a Department of Cardiology, HadassahHebrew University Medical Center, Jerusalem, Israel
b Department of Medicine on Mount Scopus, HadassahHebrew University Medical Center, Jerusalem, Israel
c Department of Ophthalmology, HadassahHebrew University Medical Center, Jerusalem, Israel
d Authority for Computation and Information (Ein Kerem Branch), Hebrew University, Jerusalem, Israel
e Endocrinology and Metabolism Service, HadassahHebrew University Medical Center, Jerusalem, Israel
* Correspondence to: Arthur Pollak, MD, Department of Cardiology, HadassahHebrew 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- (ER-
) 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- gene promoter region is associated with the angiographic severity of CAD in young patients.
Key Words: Oestrogen receptor- 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.35The effects of oestrogens on the vascular system are mediated by two distinct oestrogen receptors (ERs), ER- 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-
is the major mediator of the atheroprotective effect of oestrogens.710Diminished expression of ER-
wasassociated with the occurrence of premature atherosclerosis in premenopausal women.8Similarly, methylation-dependent inactivation of ER-
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-
gene, resulting in a premature stop codon.10Thus, alternation in ER-
expression and function may attenuate the atheroprotective role of oestrogens.
The ER- gene, located at chromosome 6q24.1, has six domains encoded by eight exons. Associations between a number of polymorphisms in the ER-
gene and various clinical phenotypes have been studied including therisk of breast cancer,11osteoporosis,1214hypertension,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- 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-
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- 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-
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- genotype.
2.3. Determination of ER- 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 [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 3298 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 03 in both age groups, P<0.001). Mean NCV was 2.8±1.9 (range 08) in younger and 3.2±1.6 (range 09) in older subjects (P=0.005). Mean NCS was 3.5±2.4 (range 09) in younger and 4.3±2.2 (range 013) 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 1316 and at 2224 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 926 and 728 in younger and older subjects, respectively, with a median of 18 repeats in both groups. The (TA)nrepeat polymorphism was in HardyWeinberg equilibrium (
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
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4. Discussion
In this study the length of the (TA)ndinucleotide repeat in the regulatory region of the ER- 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- gene polymorphisms. The ER-
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 1020%.26In addition, the frequency of the ER-
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- 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-
gene.27Their resulting transcripts differ in their 5'-untranslated regions (UTRs), but not in their coding regions. Different ER-
promoters have been identified in breast tissue, endometrium, osteoblasts, liver cells and testis.2729Human vascular smooth muscle cells (VSMC) were shown to express ER-
mRNA and protein, but it is unknown which promoter is expressed in these cells. Variant ER-
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-
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-
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- 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-
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- transcripts in the endothelium of coronary arteries. Expression of ER-
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