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Estrogen Receptor  Gene Polymorphisms and Risk of Myocardial Infarction
Stephanie C. E. Schuit, MD;
Hok-Hay S. Oei, MD;
Jacqueline C. M. Witteman, PhD;
Corine H. Geurts van Kessel, MSc;
Joyce B. J. van Meurs, PhD;
Rogier L. Nijhuis, MD;
Johannes P. T. M. van Leeuwen, PhD;
Frank H. de Jong, PhD;
M. Carola Zillikens, MD;
Albert Hofman, MD, PhD;
Huibert A. P. Pols, MD, PhD;
André G. Uitterlinden, PhD
JAMA. 2004;291:2969-2977.
ABSTRACT
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Context The role of estrogens in ischemic heart disease (IHD) is uncertain. Evidence suggests that genetic variations in the estrogen receptor  (ESR1) gene may influence IHD risk, but the role of common sequence variations in the ESR1 gene is unclear.
Objective To determine whether the ESR1 haplotype created by the c.454-397T>C (PvuII) and c.454-351A>G (XbaI) polymorphisms is associated with myocardial infarction (MI) and IHD risk.
Design, Setting, and Participants In 2617 men and 3791 postmenopausal women from The Rotterdam Study (enrollment between 1989-1993 and follow-up to January 2000), a population-based, prospective cohort study of participants aged 55 years and older, ESR1 c.454-397T>C and c.454-351A>G haplotypes were determined. Detailed interviews and physical examinations were performed, blood samples were obtained, and cardiovascular risk factors were assessed.
Main Outcome Measure The primary outcome was MI and IHD defined as MIs, revascularization procedures, and IHD mortality.
Results Approximately 29% of women and 28.2% of men were homozygous carriers of the ESR1 haplotype 1 (–397 T and –351 A) allele, 49% of women and 50% of men were heterozygous carriers, and 22% of women and 21.4% of men were noncarriers. During a mean follow-up of 7.0 years, 285 participants (115 women; 170 men) had MI, and 440 (168 women; 272 men) had an IHD event, of which 97 were fatal. After adjustment for known cardiovascular risk factors, female heterozygous carriers of haplotype 1 had an increased risk of MI (event rate, 2.8%; relative risk [RR], 2.23; 95% confidence interval [CI], 1.13-4.43) compared with noncarriers (event rate, 1.3%), whereas homozygous carriers had an increased risk (event rate, 3.2%; RR, 2.48; 95% CI, 1.22-5.03). For IHD events, we observed a similar association. In women, the effect of haplotype 1 on fatal IHD was larger than on nonfatal IHD. In men, the ESR1 haplotypes were not associated with an increased risk of MI (event rate, 5.7%; RR, 0.93; 95% CI, 0.59-1.46 for heterozygous carriers; and event rate, 5.1%; RR, 0.82; 95% CI, 0.49-1.38 for homozygous carriers) compared with noncarriers (event rate, 5.8%) and were not associated with an increased risk of IHD.
Conclusions In this population-based, prospective cohort study, postmenopausal women who carry ESR1 haplotype 1 (c.454-397 T allele and c.454-351 A allele) have an increased risk of MI and IHD, independent of known cardiovascular risk factors. In men, no association was observed.
INTRODUCTION
Ischemic heart disease (IHD) has a strong genetic component, but the identity of the genetic risk factors is unknown. There is a large ongoing effort to find genes involved in cardiovascular disease. In a large case-control study that examined 112 polymorphisms in 71 candidate genes for myocardial infarction, 3 associated gene variants were identified.1 However, genes involved in the pathways of sex steroids were not considered.
Several lines of evidence implicate sex hormones in cardiovascular disease risk, such as the difference in disease risk between men and women. The risk of IHD in women between puberty and menopause is lower than that in age-matched men. However, this sex difference diminishes when postmenopausal women and men of similar age are compared.2-3 These observations have led to the suggestion that decreasing endogenous estrogen after menopause may be the critical factor in removing the relative protection against IHD that women have in their premenopausal years.
Estrogen exerts its effects by binding to the estrogen receptors and  that, once activated, regulate the expression of multiple genes. A large body of data implicates the estrogen receptor gene (ESR1) in cardiovascular disease. In 1997, Sudhir et al described a man with a null mutation in the ESR1, leading to unresponsiveness to estrogen. This 31-year-old man was reported to have premature atherosclerotic coronary artery disease and endothelial dysfunction despite the presence of high levels of circulating estrogen.4 Furthermore, ESR1 has been identified in most cardiovascular tissues such as the coronary arterial wall in smooth muscle cells,5-6 endothelial cells,7 and myocardial cells.8 In addition, fewer estrogen receptors were found in premenopausal women with atherosclerotic coronary arteries than in those with normal coronary arteries.5 Finally, variant ESR1 transcripts are extensively expressed in human vascular tissues.9
It is conceivable that common sequence variations (polymorphisms) in the ESR1 gene affect cardiovascular disease risk in the general population. Several single-nucleotide polymorphisms (SNPs) and variable-number tandem repeat polymorphisms have been identified in the ESR1 gene (http://www.ncbi.nlm.nih.gov). Cross-sectional studies have reported associations between a number of these polymorphisms in the ESR1 gene and cardiovascular risk factors and phenotypes, including body mass index (BMI),10 hypertension,11 coronary flow reserve,12 and coronary atherosclerosis.13-14
Of the polymorphisms identified in the ESR1 gene, the c.454-397T>C (also known as IVS1-397 T/C, rs2234693, and the PvuII restriction site) and c.454-351A>G SNPs (also known as IVS1-351 A/G, rs9340799, and the XbaI restriction site) are the most widely studied so far. These polymorphisms are located in the first intron of the ESR1 gene, 397 and 351 base pairs upstream of exon 2. Herrington et al15 have recently shown a potential functional significance for the c.454-397T>C polymorphism. The aim of this study was to determine whether these polymorphisms in the ESR1 gene are associated with incident IHD and myocardial infarction.
METHODS
Study Population
The Rotterdam Study is a population-based prospective cohort study of men and women that was initiated to assess prevalence, incidence, and determinants of diseases in the elderly.16 The main focus of the Rotterdam Study was on cardiovascular, neurogeriatric, ophthalmologic, and locomotor diseases. Between July 1, 1989, and May 17, 1993, all individuals aged 55 years or older and who were residents of Ommoord, a district of Rotterdam, the Netherlands, were invited to participate. A total of 7983 men and women (78% of those eligible) entered the study, and 7085 participants visited the research center. Baseline examinations took place between July 5, 1989, and September 21, 1993, and included an initial home visit and interview by a trained research assistant and an extensive physical examination at the research center. The Rotterdam Study was approved by the medical ethics committee of the Erasmus Medical Center. Each eligible person received written and oral information about the goals and research methods of the study, together with a description of the examinations involved. Written informed consent was obtained from all participants.
Our study population of 6408 participants (3791 women) included white men and postmenopausal white women who were able to visit the research center and who completed all parts of the baseline examination, including a blood sample.
Cardiovascular Risk Factors
Cardiovascular risk factors were obtained by interview and physical examination at baseline. Interview information, including smoking habits, age at menopause, and use of hormone therapy, was obtained by a trained research assistant. Hormone therapy was defined as current or former user and never user. Smoking was categorized as current, past, or never smoker.
Anthropometric measurements were obtained at the research center. Body mass index was calculated as weight in kilograms divided by height in meters squared. Two standardized blood pressure measurements were taken by using a random zero sphygmomanometer, with the participant in sitting position, and averaged. Hypertension was defined as a systolic pressure of at least 160 mm Hg or a diastolic pressure of at least 100 mm Hg or use of antihypertensive medication, encompassing grades 2 and 3 hypertension, according to the World Health Organization (WHO) criteria.17 After an overnight fast, blood samples were obtained. Serum total cholesterol level was determined by an enzymatic procedure.18 High-density lipoprotein (HDL) level was measured similarly, after precipitation of the non-HDL fraction. Diabetes mellitus was considered present with current use of antidiabetic medication or a nonfasting or postload glucose level above 198 mg/dL (11 mmol/L), according to the WHO.19
A history of myocardial infarction was based on self-reported information verified with the general practitioner's or hospital records, which included written information on diagnosis and treatment or electrocardiographic (ECG) evidence. Infarctions detected by the Modular ECG Analysis System without evidence of symptoms (silent myocardial infarctions) were verified by an experienced cardiologist.20-21
Myocardial Infarction and IHD
Follow-up started at inclusion into the study and ended either on January 1, 2000, or at the participant's death, whichever was earlier. Research assistants collected follow-up data on cardiovascular disease morbidity and mortality from the general practitioners, and in case of treatment by a specialist, hospital records were retrieved. Information on vital status of the participants was obtained regularly from the municipal health authorities in Rotterdam.
All collected events were verified by review of hospital discharge reports and letters from medical specialists. Two research physicians independently coded events according to the International Classification of Diseases, 10th Revision (ICD-10).22 In case of discrepancy, consensus was attained in a separate session. A medical expert in cardiovascular disease also reviewed all coded events for final classification. In the analyses, we used the following outcome measurements: myocardial infarction (I21) and IHD (defined as myocardial infarction [I21], percutaneous transluminal coronary angioplasty [PTCA; Z95.5], coronary artery bypass graft surgery [CABG; Z95.1], and death from IHD [I20-I25]). In identifying myocardial infarctions, general practitioner and hospital records were reviewed, and all available information, which included ECG, cardiac enzyme levels, and the clinical judgment of the treating specialist, was used to code the events. Silent myocardial infarctions were not included in the analysis. Revascularization procedures were identified by review of hospital discharge letters from the medical specialist. For further analyses, myocardial infarction and IHD were also classified as fatal and nonfatal. In addition, all-cause mortality was also documented during follow-up.
Genotyping
All participants were genotyped for the c.454-397T>C and c.454-351A>G polymorphisms. We described the polymorphisms in relation to a specific human ESR1 complementary DNA sequence (accession number NM_000125), in which position 454 of the protein coding sequence is the first nucleotide of the start of the next closest exon to the polymorphisms studied (exon 2). The variations were 397 and 351 nucleotides upstream in the intron. These polymorphisms have also been described at http://www.ncbi.nlm.nih.gov/SNP under identification numbers rs2234693 (c.454-397T>C) and rs9340799 (c.454-351A>G).
DNA was extracted with proteinase K and sodium dodecyl sulfate digestion at 37°C overnight and purified with phenol-chloroform extractions. The extracted DNA was then precipitated with NaCl at 4 mol/L and 2 volumes of cold absolute ethanol. DNA was solubilized in double-distilled water and stored at –20°C until used for DNA amplification. Genotypes were determined in 5-ng genomic DNA with the Taqman allelic discrimination assay (Applied Biosystems, Foster City, Calif). Primer and probe sequences were optimized by using the SNP assay-by-design service of Applied Biosystems (for details, see http://store.appliedbiosystems.com). Reactions were performed with the Taqman Prism 7900HT 384 wells format. We used the genotype data for each of the 2 polymorphisms to infer the haplotype alleles present in the population by using the program PHASE, which implements a Bayesian statistical method for reconstructing haplotypes from population genotype data.23 The alleles were defined as haplotypes such as "T-A," representing a thymidine (T) nucleotide at the c.454-397T>C polymorphic site and an adenosine (A) nucleotide at the c.454-351A>G polymorphic site. Haplotype alleles were coded as haplotype numbers 1 through 4 in order of decreasing frequency in the population (1 = T-A, 2 = C-G, 3 = C-A, and 4 = T-G).
Statistical Analysis
To compare possible confounders between participants grouped by the ESR1 haplotype of interest, 1-way analysis of variance was used for continuous variables; Pearson  2, for dichotomous variables.
The association between the ESR1 haplotypes and IHD events was evaluated by stratifying participants by sex and allele copy number (0, 1, or 2) for the haplotype of interest and using a standard age-adjusted Cox proportional hazards model (model 1). The proportional hazards assumption was tested and met for the Cox proportional hazards models. According to previous analyses, we chose haplotype 1 as the risk allele.24-27
Hazard ratios of events were computed as estimates of relative risk. To account for possible confounding, we excluded all participants with previous myocardial infarctions at baseline (model 2) and computed relative risks in a multivariate model containing the following predictors of coronary heart disease28: age, BMI, age at menopause, use of hormone therapy, diastolic blood pressure, smoking, diabetes mellitus, and total and HDL cholesterol levels (model 3). For the analysis of fatal and nonfatal IHD, participants who did not have an IHD event during follow-up were categorized as the reference group. To study the risk of experiencing a fatal IHD event between carriers and noncarriers of the ESR1 haplotypes, a logistic regression model was used with the above-mentioned cardiovascular risk factors as covariates.
For missing data on categorical covariates, we used a missing value indicator, whereas for missing data on continuous covariates, we used the median value of the respective value, as calculated from the total sample. Missing values did not exceed 3.5% for any covariate. For all statistical analyses, P<.05 was considered statistically significant. All statistical analyses were performed using SPSS version 11.0.1 (SPSS Inc, Chicago, Ill).
RESULTS
We observed the 4 possible c.454-397T>C to c.454-351A>G haplotype alleles in the following frequencies: haplotype 1 (T-A), 53.5%; haplotype 2 (C-G), 34.7%; haplotype 3 (C-A), 11.8%; and haplotype 4 (T-G) was present in 1 allele in 12 816 chromosomes. Genotype distributions were in Hardy-Weinberg equilibrium.
The baseline characteristics of the study population are shown in Table 1. ESR1 haplotype 1 (c.454-397 T allele and c.454-351 A allele) was associated with diastolic blood pressure in women (P = .03).
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Table 1. Baseline Characteristics and ESR1 Haplotype 1 (T-A) in 3791 Women and 2617 Men From the Rotterdam Study
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During a mean follow-up of 7.0 years (SD, 2.0 years; range, 18 days to 10.5 years), 1474 of the 6408 participants (23.0%) died of various causes and 167 (2.6%) were lost to follow-up. Two-hundred eighty-five (4.4%) participants had myocardial infarction during follow-up, of which 53 (18.6%) were fatal and 232 (81.4%) nonfatal. Four hundred forty (6.9%) participants had an IHD event, of which 97 (22.0%) were fatal and 343 (78.0%) nonfatal.
For the 3791 postmenopausal women in our study, ESR1 haplotype 1 was significantly associated with increased risk of myocardial infarction, as well as IHD (Table 2). Exclusion of 303 women with prevalent myocardial infarctions at baseline and adjustment for age (Table 2, model 2) and subsequently for BMI, age at menopause, use of hormone therapy, diastolic blood pressure, smoking, diabetes, and total and HDL cholesterol levels did not significantly change the results (Table 2, model 3). Compared with noncarriers, heterozygous carriers of haplotype 1 had 2.23 times increased risk of myocardial infarction (95% confidence interval [CI], 1.13-4.43), whereas homozygous carriers had 2.48 times increased risk (95% CI, 1.22-5.03). For IHD, the risk for heterozygous carriers was increased 2.04 times (95% CI, 1.16-3.58), and for homozygous carriers, the risk was 2.41 times higher (95% CI, 1.35-4.31). Adjustment for current use of hormone therapy, as opposed to any previous use, did not influence the estimates. In women, ESR1 haplotype 2 showed an opposite but nonsignificant effect on IHD risk compared with haplotype 1: for incident myocardial infarctions, the hazard ratio was 0.76 (95% CI, 0.55-1.05) per copy of the C-G allele and 0.78 (95% CI, 0.59-1.01) per allele copy for IHD. No association with myocardial infarction or IHD was observed for the ESR1 haplotype 3 (S. C. E. S., unpublished data, 2003).
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Table 2. Risk of Myocardial Infarction and IHD and ESR1 Haplotype 1 (T-A) in Women and Men From the Rotterdam Study
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For the 2617 men in our study, the ESR1 haplotypes were not significantly associated with incident myocardial infarctions or IHD (Table 2 for haplotype 1). Compared with noncarriers, male heterozygous carriers of haplotype 1 had 0.90 (95% CI, 0.62-1.30) times increased risk of myocardial infarction, whereas homozygous carriers had 0.78 (95% CI, 0.51-1.21) times increased risk. Exclusion of men with prevalent myocardial infarctions at baseline and adjustment for age (Table 2, model 2) and subsequently for BMI, diastolic blood pressure, smoking, diabetes, and total and HDL cholesterol levels did not change the results (Table 2, model 3).
The 2 polymorphisms were also analyzed separately. Given the strong linkage disequilibrium between the c.454-397T>C and 351A>G polymorphisms and the virtual nonexistence of haplotype 4, haplotype 1 fully represents the c.454-397T>C polymorphism. Therefore, the results for haplotype 1 presented here are the same as for the c.454-397T>C polymorphism alone. The TT-genotype group is represented by the absence of haplotype 1 and the TC- and CC-genotypes by the presence of 1 or 2 copies of haplotype 1, respectively. The c.454-351 A allele was nonsignificantly associated with myocardial infarction risk in women (hazard ratio, 1.31 [95% CI, 0.96-1.80] per copy of the A allele) and IHD (hazard ratio, 1.29 [95% CI, 0.99-1.68] per copy of the A allele). In men, no association was observed.
Figure 1 shows the association between ESR1 haplotype 1 and the cumulative proportional hazard of incident myocardial infarction during follow-up in women and men after exclusion of participants with a prevalent myocardial infarction at baseline and adjustment for cardiovascular risk factors. Table 3 shows that, for postmenopausal women, the effect of haplotype 1 on fatal IHD was larger than on nonfatal IHD; the hazard ratio for fatal IHD was 6.13 (95% CI, 1.41-26.68) for homozygous genotypes and 1.86 (95% CI, 0.97-3.56) for nonfatal IHD. For the effect of haplotype 1 on fatal and nonfatal myocardial infarctions, results were similar. For women who did not carry haplotype 1, 13.3% of IHD events were fatal; for heterozygous carriers, this percentage was 26.9% and was 34.0% for homozygous carriers. This resulted in cardiovascular risk factor–adjusted odds ratios of 3.66 (95% CI, 0.56-23.80) for heterozygous and 4.39 (95% CI, 0.69-28.06) for homozygous carriers vs noncarriers. For myocardial infarctions, the odds ratios were similar. In men, no association with fatal or nonfatal IHD was observed.
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Figure 1. Myocardial Infarction Hazard Curve According to ESR1 Haplotype 1 (T-A) in 3488 Women and 2172 Men
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Table 3. Risk of Fatal and Nonfatal IHD and ESR1 Haplotype 1 (T-A) in Women and Men From the Rotterdam Study
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In addition, we analyzed the first-year cumulative mortality for the 168 women who had an IHD event during follow-up. Although these results did not reach statistical significance, Figure 2 shows that the first-year all-cause mortality in female homozygous carriers of haplotype 1 was approximately twice that of noncarriers.
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Figure 2. First-Year Cumulative Mortality in 168 Women and 272 Men With an Ischemic Heart Disease (IHD) Event
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COMMENT
In this prospective population-based study, we observed an increased risk of myocardial infarction in postmenopausal women who carry ESR1 haplotype 1 (c.454-397 T allele and c.454-351 A allele). The risk estimates did not change after adjustment for clinically relevant cardiovascular risk factors, indicating that ESR1 haplotype 1 is an independent risk factor. Heterozygous carriers of haplotype 1 had a 2.23 times increased risk of myocardial infarction compared with noncarriers, whereas homozygous carriers had a 2.48 times increased risk. We also analyzed the risk of IHD events by taking together myocardial infarctions and revascularization procedures (PTCA and CABG) and IHD mortality. With this approach, we included approximately 50% more events and observed a similar increased risk in female carriers of haplotype 1. For men, no association between the ESR1 haplotypes and myocardial infarction or IHD risk was observed.
For women, the effect of haplotype 1 on fatal IHD was larger than on nonfatal IHD, and IHD resulted in death more often in carriers of haplotype 1. Furthermore the first-year cumulative mortality after IHD was 2 times higher for women homozygous for haplotype 1 compared with noncarriers, mainly because of genotype-dependent differences in mortality within the first month after an IHD event. Although the latter 2 analyses did not reach statistical significance, these results suggest that postmenopausal women who carry ESR1 haplotype 1 have not only an increased risk of having an IHD event but also an increased risk of death from such an event.
A number of possible indirect and direct ESR1-dependent mechanisms through which estrogens may exert their cardioprotective effects have been presented in the literature.29-30 Some of the protective effects of estrogens could be mediated through systemic effects, such as changes in lipid profile, coagulation, and fibrinolytic systems.29 However, in our study the ESR1 c.454-397T>C and c.454-351A>G genotypes were not associated with differences in a number of cardiovascular risk factors at baseline, such as hypertension, hypercholesterolemia, and diabetes mellitus, which is in accordance with 2 previous studies that have shown that the c.454-397T>C and c.454-351A>G genotypes were not associated with baseline cholesterol levels in healthy men and postmenopausal women, as well as in men and women with preexisting coronary artery disease.31-32 Furthermore, when our analyses were adjusted for the presence of these and other common cardiovascular risk factors, the observed hazard ratio did not fundamentally change, which suggests that it is not through these pathophysiologic pathways that ESR1 gene polymorphisms influence IHD disease but that this haplotype is an independent risk factor.
Direct actions of estrogen on blood vessels could contribute substantially to the cardioprotective effects of estrogen.29, 33-34 One of these direct actions on the blood vessel wall that may be essential in IHD pathology is the influence of estrogen on nitric oxide production. Endothelial cell–derived nitric oxide plays a critical role in cardiovascular disease pathology. Nitric oxide, a primary vascular target of estrogens, not only causes the relaxation of the vascular smooth muscle cells but also inhibits platelet activation.35 Estrogen increases nitric oxide production in vessels such as the aorta by increasing the expression and enzymatic activity of the enzyme responsible for nitric oxide synthesis, endothelial nitric oxide synthase, as well as by inducing the release of nitric oxide. Several studies have shown that ESR1 is essential to all 3 of these estrogen effects on vascular nitric oxide production.36-39 This result is further supported by the observation that basal production of endothelium-derived nitric oxide was significantly lower in the aorta of ESR1 knockout mice compared with wild-type mice.40 Studies have shown that ESR1 also mediates 2 other effects of estrogen in the vessel wall: acceleration of reendothelialization and inhibition of the vascular injury response.41-42 Particularly the latter 2 mechanisms may explain why the ESR1 polymorphisms are most strongly associated with fatal IHD.
How could these specific polymorphisms in the ESR1 gene influence myocardial infarction risk in postmenopausal women? The c.454-397T>C and c.454-351A>G polymorphisms have been an important area of research in diseases such as osteoporosis,25, 43-44 cardiovascular disease,31 and cancer,45 and a number of hypotheses for the functional significance of these polymorphisms have been reported in the literature. Given their location, 397 and 351 base pairs upstream from the start of exon 2, possible functional mechanisms include altering ESR1 expression by altered binding of transcription factors and influencing alternative splicing of the ESR1 gene. Both of these mechanisms can be a direct result of either of these polymorphisms or through linkage disequilibrium with a truly functional, but so far unknown, sequence variation elsewhere in the ESR1 gene.
The first mechanism was recently supported by findings of Herrington et al,15 which were confirmed in our own laboratory (S. C. E. S., unpublished data, 2003). Herrington et al15 showed that the c.454-397 T allele eliminates a functional binding site for the transcription factor B-myb, which suggests that the presence of this allele may result in lower ESR1 transcription. In the presence of a decreased number of estrogen receptors, estrogen signaling may be less effective and, therefore, estrogen actions may be decreased.
These findings are further supported by the observation in our study population, as well as in others, that this c.454-397 T allele has been associated with a number of phenotypes that are known to be related to low estradiol levels and therefore low estrogen activity, such as increased risk of osteoporosis,25 decreased risk of osteoarthritis24 and hysterectomy,26 lower BMI,10 shorter stature,27 and later age at menopause.26 In our study, we found the T allele to be associated with increased risk of myocardial infarction and IHD events in postmenopausal women, which suggests that the potentially lower ESR1 expression caused by the presence of the T allele at the –397 polymorphic site may lead to a higher susceptibility to IHD. The c.454-351 A allele is also associated with IHD, which may be due to linkage disequilibrium with the c.454-397T>C SNP or to functional significance of the c.454-351A>G polymorphism itself.
Given the substantial differences in hormone dynamics between men and postmenopausal women, we chose to stratify our analysis by sex. An intriguing aspect of this study is that ESR1 haplotype 1 is significantly associated with an increased risk of IHD in women but not men. Similar results were also found for the associations between the ESR1 haplotype 1 with height and osteoporosis.25, 27 In these studies, the associations were also found only in women.
Recently, Shearman et al46 reported an association between the c.454-397T>C polymorphism and cardiovascular disease in the Framingham Heart Study. They found that the c.454-397 T allele prevented IHD in men, which is seemingly in contrast to our findings that the T allele prevents IHD in women. However, there is no clear conflict with the seemingly opposite results reported by Shearman et al,46 because the men in our study showed a nonstatistical trend opposite to that of the women. Furthermore, the men in the Rotterdam Study were much older than the men in the Framingham Heart Study, and the effects of risk factors are known to change in older cohorts. Furthermore, the Framingham Heart Study had few cardiovascular events in women; for example, only 3 cases of myocardial infarction were documented in the female participants of that study.
An explanation for these opposing results in men and women is not immediately apparent. The most obvious difference between men and postmenopausal women is the cessation of gonadal function in women. Perhaps the presence of an intact hypothalamic-pituitary-gonadal axis in men leads to protection of the ERa PvuII T allele in cardiovascular disease, whereas in postmenopausal women, who are completely dependent on peripheral conversion to estradiol, an opposing effect occurs. In men, compared with postmenopausal women of the same age, estradiol levels are approximately 3 times higher (S. C. E. S., unpublished data, 2003). In the presence of sufficiently high estradiol levels, differences in ESR1 expression may not have clinical consequences. However, estrogen deficiency after menopause in combination with lower ESR1 expression caused by the c.454-397 T allele may lead to a higher susceptibility to IHD, which could explain why we did not observe an association between ESR1 gene polymorphisms and IHD in men.
There are limitations to genetic-association studies. They can be influenced by population stratification or heterogeneity, especially case-control studies in a population of mixed racial origin. However, for our study a population-based prospective cohort design was chosen, and all participants were of Dutch white origin. Furthermore, the c.454-397T>C and c.454-351A>G genotypes were in Hardy-Weinberg equilibrium, and haplotype frequencies were similar to those found in other studies of white individuals.47 Therefore, our study population may be considered ethnically homogeneous and representative of the Dutch population. Another limitation of association studies is the definition of the phenotype of interest and the occurrence of phenotype heterogeneity. We diagnosed myocardial infarctions and IHD in strict adherence to the ICD-10 guidelines and reviewed hospital discharge letters and reports from treating specialists to identify cardiovascular disease events. Therefore, we believe that phenotype heterogeneity did not influence our results. Furthermore, in the Netherlands the only way to access specialist and hospital care is by consulting a general practitioner. Therefore, checking the general practitioners' medical records for all participants should have resulted in a nearly complete follow-up.
As is true for all association studies, the validity of genetic-association studies is greatly strengthened by confirmation of the results. The findings reported in this study are supported by not only functional studies but also associations with other phenotypes (pleiotropy) in women, such as osteoporosis, osteoarthritis, hysterectomy, BMI, stature, and age at menopause. The presented body of data supports the theory that in women, the presence of the c.454-397 T allele leads to lower estrogen action.
Selective nonresponse of individuals with impaired health or otherwise at an increased risk of cardiovascular disease may have occurred. However, such a nonresponse bias will presumably not be genotype dependent and will not lead to overestimation of the hazard ratios.
In interpreting the clinical implications of these results, we must consider that 78% of the population carries the ESR1 haplotype 1 risk allele and that heterozygous and homozygous carriers of haplotype 1 have a 2-fold increased risk of IHD. Perhaps we should view this not as a "risk" allele but consider the noncarriers as having a protective allele, which implies that noncarriers (22% of the population) have a 50% reduced risk.
In conclusion, this population-based prospective cohort study shows a significant 2-fold increased risk of myocardial infarction, as well IHD events, in postmenopausal women who carry ESR1 haplotype 1 (c.454-397 T allele and c.454-351 A allele). The association was not explained by known cardiovascular risk factors such as age, previous myocardial infarction, BMI, age at menopause, use of hormone therapy, blood pressure, smoking, diabetes, and cholesterol level, suggesting that haplotype 1 is an independent risk factor for IHD. Furthermore, our results also suggest that postmenopausal women who carry ESR1 haplotype 1 have not only an increased risk of having an IHD event but also an increased risk of death from such an event.
AUTHOR INFORMATION
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Corresponding Author: Huibert A. P. Pols, MD, PhD, Department of Internal Medicine, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, the Netherlands (h.pols{at}erasmusmc.nl).
Author Contributions: Dr Schuit had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Schuit, Oei, de Jong, Hofman, Pols, Uitterlinden.
Acquisition of data: Schuit, Oei, Witteman, Geurts van Kessel, van Meurs, Nijhuis, Pols, Uitterlinden.
Analysis and interpretation of data: Schuit, Oei, Witteman, Geurts van Kessel, van Meurs, van Leeuwen, de Jong, Zillikens, Pols, Uitterlinden.
Drafting of the manuscript: Schuit, Oei.
Critical revision of the manuscript for important intellectual content: Oei, Witteman, Geurts van Kessel, van Meurs, Nijhuis, van Leeuwen, de Jong, Zillikens, Hofman, Pols, Uitterlinden.
Statistical expertise: Schuit, Oei, Witteman, Geurts van Kessel, van Meurs, Pols, Uitterlinden.
Obtained funding: Hofman, Pols.
Administrative, technical, or material support: Schuit, Geurts van Kessel, Nijhuis.
Supervision: Witteman, van Meurs, van Leeuwen, de Jong, Zillikens, Hofman, Pols, Uitterlinden.
Funding/Support: This study was funded by the Netherlands Organization for Scientific Research under the Research Institute for Diseases in the Elderly (grant 014-90-001).
Role of the Sponsor: The Netherlands Organization for Scientific Research had no role in the design and conduct, data management and analysis, manuscript preparation, or in the review or authorization for submission.
Author Affiliations: Departments of Internal Medicine (Drs Schuit, van Meurs, van Leeuwen, de Jong, Zillikens, Pols, and Uitterlinden and Ms Geurts van Kessel) and Epidemiology & Biostatistics (Drs Schuit, Oei, Witteman, Nijhuis, Hofman, and Pols), Erasmus Medical Center, Rotterdam, the Netherlands.
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