduces the dimensionality of association tests and increases the statistical power (36). The present study revealed an increased risk of MI in men carrying the haplotype named TGG. The risk was found also in the overall population but did not appear in women, which could be due to the small number of female cases available in the study. The TGG haplotype represents a unique change in the first SNP, rs1271572, from the major G allele to the minor T allele. The other SNPs involved are represented in the haplotype by their major allele. Therefore, the rs1271572 SNP could be a genetic risk factor for MI.
A few hypotheses may explain the differences in risk between men and women that are conferred by this haplotype. First, ER[beta] is more highly expressed in women than in men; thus, depending on the number of copies of the haplotype, the increased quantity of normal ER[beta] protein produced in women could compensate for the defect. Another possibility is that not only the expression and regulation of ER[beta], but also its activity (as determined by a complex process that is modulated by estrogens and antiestrogens), could be influenced by the differences in concentrations of circulating hormones between the sexes. Interactions with other genes or nongenetic factors correlated with sex might also explain this phenomenon (37). ER[beta] may also mediate sex differences in ischemia-reperfusion injury, as ER[beta] knockout female mice display significantly less functional recovery (and more necrosis) than wild-type female control mice; thus ER[beta] may have a greater cardioprotective role in females (38).
A study on the same set of 3 polymorphisms in the ESR2 gene was recently reported by Rexrode et al. (11 ). In that study, an association between rs1271572 and MI was also described, but the association was seen only in women, whereas in our study it was found only in men. The contradictory findings between the Rexrode study and ours might be due to differences between populations. Patients of the Rexrode study were mainly whites from North America, and the frequencies described in the HapMap project for the rs1271572 polymorphism are very different in African and Asian populations. Therefore, it is highly possible that even a small proportion of people from ethnic backgrounds other than white could lead to some differences in observed frequencies. Rexrode et al. also described an association between the rs1256049 SNP and MI in women (11 ), but this association was not observed in our population. The differences between these 2 studies on the same subject suggest that a more detailed examination of the ESR2 gene will be important.
Techniques available today permit analysis of the entire ESR2 gene using SNP tagging, genome-wide associations, copy number variants, microsatellite analysis, and many other approaches. It would be useful to investigate whether the SNPs considered in this study are in linkage disequilibrium with other functional variants in the ESR2 gene or other genes in close proximity on the chromosome, such as KCNHS [potassium voltage-gated channel, subfamily H (eag-related), member 5], and other functionally-related genes such as ER[alpha]. Functional studies should be performed to analyze the levels of RNA and protein expression and their activity.
A recent genome-wide association study with various cardiovascular disease outcomes in the context of the Framingham Heart Study (39) did not identify ESR2 as a candidate gene. Instead, these investigators identified 4 SNPs located on the same chromosome, 14. Three of the SNPs were associated with atrial fibrillation and 1 with major cardiovascular disease. In particular, the gene GJA4 (gap junction protein, [alpha]4, 37 kDa) was described as associated with cardiovascular and coronary heart disease outcomes. In this regard, a genome-wide association study in our population would be necessary to confirm and support those results.
In summary, the present study is the largest reported investigation of the relationship of ESR2 genetic variants and MI. The findings of this study suggest that a variant of ESR2 may contribute to cardiovascular disease; this knowledge may be helpful in elucidating the complex mechanisms of cardiovascular diseases and their genetic components. Functional studies, as well as the analysis of nearby polymorphisms that could explain the phenotype observed, may help in understanding the exact contribution of the variant described to the susceptibility to cardiovascular disease.
Grant/Funding Support: This study was supported by the HERACLES Network from el Fondo de Investigaciones Sanitaris (ref ISCIII G03/045).
Financial Disclosures: None declared.
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Sophie Domingues-Montanari,  Isaac Subirana,  Marta Tomas,  Jaume Marrugat, [1,2] and Mariano Senti [1,3] *
 Unitat de Lipids i Epidemiologia Cardiovascular, Institut Municipal d'Investigacio Medica (IMIM), Barcelona, Spain;  Universitat Autonoma de Barcelona, Barcelona, Spain;  Universitat Pompeu Fabra, Barcelona, Spain.
 Nonstandard abbreviations: MI, myocardial infarction; ND, cardiovascular disease; ER, estrogen receptor; SNP, single nucleotide polymorphism; BMI, body mass index; OR, odds ratio.
 Human genes: ESR1, estrogen receptor 1 (formerly ESR); ESR2, estrogen receptor 2 (ER R); KCNHS, potassium voltage-gated channel, subfamily H (eag-related), member 5; GJA4, gap junction protein, [alpha]4, 37 kDa (formerly CX37).
* Address correspondence to this author at: Unitat de Lipids i Epidemiologia Cardiovascular, Institut Municipal d'Investigacio Medica, c/ Dr Aiguader 88, Barcelona 08003, Spain. E-mail email@example.com.
Received December 19, 2007; accepted March 10, 2008.
Previously published online at D01: 10.1373/clinchem.2007.102400
Table 1. Baseline characteristics of MI cases and controls. Controls MI Cases Subjects, n (%) 2379 (77) 710 (23) Age, years 49.2 (12.9) 61.5 (11.0) BMI, k/[m.sup.2] 26.7 (5.0) 27.5 (4.3) Men, n (%) 640 (27) 566 (79.7) Systolic blood pressure, mmHg 127.5 (20.5) 117.4 (36.4) Diastolic blood pressure, mmHg 77.5 (10.9) 65.2 (11.1) Hypertension, n (%) 811 (34.3) 287 (40.4) Treatment for hypertension, n (%) 287 (12.1) 255 (36.0) Smokers 630 (27.1) 306 (47.1) Diabetes, n (%) 249 (10.5) 320 (48.7) HDL cholesterol, mmol/L 52.3 (14.8) 43.6 (12.5) LDL cholesterol, mmol/L 147.7 (39.1) 113.3 (37.6) Total cholesterol, mmol/L 221.3 (43.6) 182.3 (42.9) Triglycerides, mmol/L 108.9 (75.1) 128.3 (70.4) Data are mean (SD) unless noted otherwise. All differences are significant (P < 0.001) after Bonferroni correction. Table 2. Genotype distribution and allele frequencies of ESR2 polymorphisms among MI cases and controls. Control MI P n 2379 710 rs1271572 GG 849 (35.7) 185 (26.1)} GT 1128 (47.4) 411 (57.9)} <0.001 (a) TT 402 (16.9) 114 (16.1)} G 1413 (59.4) 390 (55)} <0.001 (a) T 966 (40.6) 320 (45)} rs1256049 GG 2174 (91.4) 656 (92.4)} AG 203 (8.5) 52 (7.3)} 0.262 AA 2 (0.1) 2 (0.3)} G 2276 (95.6) 682 (96.1)} 0.220 A 103 (4.4) 28 (3.9)} rs4986938 AA 436 (18.3) 135 (19.0)} AG 1106 (46.5) 338 (47.6)} 0.672 GG 837 (35.2) 237 (33.4)} A 989 (41.6) 304 (42.8)} 0.700 G 1390 (58.4) 406 (57.2)} Data are n (%). (a) Significant after Bonferroni correction. Table 3. Estimated population frequencies of ESR2 haplotypes. Haplotype rs1271572 rs1256049 rs4986938 Frequency, % 1 G G A 39.8 2 T G G 38.2 3 G G G 15.3 4 G A G 4.3 5 T G A 2.3 6 G A A 0.1 7 T A G 0.0 8 T A A 0.0 Table 4. Adjusted ORs and 95% CIs for the effect of ESR2 polymorphisms on the risk of MI. OR 95% CI P rs1271572 GG/T Overall 1.65 1.18-2.30 0.003 (a) Men 1.76 1.21-2.55 0.003 (a) Women 1.12 0.55-2.28 0.754 rs1256049 AA/G Overall 1.17 0.68-2.02 0.551 Men 1.13 0.62-2.06 0.683 Women 1.03 0.32-3.32 0.956 rs4986938 GG/A Overall 1.27 0.92-1.77 0.142 Men 1.18 0.79-1.75 0.411 Women 1.75 0.80-3.84 0.158 Logistic regression adjusted for sex, age, BMI, diabetes, smoking, hypertension, and HDL cholesterol. Overall, n = 2489; men, n = 872; women, n = 1621. (a) Significant after Bonferroni correction. Table 5. Association of ESR2 TGG haplotype and MI. OR 95% CI P Overall 1.41 1.06-1.87 0.020 (a) Men 1.57 1.12-2.21 0.009 (a) Women 1.04 0.63-1.74 0.867 Logistic regression adjusted for sex, age, BMI, diabetes, smoking, hypertension, and HDL levels. TGG haplotype defined as T allele for rs1271572, G allele for rs1256049, and G allele for rs4986938. Overall, n = 2489; men, n = 872; women, n = 1621. (a) Significant after Bonferroni correction.
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|Title Annotation:||Lipids, Lipoproteins, and Cardiovascular Risk Factors|
|Author:||Domingues-Montanari, Sophie; Subirana, Isaac; Tomas, Marta; Marrugat, Jaume; Senti, Mariano|
|Article Type:||Clinical report|
|Date:||Jul 1, 2008|
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