Association of oestrogen receptor alpha gene polymorphism with the angiographic extent of coronary artery disease

Arthur Pollaka,*, Ariel Rokachb, Anat Blumenfeldc, Laura J Rosend, Luba Resnike and Rivka Dresner Pollake

a Department of Cardiology, Hadassah–Hebrew University Medical Center, Jerusalem, Israel
b Department of Medicine on Mount Scopus, Hadassah–Hebrew University Medical Center, Jerusalem, Israel
c Department of Ophthalmology, Hadassah–Hebrew University Medical Center, Jerusalem, Israel
d Authority for Computation and Information (Ein Kerem Branch), Hebrew University, Jerusalem, Israel
e Endocrinology and Metabolism Service, Hadassah–Hebrew University Medical Center, Jerusalem, Israel

* Correspondence to: Arthur Pollak, MD, Department of Cardiology, Hadassah–Hebrew University Medical Center, P.O. Box 12000, 91120 Jerusalem, Israel. Tel: +972-2-6778910; Fax: +972-2-6510804
E-mail address: apollak{at}hadassah.org.il

Received 15 January 2003; revised 28 September 2003; accepted 29 October 2003

Abstract

Aims To investigate the association between sequence variants in the promoter region of the oestrogen receptor-{alpha} (ER-{alpha}) gene and the angiographic severity of coronary artery disease (CAD).

Methods and results We studied 503 subjects undergoing coronary angiography (mean age 63±12 years, 72% men, 28% women). Coronary artery disease extent was assessed by the number of: (1) major coronary vessels with >50% narrowing (NMCV); (2) coronary vessels with any narrowing (NCV); (3) narrowed coronary segments (NCS). The number of thymine and adenine dinucleotide repeats [(TA)n], 1174 base-pairs upstream exon 1, was determined by PCR. The median number of (TA)n(18) was used to categorize subjects into long, short and mixed allele genotypes. Poisson regression was used to analyse the association between genotypes and CAD extent, with age category (age <=55 vs >55), sex, risk factors and age at onset of CAD as covariates. In young subjects, (TA)nlength had a significant effect on NCS (P=0.047) and a borderline significant effect on NCV (P=0.066). Young subjects homozygous for long alleles had higher NCV and NCS compared to those homozygous for short alleles (NCV 3.7±2.4 vs 2.4±1.8, NCS 4.4±2.7 vs 3.1±2.3, respectively, P<=0.034).

Conclusion The (TA)nlength in the ER-{alpha} gene promoter region is associated with the angiographic severity of CAD in young patients.

Key Words: Oestrogen receptor-{alpha} gene polymorphism • Coronary artery disease • Risk factors • Angiography

1. Introduction

Oestrogens have vasodilatory, anti-inflammatory and anti-proliferative effects on the cardiovascular system, as well as favourable effects on the lipid profile.1,2Despite these beneficial effects, clinical trials have failed to demonstrate a lower rate of coronary artery disease (CAD) in postmenopausal women receiving hormone replacement therapy.3–5The effects of oestrogens on the vascular system are mediated by two distinct oestrogen receptors (ERs), ER-{alpha} and ER-ß, encoded by two separate genes, which are both expressed in endothelial cells and vascular smooth muscle cells.2,6Animal and human studies have shown that ER-{alpha} is the major mediator of the atheroprotective effect of oestrogens.7–10Diminished expression of ER-{alpha} wasassociated with the occurrence of premature atherosclerosis in premenopausal women.8Similarly, methylation-dependent inactivation of ER-{alpha} is increased in vascular cells from human atherosclerotic lesions.9Furthermore, premature CAD was reported in a young man homozygous for a mutation in exon 2 of the ER-{alpha} gene, resulting in a premature stop codon.10Thus, alternation in ER-{alpha} expression and function may attenuate the atheroprotective role of oestrogens.

The ER-{alpha} gene, located at chromosome 6q24.1, has six domains encoded by eight exons. Associations between a number of polymorphisms in the ER-{alpha} gene and various clinical phenotypes have been studied including therisk of breast cancer,11osteoporosis,12–14hypertension,15lipid levels,16and lipid response to hormone replacement therapy.17Most of these studies focused on the PvuII polymorphism, caused by a T/C transition in intron 1 of the gene, and the XbaI polymorphism, caused by a G/A transition located 50 base pairs downstream of the PvuII polymorphic site. These studies yielded inconsistent relationships with clinical phenotypes.

Data regarding the relationship between ER-{alpha} gene polymorphisms and CAD is sparse. The first reported study by Matsubara et al. found no relationship between the angiographic severity of CAD and the PvuII and XbaI polymorphisms in Japanese men and women.16On the other hand, an autopsy study in Finnish men demonstrated an association between the ER-{alpha} PvuII polymorphism and autopsy-verified coronary artery wall atherosclerosis and thrombosis.18

The length of a simple sequence repeat of the dinucleotide thymine and adenine [(TA)nrepeat],located in the promoter region of the ER-{alpha} gene upstream of exon 1, was reported to be associated with the severity of CAD in a necropsy study in Finnish men.19A recent study also found an association between the length ofthe (TA)nrepeat and CAD in a selected population of Japanese patients with heterozygous familial hypercholesterolaemia.20In the present study we aimed to assess the relationship between the (TA)nrepeat polymorphism in the regulatory region of the ER-{alpha} gene and the angiographic severity of CAD in an unselected population of Caucasian men and women undergoing coronary angiography.

2. Methods

2.1. Subjects
Consecutive subjects who had undergone coronary angiography at the Hadassah University Hospital between October 2001 and March 2002 were recruited for this study. Clinical data included: sex, age, age at onset of CAD symptoms, ethnic origin, premature CAD in first-degree relatives, hypertension, hyperlipidaemia, diabetes mellitus, smoking habits, height, weight, and the use of medications. The following definitions were utilized for coronary risk factors: hypertension, blood pressure >=140/90mmHg (confirmed by measurements on several occasions) or antihypertensive therapy; obesity, body mass index (BMI) >=29kg/m2; dyslipidaemia, LDL >=130mg%, HDL <35mg%, triglycerides >=200mg% or use of lipid lowering medications; diabetes, fasting plasma glucose >126mg% or glucose lowering treatment; smoking, >10cigarettes/day if current or recent (<2 years before CAD onset). Accordingly, each risk factor was coded as either present or absent, and the total number of coronary risk factors ranged from 0 to 6. Ethnic origin was defined based on place of birth of four grandparents.

The Ethics Committee of the Hadassah University Hospital approved the study. Informed consent was obtained from all participants.

2.2. Documentation of CAD
The extent of CAD was graded by three methods based on the angiographic findings: (1) NMCV, the number of major coronary vessels (LAD, LCX, RCA) with at least one >50% stenosis, classifying grades between 0 and 3 (namely one-, two-, or three-vessel disease). A >50% lesion in the left main coronary artery was regarded as 2-vessel disease; (2) NCV, the number of major coronary vessels (LM, LAD, LCX, RCA) and/or their second-order branches (up to two large diagonals, two large marginals and the PDA) in which any degree of atherosclerotic narrowing was identified, classifying grades between 0 and 9; (3) NCS, the number of coronary segments in which any degree of atherosclerotic narrowing was identified, using a 15-segment scheme,20,21classifying grades between 0 and 15.

Coronary angiograms were interpreted by two investigators blinded to the patient's risk factors and ER-{alpha} genotype.

2.3. Determination of ER-{alpha} genotype
DNA was purified from peripheral blood according to standard protocols. The DNA region containing the polymorphic (TA)nrepeat at 1174 base-pairs upstream of exon 1 was amplified by polymerase chain reaction (PCR) with the primers described by Sano et al.13The PCR reaction was performed in the presence of [{alpha}32P]dGTP, separated on 6% acrylamide gel, and autoradiographed. The PCR products were sequenced by ABI 3700 DNA analyser and analysed with sequence analysis software (ABI). The number of TA repeats was determined accordingly.

2.4. Statistical analysis
The median number of TA repeats was used to classify the study population into three (TA)ngenotypes: those with long allele genotype (both alleles >= median TA length), those with short allele genotype (both alleles < median TA length), and those with a mixed genotype (one short and one long allele). Poisson regression was used to evaluate the association between (TA)ngenotypes and the angiographic extent of CAD.22Since the contribution of a single gene polymorphism is expected to manifest itself in younger rather than in older patients, the population was categorized by age into younger (age <=55 years) and older (age >55 years) patients, based on the cut-off age of55 years used for the definition of family history of premature CAD as a risk factor.23,24Age category, sex, the number of coronary risk factors and the age at onset of CAD were included as covariates in the Poisson regression model. When applied to the complete dataset (younger and older patients combined), a significant interaction was found between age category and (TA)ngenotype for two out of three response variables (the P-values for these interactions for NMCV, NCV, and NCS were 0.5390, 0.0126, and 0.0033, respectively). Therefore, a stratified analysis was performed for younger and older patients. The stratified analysis was performed for all three variables to preserve the unity of the analytic approach.

Data are presented as mean±SD, if not stated otherwise. Two-sample t-test and chi-square analysis were used, asappropriate, to compare clinical and angiographic variables in younger and older subjects. ANOVA was used to compare clinical variables among the three-genotype groups within each age category. A P value of <0.05 was considered statistically significant. SAS Version 8.2 was used to perform the analysis.

3. Results

3.1. Characteristics of the study population
Five hundred and fifteen out of 576 (89%) consecutive patients who underwent coronary angiography, agreed to participate in the study. Genotyping was successful in 503/515 patients (97%), who comprised the study population. The mean age was 62.8±11.7 years (range 32–98 years). There were 364 men (72%, mean age 61±12 years) and 139 women (28%, mean age 67±10 years). The study population included 41% Ashkenazi Jews, 31% non-Ashkenazi Jews (17% from Iran and Iraq, 14% from North Africa), 10% of mixed origin and 18% Muslim Arabs. There were no significant differences in the mean number of coronary risk factors among the various ethnic groups. Mean age at onset of CAD in men and women was 56±11 and 64±10 years, respectively.

There were 372 patients over age 55 and 131 patients who were 55 years of age or younger, comprising the older and younger age categories (mean age 68.2±7.9 and 47.4±5.2, respectively). The prevalence of diabetes was significantly higher in older patients (43% in older vs 28% in younger subjects, P<0.003), while smoking and family history of CAD were more prevalent in younger patients (60% vs 33% smoking and 46% vs 31% family history in younger vs older subjects, respectively, P<0.002). There was a significant difference in gender ratio between younger and older patients (86% vs 67% male, 14% vs 33% female, respectively, P<0.001). The total number of CAD risk factors was similar in younger and older subjects (2.9±1.3 vs 2.8±1.3, respectively, P=0.62).

3.2. Angiographic extent of CAD
Mean NMCV was 1.7±1.0 in younger and 2.2±0.9 in older subjects (range 0–3 in both age groups, P<0.001). Mean NCV was 2.8±1.9 (range 0–8) in younger and 3.2±1.6 (range 0–9) in older subjects (P=0.005). Mean NCS was 3.5±2.4 (range 0–9) in younger and 4.3±2.2 (range 0–13) in older subjects (P<0.001). Patent coronary arteries and single vessel disease were more prevalent in younger patients (15.3% vs 7.0% patent coronaries and 28.2% vs 17.2% single vessel disease in younger vs older subjects, respectively, P<0.01), while older patients had a significantly higher rate of triple vessel disease (47.3% vs 26.0% in older and younger subjects respectively, P<0.001).

3.3. Distribution of (TA)nrepeat polymorphism
The frequency distribution of the (TA)ndinucleotide repeat polymorphism in 503 subjects (1006 chromosomes) is plotted in Fig. 1a, showing two peaks: at 13–16 and at 22–24 repeats. The number of TA repeats ranged from 7 to 28 (median=18). Accordingly, patients were categorized as long allele genotype (both alleles >=18 repeats, n=108), short allele genotype (both alleles<18 repeats, n=152), and mixed allele genotype (a long and a short allele, n=243). The frequency distributions of the (TA)nrepeat polymorphism in younger and older subjects were similarly bimodal (Fig. 1b and c). The range of TA repeats was 9–26 and 7–28 in younger and older subjects, respectively, with a median of 18 repeats in both groups. The (TA)nrepeat polymorphism was in Hardy–Weinberg equilibrium ({chi}2=0.349, P=0.96). There were neither gender nor ethnic differences in the (TA)nrepeat frequency distribution. The (TA)nrepeat frequency distribution in our population was not significantly different from European (Italian and Finnish) and Asian (Japanese) populations,13,14,20,25in which a double-peak distribution with a median of 19 and 17 repeats, respectively, was observed.19,20



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1 Frequency distribution of the oestrogen receptor-{alpha} gene (TA)ndinucleotide repeat polymorphism in subjects undergoing coronary angiography. (a) all subjects; (b) younger subjects; (c) older subjects.

 
Clinical characteristics of the three-genotype groups, stratified by age category, are presented in Table 1. There were no significant differences among the three genotype groups in both age categories, except for an increased frequency of obesity in older subjects homozygous for long allele genotype compared to older subjects with short allele genotype (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1 Subject clinical characteristics by ER-{alpha} (TA)ngenotype and age category

 
3.4. Effect of (TA)nrepeat polymorphism on angiographic extent of CAD
The length of (TA)nrepeat had a significant effect on the angiographic extent of CAD for NCS (P=0.047) and a borderline significant effect for NCV (P=0.066) in younger patients, independent of sex, number of CAD risk factors and age at disease onset. Young patients with two long alleles had a higher number of narrowed coronary vessels and a higher number of narrowed coronary segments compared to patients with two short alleles (NCV 3.7±2.4 vs 2.4±1.8, NCS 4.4±2.7 vs 3.1±2.3, respectively, P<=0.034) (Fig. 2). A graded effect for NCV and NCS was also noted such as younger patients with two long alleles had a higher number of narrowed coronary vessels and narrowed coronary segments compared to those with mixed allele genotype (P=0.028 and P=0.022, respectively) (Fig. 2).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2 Association of oestrogen receptor-{alpha} genotype category according to (TA)nlength with the angiographic extent of coronary artery disease in younger subjects. Graphs show means (±SEM). P values are derived from the Poisson regression model, adjusted for sex, age at onset of coronary artery disease and the number of coronary risk factors.

 
In older patients there was no association between the (TA)nlength and NMCV or NCV (P=0.6 and P=0.5, respectively) (Table 2). Nor was there a graded effect of (TA)nlength on any parameter of CAD severity. There was an association with NCS (P=0.03) such as older patients with short allele genotype had higher number of narrowed coronary segments compared to those with long allele genotype (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2 Angiographic extent of CAD by ER-{alpha} (TA)ngenotype in older subjects

 
There was no interaction between the length of the (TA)nrepeat and gender (P=0.58, 0.31 and 0.11, for NMCV, NCV and NCS in younger subjects, and P=0.97, 0.76 and 0.70 in older subjects, respectively).

4. Discussion

In this study the length of the (TA)ndinucleotide repeat in the regulatory region of the ER-{alpha} gene was found to be associated with measures of angiographic extent of CAD. Young subjects homozygous for long alleles had a significantly higher number of narrowed atherosclerotic coronary segments and borderline statistically significant higher number of narrowed coronary vessels compared to subjects homozygous for short alleles. Associations found were apparent after controlling for gender, age of disease onset and the major coronary risk factors.

Our findings in young subjects are in agreement with the reports of Kunnas et al.19and Lu et al.20In the study of Kunnas et al. men with long allele genotype had a significantly greater number of severely narrowed coronary arteries, larger areas of complicated lesions and more coronary artery calcifications compared to men with short allele genotype. However, their study was a necropsy study in men only in which not all coronary risk factors were taken into account. The study of Lu et al. was conducted in a selected population of Japanese patients heterozygous for familial hypercholesterolaemia. They found a significantly higher frequency of long (TA)nrepeat in postmenopausal women with CAD than in those without CAD. However, findings in the Japanese population may not be applicable to other populations, since the Japanese population was found to have a significantly different genotype distribution of other ER-{alpha} gene polymorphisms. The ER-{alpha} B-variant gene polymorphism, previously reported to be associated with hypertension, was not detected in the Japanese population, although its allele frequency in white postmenopausal women was found to be 10–20%.26In addition, the frequency of the ER-{alpha} PvuII polymorphism was also reported to be lower in the Japanese population compared to Caucasian populations.17

To our knowledge, this is the first report of the effect of the (TA)nrepeat polymorphism on the angiographic severity of CAD in an unselected Caucasian population undergoing coronary angiography. The age and sex distribution as well as the coronary risk factor profile and the angiographic findings in our patients indicate that our study population represents the typical patient population referred for coronary angiography. In addition, the distribution of the (TA)nlength in our population was similar to its previously reported distribution.13,14,25

The association between (TA)nlength and CAD was observed mainly in younger patients. Since the majority of younger subjects in this study were men, the positive finding was largely driven by the results in younger men. The significant, though relatively small, contribution of a single gene polymorphism is expected to manifest itself in younger rather than in older patients, in whom there is a dominant effect of the major CAD risk factors. Furthermore, since the outcome measure in this study was the angiographic extent of atherosclerosis in the coronary tree, which is largely determined by age and disease duration, the contribution of the (TA)nrepeat polymorphism in older patients could be masked by the effect of these indices on the angiographic findings. At this stage we cannot explain the association observed between short allele genotype and narrowed coronary segments in older subjects. However, since no similar trend or association with other measures of CAD severity was observed, we believe this finding may be incidental.

The molecular mechanism by which the (TA)nrepeat polymorphism may be associated with CAD is unclear. The (TA)nrepeat is located in the regulatory region of the ER-{alpha} gene, between promoters A and B, ~1174 base-pairs upstream of the first proposed transcribed site in exon 1.13At least seven different promoters have been identified in the ER-{alpha} gene.27Their resulting transcripts differ in their 5'-untranslated regions (UTRs), but not in their coding regions. Different ER-{alpha} promoters have been identified in breast tissue, endometrium, osteoblasts, liver cells and testis.27–29Human vascular smooth muscle cells (VSMC) were shown to express ER-{alpha} mRNA and protein, but it is unknown which promoter is expressed in these cells. Variant ER-{alpha} mRNAs were shown to be present in VSMC,30including variants with deletions of exons responsible for hormone binding, yielding truncated receptors. However, the promoter region was not cloned in these variants. Thus, the association between these ER-{alpha} variants and promoter structure or use is unknown. Interestingly, short allele (TA)ngenotype (TA <15 repeats) rather than long (TA)ngenotype was reported to be associated with low bone mineral density and increased vertebral fracture risk in postmenopausal Italian women,14possibly suggesting different promoter use in bone tissue and in the vascular system. We speculate that (TA)nlength may effect promoter usage, thereby influencing the expression of the ER-{alpha} gene.

The (TA)nrepeat sequence may be linked to another causative sequence variant. Linkage disequilibrium between the (TA)nrepeat and the PvuII intronic polymorphism of the ER-{alpha} gene, was previously reported.14The PvuII polymorphic site was shown to produce a functional binding site for the transcription factor B-myb,17,31and thus may be associated with a reduction in ER-{alpha} transcription. An association between the PvuII polymorphism and the magnitude of the increase inHDL-cholesterol in response to hormone-replacement therapy was observed in postmenopausal women.17In addition, a high degree of linkage disequilibrium between the variable length of the (TA)nrepeat and another polymorphic site, the –1989T/G polymorphism in promoter B region, was reported in Japanese individuals with heterozygous familial hypercholesterolaemia.20However, the significance of this polymorphism is still unknown. Linkage of the (TA)nrepeat locus to other, as-yet-unidentified, causative variants is also possible.

Our findings suggest that young carriers of the long (TA)nvariant may benefit less from the cardiovascular protective effects of oestrogen receptors, thus having more extensive CAD. Future studies are indicated to assess the relationship between (TA)nlength and the quantity and quality of ER-{alpha} transcripts in the endothelium of coronary arteries. Expression of ER-{alpha} should be studied in subjects who vary by the (TA)nrepeat genotype. Finally, the effect of the (TA)nlength on clinical cardiovascular events and mortality needs to be evaluated in different populations.

Acknowledgments

This work was supported, in part, by the HWZOA Research Fund for Women's Health. We are grateful to Prof. Rodney H. Falk, Director of Clinical Cardiac Research at the Boston University Medical Center (Boston, USA), for critically reviewing the manuscript and for his thoughtful suggestions.

References

  1. Farhat MY, Lvigne MC, Ramwell PW. The vascular protective effects of estrogen. FASEB J. 1996;10:615–624.[Abstract/Free Full Text]
  2. Gruber CJ, Tschugguel W, Schneeberger C et al. Production and actions of estrogens. N Engl J Med. 2002;346:340–352.[Free Full Text]
  3. Women's Health Initiative Trial. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomized controlled trial. JAMA. 2002;288:321–333.[Abstract/Free Full Text]
  4. Manson JE, Hsia J, Johnson KC et al. Estrogen and Progestin and the risk of coronary heart disease. N Engl J Med. 2003;349:523–534.[Abstract/Free Full Text]
  5. Hodis HN, Mack WJ, Azen SP et al. Hormone therapy and the progression of coronary- artery atherosclerosis in postmenopausal women. N Engl J Med. 2003;349:535–545.[Abstract/Free Full Text]
  6. Mendelsohn ME. Genomic and nongenomic effects of estrogen in the vasculture. Am J Cardiol. 2002;90(1A):3F–6F.[CrossRef][Medline]
  7. Hodgin JB, Krege JH, Reddick RL et al. Estrogen receptor {alpha} is a major mediator of 17ß-estradiol's atheroprotective effects on lesion size in Apoe-/- mice. J Clin Invest. 2001;107:333–340.[Abstract/Free Full Text]
  8. Losordo DW, Kearney M, Kim EA et al. Variable expression of the estrogen receptor in normal and atherosclerotic coronary arteries of premenopausal women. Circulation. 1994;89:1501–1510.[Abstract]
  9. Post WS, Goldschmidt-Clermont PJ, Wilhide CC et al. Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system. Cardiovasc Res. 1999;43:985–991.[CrossRef][Medline]
  10. Sudhir K, Chou TM, Chatterjee K et al. Premature coronary artery disease associated with a disruptive mutation in the estrogen receptor gene in a man. Circulation. 1997;96:3774–3777.[Abstract/Free Full Text]
  11. Andersen TI, Heimdal KR, Skrede M et al. Oestrogen receptor (ESR) polymorphisms and breast cancer susceptibility. Hum Genet. 1994;94:665–670.[Medline]
  12. Kobayashi S, Inoue S, Hosoi T et al. Association of bone mineral density with polymorphism of the estrogen receptor gene. J Bone Miner Res. 1996;11:306–311.[Medline]
  13. Sano M, Inoue S, Hosoi T et al. Association of estrogen receptor dinucleotide repeat polymorphism with osteoporosis. BiochemBiophys Res Commun. 1995;217:378–383.[CrossRef][Medline]
  14. Becherini L, Gennari L, Masi L et al. Evidence of a linkage disequilibrium between polymorphisms in the human estrogen receptor a gene and their relationship to bone mass variation in postmenopausal Italian women. Hum Mol Genet. 2000;9:2043–2050.[Abstract/Free Full Text]
  15. Lehrer S, Rabin J, Kalir T et al. Estrogen receptor variant and hypertension in women. Hypertension. 1993;21:439–441.[Abstract]
  16. Matsubara Y, Murata M, Kawano K et al. Genotype distribution of estrogen receptor polymorphisms in men and postmenopausal women from healthy and coronary populations and its relation to serum lipid levels. Arterioscler Thromb Vasc Biol. 1997;17:3006–3012.[Abstract/Free Full Text]
  17. Herrington DM, Howard TD, Hawkins GA et al. Estrogen-receptor polymorphisms and effects of estrogen replacement on high-density lipoprotein cholesterol in women with coronary disease. N Engl J Med. 2002;346:967–974.[Abstract/Free Full Text]
  18. Lehtimaki T, Kunnas TA, Mattila KM et al. Coronary artery wall atherosclerosis in relation to the estrogen receptor 1 gene polymorphism: an autopsy study. J Mol Med. 2002;80:176–180.[CrossRef][Medline]
  19. Kunnas TA, Laippala P, Penttila A et al. Association of polymorphism of human {alpha} oestrogen receptor gene with coronary artery disease in men: a necropsy study. BMJ. 2000;321:273–274.[Free Full Text]
  20. Lu H, Higashikata T, Inazu A et al. Association of estrogen receptor-{alpha} gene polymorphisms with coronary artery disease in patients with familial hypercholesterolemia. Arterioscler Thromb Vasc Biol. 2002;22:817–823.[Abstract/Free Full Text]
  21. Mabuchi H, Koizumi J, Shimizu M et al. Development of coronary heart disease in familial hypercholesterolemia. Circulation. 1989;79:225–232.[Abstract]
  22. Armitage P, Berry G, Matthews J. Statistical Methods in Medical Research. : Blackwell Science Ltd; 2002. .
  23. Roncaglioni MC, Santoro L, D'Avanzo B et al. Role of family history in patients with myocardial infarction. An Italian case-control study. GISSI-EFRIM Investigators. Circulation. 1992;85:2065–2072.[Abstract]
  24. National Cholesterol Education Program (NCEP). Second report of the expert panel on detection, evaluation and treatment of high blood cholesterol in adults (Adult Treatment Panel II). Circulation. 1994;89:1329–1445.
  25. Kunnas TA, Holmberg-Marttila D, Karhunen PJ. Analysis of estrogen receptor dinucleotide polymorphism by capillary gel electrophoresis with a population genetic study in 180 Finns. Hum Hered. 1999;49:142–145.[CrossRef][Medline]
  26. Fujimoto J, Hirose R, Ichigo S et al. DNA polymorphism in B-domain of the estrogen receptor-{alpha} among Japanese women. Steroids. 1998;63:146–148.[CrossRef][Medline]
  27. Kos M, Reid G, Denger S et al. Minireview: genomic organization of the human ER{alpha} gene promoter region. Mol Endocrinol. 2001;15:2057–2063.[Abstract/Free Full Text]
  28. Grandien K. Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor-mRNA isoform. Mol Cell Endocrinol. 1996;116:207–212.[CrossRef][Medline]
  29. Grandien K, Backdahl M, Ljunggren O et al. Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines. Endocrinology. 1995;136:2223–2229.[Abstract]
  30. Hodges YK, Richer JK, Horwitz KB et al. Variant estrogen and progesterone receptor messages in human vascular smooth muscle. Circulation. 1999;99:2688–2693.[Abstract/Free Full Text]
  31. Herrington DM, Howard TD, Brosnihan B et al. Common estrogen receptor polymorphism augments effects of hormone replacement therapy on E-selectin but not C-reactive protein. Circulation. 2002;105:1879–1882.[Abstract/Free Full Text]