Archives of Medical Research 36 (2005) 511–517

ORIGINAL ARTICLE

Genetic Polymorphisms of Estrogen Receptors inPatients with Premature Coronary Artery Disease

Antonio de Padua Mansur, Carlos Ce´sar Montezino Nogueira, Ce´lia M.C. Strunz,JoseMendes Aldrighi and Jose´ Antonio F. Ramires

Heart Institute (InCor), University of Sa˜o Paulo Medical School, Sa˜o Paulo, Brazil

Received for publication November 23, 2004; accepted February 21, 2005 (D-04-00149).

Background.Estrogen protects against atherosclerosis through its genomic/nongenomiceffects. Estrogen receptors (ESR)α (1) andβ (2) mediate much estrogen action. Bothreceptors exist in the arterial wall, but the extent of their distribution in arterial layers isunknown. Allelic variants of the gene encoding ESR1 and ESR2 may alter their expressionand function, resulting in genetic variability.

Methods. In the present age-adjusted, case-control study, the prevalence of four mutationsin estrogen receptors was analyzed in patients with premature CAD and controls.

Results.Mutation in the ESR1 (PvuII) was more prevalent in the controls (18 vs.11%; p � 0.062) than in CAD patients, and the mutation identified by theXbaI enzymein the same receptor was associated with reduced apolipoprotein B levels and low bodymass index. Mutation of the ESR2 (AluI) was more prevalent in CAD patients (0.6 vs.18%; p � 0.008). Homozygosis for this mutation involved increased body mass index,elevated serum triglycerides and apolipoprotein B, and reduced HDL-cholesterol.Multivariate logistic regression analysis showed dyslipidemia, low serum HDL levels,and ESR2 polymorphism (AluI) [OR � 1.89 (95% CI: 1.08–3.34);p � 0.034] to beindependent risk factors for CAD.

Conclusions.Our data suggest that mutation of the ESR2 is an independent risk markerfor premature CAD. � 2005 IMSS. Published by Elsevier Inc.

Key Words:Coronary artery disease in young, Estrogen receptor, Polymorphisms, Risk factors.

n,

nshes

-

-

n-

-e.

Introduction

Estrogen influences the growth, differentiation, and functionof many target tissues, notably of the male and female reproductive systems. Estrogen also plays an important role ithe cardiovascular system, where it has certain cardioprotetive effects. The physiological function of estrogen is medi-ated via its two specific receptors, estrogen receptorα(ESR1) and estrogen receptorβ (ESR2)(1). Polymorphicalterations of ESR1 are associated with diseases suchbreast cancer(2,3), repeated abortion(4,5), arterial hyperten-sion(6), altered serum lipid levels(7), and coronary athero-sclerosis(8). Polymorphisms of ESR2 have been reportedto be associated with ovulatory dysfunction and with change

Address reprint requests to: Antonio P. Mansur, Heart Institute (InCor)University of Sao Paulo Medical School, Av. Dr. Ene´as C. Aguiar 44, CEP:05403-000, Sa˜o Paulo, Brazil; Phone and FAX: 55-11-30695299;E-mail: corantonio@incor.usp.br

0188-4409/05 $–see front matter. Copyright� 2005 IMSS. Published by Eldoi : 10.1016/ j .arcmed.2005.04.002

-nc-

as

s

,

in the plasma levels of some female sex hormones (estrogeprogesterone, follicle-stimulating hormone, and luteinizinghormone)(9). ESR2 has also been reported to be present ithe cells and tissues of the cardiovascular system, but it inot known whether ESR polymorphisms are associated witcoronary artery disease (CAD). Observational studies havshown that hormone replacement therapy (HRT) reducethe risk of atherosclerotic disease(10–13). On the otherhand, randomized studies in which equine estrogen conjugates were administered to women with coronary arterydisease (CAD) did not confirm the results of the observational studies(14,15). Many estrogen actions are executedby estrogen receptors that regulate the transcription and sythesis of ribonucleic acid (RNA) and its proteins(16,17).The objective of the present study was to investigate theassociation of ESR1 and ESR2 polymorphisms with premature CAD, where a genetic component probably may be morimportant and with the major risk factors for the disease

sevier Inc.

Mansur et al. /Archives of Medical Research 36 (2005) 511–517512

nl

e

e

u

r

c,to

e

r

Patients and Methods

A case-control study adjusted for age was conducted in 2subjects from March 2000 to December 2003. The subjecwere divided into two groups: 1) a control group consistinof 142 healthy volunteers (no personal history of CADnormal resting and exercise electrocardiograms), and 2) 1patients with premature CAD aged�55 years old. Clinicaldata obtained were age, sex, race, body mass index [wei(kg)/height (m2)], smoking history, arterial hypertension,diabetes, and the use of medications. Assessment of the liprofile included triglycerides, total cholesterol and its HDLLDL, apolipoprotein AI, and apolipoprotein B fractions. Afamily history of CAD was defined as CAD occurring inparents (before age 55 for men and 65 for women) asiblings. Dyslipidemia was divided into hypercholesteroemia (total cholesterol�6.2 mmol/L) and hypertriglyceride-mia (triglycerides �2.8 mmol/L) (18). Diabetes wasdiagnosed when the patient was taking hypoglycemic druor blood glucose levels were�7.0 mmol/L (19). Arterialhypertension was considered present when diastolic presslevels were�90 mmHg(20). No patients were using HRT.

Lipid analysis.Plasma lipid levels were determined by usingvenous blood samples obtained after a 12-h overnight faFor patients with acute myocardial infarction, samples wetaken on the first morning of hospitalization. Total cholesterol (TC) and triglycerides were determined with standarenzymatic methods. HDL cholesterol was assayed by seltive precipitation with dextran�magnesium chloride, andLDL cholesterol was determined with the Friedewald formula: LDL � TC � [HDL � (triglycerides/2.2)] (21).Apolipoproteins AI and B were determined by using thimmunoturbidimetric method.

DNA amplification and genotyping analysis.DNA was ob-tained from peripheral blood lymphocytes of patients ancontrols after informed consent was obtained. The Instittional Review Committee of our institution approved thisstudy. The polymorphisms were analyzed by the polymerachain reaction (PCR) and based on fragment size analysPCR amplification of the intron-1 region of ESR1 was peformed according to the method of Yaich et al.(22)by usingthe restriction enzymesPvuII andXbaI. PvuII polymorphismresults from the T/C transition in intron-1 located approximately 400 base pairs (bp) above exon-2. PCR produwithout thePvuII site only generate the 1.3-kb fragmentand those with the restriction site result in two fragmenof 850 and 450 bp. The second polymorphism is thatXbaI and is located approximately 50 bp from thePvuIIpolymorphism. The presence of this site for this enzymresults in two fragments of 900 and 400 bp (Figure 1).

The domain of the binding region of exon-5 and thuntranscribed region of exon-8 of the gene of the ESRwere amplified by the PCR technique described by Sundarjan et al.(9). Amplification of exon-5 and exon-8 resulted

95ts

g,53

ght

pid,

d-

gs

ure

st.re-dc-

-

d-

seis.-

-ts

sf

e

2a-

in products of 156 and 307 bp, respectively. The exchangeof nucleotide G (guanine) with A (adenosine) at position1082 of exon-5 creates a site for the restriction enzymeRsaI,which generates two fragments of 125 and 31 bp from thePCR product. This same exchange at position 1730 ofexon-8 introduces a site of recognition of the enzymeAluI,resulting in two fragments of 240 and 67 bp (Figure 2).

Cardiac catheterization.Cardiac catheterization was per-formed according to Sones and Shirey’s technique(23).Coronary artery disease severity was based on the numberof major epicardial coronary arteries affected.

Statistical analysis.Statistical analysis was performed withSAS software version 8.0. We determined that a samplesize of 141 patients would have an 80% power to detect adifference of 15% between genotype proportions previouslyrelated to CAD, atα � 0.05. Age, body mass index, andlipid profile were analyzed by using Student’st test. Variableswithout normal distribution were analyzed with the Wil-coxon test. The chi-square statistic was used for categori-cal variables, such as the prevalence of hypertension,smoking, family history, and ESR genotypes. The Kruskal-Wallis test was used for multiple comparisons of variableswithout normal distribution. Multiple regression determinedwith the logistic regression model was carried out with CAD

Figure 1. Genotypes analysis of the Pvull (A) and the Xbal polymorphism(B) in the intro 1 of the ERSI gene. (A) Lane 1, 100-bp DNA ladder; Lane2, uncleaved; Lane 3, no polymorphism (PP); Lane 4, heterozygous (Pp);Lane 5, homozygous (pp). (B) Lane 1, 100 bp DNA ladder; Lane 2,uncleaved; Lane 3, no polymorphism (XX); Lane 4, heterozygous (Xx);Lane 5, homozygous (xx).

Genetic Polymorphisms in Premature Coronary Artery Disease 513

r

r

r

in

d,

eofe-

1

o-,n

s-

-

-e

rs

d

sel

ec

inrde

de

-

Figure 2. Genotype analysis of the Alul polymorphism (A) in the 3′ UTRof exon 8 and the rsal polymorphism (B) in the exon 5 of the ERSgene. (A) Lane 1, 100-bp DNA ladder; Lane 2, uncleaved; Lane 3, npolymorphism (AA); Lane 4, heterozygous (Aa); Lane 5, homozygou(aa). (B) Lane 1, 100-bp DNA ladder; Lane 2, uncleaved; Lane 3, npolymorphism (RR); Lane 4, heterozygous (Rr); Lane 5, homozygous (r

as the dependent variable and the independent variabconsisted of those with ap �0.1 value upon bivariateanalysis, such as sex, smoking status, family history, lipprofile, and estrogen receptor genotypes(24). A p value of�0.05 was considered statistically significant.

Results

Clinical characteristics of the total population (CAD patientand controls) are listed inTable 1. Mean age, sex, anddiabetes history were similar for the two groups. Race, smoers, ex-smokers, body mass index, and subjects with artehypertension and dyslipidemia were more prevalent in thCAD group. A family history of CAD was more prevalentin the control group. In the CAD group, acute myocardiainfarction was diagnosed in 87 (57%) patients, coronaangioplasty was performed in 26 (18%), and surgery fomyocardial revascularization in 32 (22%).

Genotypes and allele frequencies.The distribution of thegenotypic frequencies of ESR1 and ESR2 is presentedTable 2. The homozygous pp genotype of the ESR1PvuIIwas more prevalent in the control group but did not reac

2oso).

les

id

s

k-iale

lyr

in

h

statistical significance (18 vs. 11%,p � 0.062). The homo-zygous aa genotype of the ESR2 was more prevalentCAD patients (18 vs. 0.6%;p � 0.008). Among the 295individuals of the total population, the frequencies of the pand P alleles of the ESR1 were 49 and 51%, respectively, anthe frequencies of the x and X alleles were 31 and 69%respectively, The frequencies of the r and R alleles of thESR2 were 6 and 94%, respectively, and the frequenciesthe a and A alleles were 34 and 66%, respectively. Thgenotypic frequencies of the ESR1 and ESR2 were in HardyWeinberg equilibrium in the population studied(25).

Genotypes and lipid profile and body mass index.Sub-jects with the mutant xx homozygous genotype of the ESRhad lower mean apolipoprotein B values and lower bodymass index. Subjects with the mutant aa homozygous gentype of the ESR2 had higher mean values of triglyceridesapolipoprotein B, and body mass index, and lower meaHDL values (Table 3). No significant differences were ob-served between the genotypic frequency of polymorphismof ESR1 and ESR2 and the number of compromised coronary arteries in CAD patients. Multivariate analysis per-formed with the logistic regression model with adjustmentfor traditional risk factors showed that the presence of dyslipidemia, low HDL levels, and polymorphism of ESR2 (AluI)[OR � 1.89 (95% CI: 1.08–3.34);p � 0.034] were indepen-dent risk factors for CAD (Table 4).

Discussion

The true importance of estrogen receptors in the development of arterial disease in women and men still needs to bproven. Our results show that the estrogen receptorβ identi-fied by the restriction enzymeAluI was associated withpremature CAD and two mutations in the estrogen receptowith the major risk factors for the disease. In addition, multi-variate analysis selected the mutation in the ESR2 identifieby the restriction enzymeAluI as an independent risk factorfor premature CAD. Losordo et al.(26) were the first toshow that the presence of the estrogen receptor in the veswall plays an important role in protection against CAD.Premenopausal women with a marked deficiency of thesreceptors in the vessel wall had significant atherosclerotidisease. However, we do not know whether polymorphicalterations in the genes responsible for ESR1 and ESR2,particular those analyzed by us, are responsible for higheor lower receptor expression. Recent studies have reportecontroversial results concerning the association of thespolymorphisms with CAD. For example, Matsubara et al.(27) found no association between CAD prevalence anseverity and the presence of ESR1 polymorphisms. Thpresent study showed that mutation of ESR1 (PvuII) wasrelated to CAD, with a higher prevalence of mutant homozygotes and that the mutation identified with the restriction

Mansur et al. /Archives of Medical Research 36 (2005) 511–517514

Table 1. Characteristics of control subjects and patients

All patients (n � 295) Controls (n � 142) CAD (n � 153) p

Age (years) 44� 10.2 44.2� 11.8 43.4� 7.5 0.431Men 160 (54%) 69 (49%) 91 (60%)Women 135 (46%) 73 (51%) 62 (40%) 0.074White 262 (89%) 120 (83%) 143 (94%)Black 29 (10%) 22 (16%) 08 (5%)Asian 04 (1%) 02 (1%) 02 (1%) 0.018Weight (kg) 76.3� 16.6 73.8� 17.2 77.1� 17.6 0.096Height (cm) 164� 0.1 163� 0.1 165� 0.09 �0.001BMI (kg/m2) 27.4 � 5 26.6� 5 27.8� 5 0.042Nonsmoker 120 (41%) 71 (50%) 49 (32%)Active smoker 98 (33%) 32 (22%) 65 (42%)Ex-smoker 64 (22%) 25 (18%) 39 (26%) �0.001SAP (mmHg) 133.4� 18.7 129.5� 5.3 135.8� 21 �0.001DAP (mmHg) 85.3� 12.1 83.2� 8.8 87.3� 14.4 �0.001Hypertension 109 (37%) 39 (27%) 70 (46%) �0.001Diabetes 56 (19%) 19 (13%) 37 (24%) 0.23Dyslipidemia 118 (40%) 32 (22%) 86 (56%) �0.001Family history 162 (55%) 90 (63%) 72 (47%) �0.001MI 87 (30%) 00 87 (57%)Angioplasty 26 (9%) 00 26 (18%)CABG 32 (11%) 00 32 (22%)

n, number of individuals; BMI, body mass index; SAP, systolic arterial pressure; DAP, diastolic arterial pressure; FH, family history; MI, myocardialinfarction; CABG, coronary artery bypass graft.p � values that correspond to statistical analysis comparing the control and CAD groups.

se

tt

ady

R

n

h

is

n

.

enzymeXbaI was associated with lower apolipoprotein Bconcentrations in peripheral blood and lower body maindex. The latter association was also observed in a recstudy on postmenopausal women, in which carriers of thmutation had a lower body mass index compared withose with other genotypes(28). These data suggest thamutation in ESR1 may be associated with protectioagainst the development of CAD. Conversely, Kunnas et(8) showed that polymorphism of ESR1 is directly relateto the development of CAD and that the presence of polmorphism deprives the cardiovascular system of the benecial effects of estrogen. Recently, Herrington et al.(7)showed that the presence of polymorphic changes in EShad a favorable effect on HDL-cholesterol levels in menopausal women on HRT.

In our study, different HDL levels were observed in patients with the ESR2 mutation identified by the restrictioenzymeAluI, with the concentrations of this lipoprotein beingreduced in women with mutant homozygosis. The presestudy was the first to investigate the association of ESRpolymorphism with premature CAD. Sundarrajan et al.(9)have already shown that polymorphism of this receptois associated with ovulatory dysfunction in patients witmenstrual disorders and with alterations in the concentrtions of various female hormones, such as estrogen aprogesterone. In our study, subjects with polymorphchanges in ESR2 (AluI) also had an increased body mas

snteh

nl.

-fi-

1-

-

nt2

r

a-ndc

index and elevated serum triglyceride and apolipoprotein Bconcentrations. These phenotypic characteristics are stronglyassociated with CAD(29–31). Apolipoprotein B is the pro-tein component of low molecular weight lipoproteins (LDL)and is considered an independent marker of CAD risk(32).The mechanism by which polymorphic changes in estrogenreceptors alter lipoprotein levels is unknown. It has been re-cently reported that genes containing polymorphisms maycause structural differences in mRNA(33), which in turnmay have different biological functions. Although theAluIandRsaI polymorphisms do not lead to changes in the aminoacids of the estrogen receptor protein, they may possiblymaintain a bond in disequilibrium with other regulatory se-quences, possibly affecting gene expression and estrogereceptorβ function(9). According to Sundarrajan et al.(9),the polymorphisms frequently detected in the sequence ofhuman DNA can be used as markers to identify geneticfactors possibly related to multifactorial diseases. Multivari-ate analysis showed that the presence of dyslipidemia, serumHDL levels, and the presence of polymorphism of ESR2(AluI) were the only independent genetic risk factors forCAD in our study. The participation of the genetic compo-nent in the development of CAD has been exhaustivelyanalyzed, with mutations involving genes linked to athero-genesis and thrombogenesis being the first to be studiedPolymorphisms of the genes of apolipoproteins AI, B, and E,of angiotensin converting enzyme, of factor V Leiden, and of

Genetic Polymorphisms in Premature Coronary Artery Disease 515

s

n

Table 2. Genotype and allele distribution in control subjects and inpatients with CAD

All patients Controls CAD p

ESR1 (PvuII) 295 142 153pp 43 (15%) 26 (18%) 17 (11%)Pp 154 (52%) 69 (49%) 85 (56%)PP 98 (33%) 47 (33%) 51 (33%) 0.062p 240 (49%) 121 (43%) 119 (39%)P 250 (51%) 163 (57%) 187 (61%) 0.409ESR1 (XbaI) 295 142 153xx 23 (%) 12 (8%) 11 (7%)Xx 139 (47%) 69 (49%) 70 (46%)XX 133 (45%) 61 (43%) 72 (47%) 0.764x 185 (31%) 93 (33%) 92 (30%)X 405 (69%) 191 (67%) 214 (70%) 0.591ESR2 (RsaI) 290 142 148rr 4 (1%) 03 (2%) 1 (1%)Rr 25 (9%) 14 (10%) 11 (8%)RR 261 (90%) 125 (88%) 136 (91%) 0.826r 33 (6%) 20 (%) 13 (%)R 547 (94%) 264 (%) 283 (%) 0.243ESR2 (AluI) 290 142 148aa 36 (12%) 09 (6%) 27 (18%)Aa 128 (44%) 66 (47%) 62 (42%)AA 126 (44%) 67 (47%) 59 (40%) 0.008a 200 (34%) 84 (30%) 116 (39%)A 380 (66%) 200 (70%) 180 (61%) 0.015

n, number of individuals; ESR1,α estrogen receptor; ESR2,β estrogenreceptor.p � values that correspond to statistical analysis comparing the control aCAD groups.

prothrombin(34,35)produce contradictory results. This wathe first time that a case-control study showed that muttions in ESR2 could be associated with premature CAD a

nd

a-d

Table 4. Multiple regression analysis of data for patientswith coronary artery disease

Variable Odds ratio 95% CI p

HDL 0.94 0.91–0.98 0.001Dyslipidemia 3.34 1.55–7.14 0.002ESR2 (Alu I) 1.89 1.08–3.34 0.034

HDL, cholesterol; ESR2 (AluI), β estrogen receptor polymorphisms identi-fied with the restriction enzymeAluI.

with its major risk factors. ESR1 and ESR2 are present invarious types of tissues, but ESR2 has been more frequentlydetected in extragenital tissues, such as the lungs, bones,bone marrow, intestinal mucosa, and in tissues of the cardio-vascular system, such as endothelium, smooth muscle cells,and cardiac myocytes. The true function of these receptors inthe various organs is unknown, but it is possible that theirpresence and the integrity of their function are importantfor full tissue development and for protection against degen-erative diseases. Despite not being tested, the different bio-logical functions of estrogen receptor polymorphisms maybe the result of linkage disequilibrium(25). Recently, studieshave shown the different responses of these receptors tosome estrogen agonists and antagonists(36,37), ESR2 beingmore prevalent in vascular tissue(38) and also perhapshaving antiproliferative effects that reduce the progressionof atherosclerotic lesions.

The type of study reported here might have some limita-tions. Because this was a case-control study, we cannotstate that a direct cause/effect relationship exists, but we can

Table 3. Genotype distribution of estrogen receptor, lipid profile, and body mass index

Genotype

ESR1 (XbaI) xx Xx XX p

TG 1.84� 1.27 2.36� 3.95 1.75� 1.09 0.56TC 5.20� 1.31 5.60� 1.47 5.41� 1.24 0.36HDL 1.10 � 0.35 1.12� 0.32 1.15� 0.31 0.59LDL 3.28 � 1.12 3.55� 1.08 3.49� 0.99 0.378Apo AI 1.23 � 0.27 1.34� 0.24 1.32� 0.3 0.566Apo B 0.95� 0.3 1.15� 0.32 1.13� 0.28* 0.03*BMI 25.34 � 4.16 27.12� 4.35 28.28� 5.91* 0.024*

ESR2 (AluI) aa Aa AATG 3.49� 7.02 1.86� 1.91 1.85� 0.95* 0.01*TC 5.62� 1.79 5.3� 1.31 5.67� 1.21 0.168HDL 0.97 � 0.25 1.16� 0.37 1.14� 0.24* 0.028*LDL 3.32 � 1.00 3.40� 1.04 3.66� 1.04 0.151Apo AI 1.25 � 0.22 1.34� 0.28 1.32� 0.24 0.45Apo B 1.23� 0.35 1.05� 0.26 1.18� 0.32* 0.018*BMI 29.59 � 4.97 26.96� 4.74 27.53� 5.1* 0.02*

ESR1,α estrogen receptor; ESR2,β estrogen receptor; TG, triglycerides; TC, total cholesterol; Apo, apolipoprotein. BMI, body mass index (kg/m2).Values are reported as means� SD as mmol/L for TG, TC, HDL and LDL, and as g/L for Apo AI and Apo B.*p � values that correspond to statistical analysis comparing the estrogen receptor homozygosis genotype (xx vs. XX and aa vs. AA).

Mansur et al. /Archives of Medical Research 36 (2005) 511–517516

yartA

esin

encol

nd

nnt

n

-d

-

cer

s

S

of

dits

n.

ry

lsey.

ety.

-f

infer that an association does exist between the presencepolymorphisms of ESR2 identified by the restriction enzymeAluI and premature CAD. This association was more evidenwhen the mutationwasalso associatedwith variables stronglrelated to CAD like lipid profile and body mass index. Thus,we may conclude that the present results suggest that polymorphism of ESR2 identified by the restriction enzymeAluIis an independent risk factor for CAD in the group of youngpatients studied here.

AcknowledgmentsThis study was supported by Fundac¸ao ao Amparo a Pesquisado Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional deDesenvolvimento Cientı´fico e Tecnolo´gico (CNPq).

References1. Clark JH, Schrader WT, O’Malley BW. Mechanisms of action of steroid

hormones. In: Wilson J, Foster DW, eds. Textbook of Endocrinology.Philadelphia: WB Saunders Company;1992. pp. 35–90.

2. Andersen TI, Heimdal KR, Skrede M, Tveit K, Berg K, Borresen AL.Oestrogen receptor (ESR) polymorphisms and breast cancer susceptbility. Hum Genet 1994;94:665–670.

3. Parl FF, Cavener DR, Dupont WD. Genomic DNA analysis of theestrogen receptor gene in breast cancer. Breast Cancer Res Tre1989;14:57–64.

4. Lehrer S, Sanchez M, Song HK, Dalton J, Levine E, Savoretti P,Thung SN, Schachter B. Oestrogen receptor B-region polymorphismand spontaneous abortion in women with breast cancer. Lance1990;335:622–624.

5. Berkowitz GS, Stone JL, Lehrer SP, Marcus M, Lapinski RH,Schachter BS. An estrogen receptor genetic polymorphism and the riskof primary and secondary recurrent spontaneous abortion. Am J ObsteGynecol 1994;171:1579–1584.

6. Lehrer S, Rabin J, Kalir T, Schachter BS. Estrogen receptor variantand hypertension in women. Hypertension 1993;21:439–441.

7. Herrington DM, Howard TD, Hawkins GA, Reboussin DM, Xu J,Zheng SL, Brosnihan KB, Meyers D, Bleecker ER. Estrogen-receptorpolymorphisms and effects of estrogen replacement on high-densitylipoprotein cholesterol in women with coronary disease. N Engl J Med2002;346:967–974.

8. Kunnas TA, Laippala P, Penttila A, Lehtimaki T, Karhanen PJ. Associa-tion of polymorphism of humanα oestrogen receptor gene with coro-nary artery disease in men: a necropsy study. BMJ 2000;321:273–274

9. Sundarrajan C, Liao WX, Roy AC, Chye NGS. Association betweenestrogen receptor-β gene polymorphisms and ovulatory dysfunctionsin patients with menstrual disorders. J Clin Endocrinol Metab 2001;86:135–139.

10. Lerner DJ, Kannel WB. Patterns of coronary heart disease morbidityand mortality in the sexes: a 26-year follow-up of the Framinghampopulation. Am Heart J 1986;111:383–390.

11. Baysal K, Losordo DW. Oestrogen receptor and cardiovascular dis-ease. Clin Exp Pharmacol Physiol 1996;23:537–548.

12. Barrett-Connor E, Grady D. Hormone replacement therapy, heart disease, and other considerations. Annu Rev Public Health 1998;19:55–72

13. Grodstein F, Manson JE, Colditz GA, Willet WC, Speizer FE,Stampfer MJ. A prospective, observational study of menopausal hor-mone therapy and primary prevention of cardiovascular disease. AnnIntern Med 2000;133:933–941.

of

ty

-

i-

at

t

t

.

-.

14. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B,Vittinghoff E. Randomized trial of estrogen and progestin for secondarprevention of coronary heart disease in postmenopausal women. Heand Estrogen/Progestin Replacement Study (HERS) group. JAM1998;280:605–613.

15. GradyD,HerringtonD,BittnerV,BlumenthalR,DavidsonM,HlatkyM,Hsia J, Hulley S, Herd A, Khan S, Newby LK, Waters D, VittinghoffE, Wenger N: HERS Research Group . Cardiovascular disease outcomduring 6.8 years of hormone therapy: Heart and Estrogen/ProgestReplacement Study follow-up (HERS II). JAMA 2002;288:49–57.

16. Katzenellenbogen BS. Mechanisms of action and cross-talk betweestrogen receptor and progesterone receptor pathways. J Soc GyneInvestig 2000;7:33–37.

17. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, NilssoS, Gustafsson JA. Comparison of the ligand binding specificity antranscript tissue distribution of estrogen receptorα andβ. Endocrinol-ogy 1997;38:863–870.

18. Summary of the second report of the National Cholesterol EducatioProgram (NCEP) Expert Panel on Detection, Evaluation, and Treatmeof High Blood Cholesterol in Adults (Adult Treatment Panel II). JAMA1993;269:3015–3023.

19. Report of the Expert Committee on the Diagnoses and Classificatioof Diabetes Mellitus. Diabetes Care 1997;20:1183–1197.

20. The sixth report of the Joint National Committee on prevention, detection, evaluation and treatment of high blood pressure. Arch Intern Me1997;157:2413–2446.

21. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without useof the preparative ultracentrifuge. Clin Chem 1972;18:499–502.

22. Yaich L, Dupont WD, Cavener DR, Parl FF. Analysis of the PvuIIrestriction fragment-length polymorphism and exon structure of theestrogen receptor gene in breast cancer and peripheral blood. CanRes 1992;52:77–83.

23. Sones FM, Shirey EK. Cinecoronary arteriography. Mod ConceptCardiovasc Dis 1962;31:735–738.

24. Rosner B. Fundamentals of Biostatistics. 2nd ed. Boston: PWPublishers;1986.

25. Thompson MW, Mcinnes RR, Willard HF. Genetics in Medicine. 5thed. Philadelphia: W.B. Saunders Company;1991. pp. 143–165.

26. Losordo DW, Kearney M, Kim EA, Jekanowski J. Variable expressionof estrogen receptor in normal and atherosclerotic coronary arteriespremenopausal women. Circulation 1994;89:1501–1510.

27. Matsubara Y, Murata M, Kawano K, Koichi Z, Zama T, Aoki N.Genotype distribution of estrogen receptor polymorphism in men anpostmenopausal women from healthy and coronary populations andrelation to serum lipid levels. Arterioscler Thromb Vasc Biol 1997;17:3006–3012.

28. Deng HW, Li J, Li JL, Dowd R, Davies KM, Johnson M, Gong G,Deng H, Recker R. Association of estrogen receptorα genotypes withbody mass index in normal healthy postmenopausal Caucasian womeClinc Endocrinol Metabol 2000;85:2748–2751.

29. Miller M, Seidler A, Moalemi A, Pearson TA. Normal triglyceridelevels and coronary artery disease events: the Baltimore CoronaObservational Long-Term Study. J Am Coll Cardiol 1998;31:1252–1257.

30. Assmann G, Schulte H. Relation of high-density lipoprotein cholesteroand triglycerides to incidence of atherosclerotic coronary artery disea(the PROCAM experience). Prospective cardiovascular Munster studAm J Cardiol 1992;70:733–737.

31. Stevens J, Cai J, Pamuk ER, Williamson DF, Thun MJ, Wood JL. Theffect of age on the association between body-mass index and mortaliN Engl J Med 1998;338:1–7.

32. Lamarche B, Moorjani S, Lupien PJ, Cantin B, Bernard PM, Dagenais GR, Despres JP. Apolipoprotein A-I and B levels and the risk oischemic heart disease during a five-year follow-up of men in theQuebec Cardiovascular Study. Circulation 1996;94:273–278.

Genetic Polymorphisms in Premature Coronary Artery Disease 517

33. Shen LX, Basilion JP, Stanton VP Jr . Single nucleotide polymorphismcan cause different structural folds of mRNA. Proc Natl Acad Sci USA1999;96:7871–7876.

34. MansurAP,Annicchi-BizzacchiJ,FavaratoD, AvakianSD,Ce´sarLAM,Ramires JAF. Angiotensin-converting enzyme and apolipoprotein gepolymorphism and different syndromes of coronary disease. ClCardiol 2000;23:335–340.

35. Arruda VR, Siqueira LH, Chiaparini LC, Coelho OR, Mansur APRamires JAF, Annichino-Bizzacchi JM. Prevalence of the prothrombin gene variant 20210G→A among patients with myocardial in-farction. Cardiovasc Res 1998;37:42–45.

s

nein

,-

36. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ,Scanlan TS. Differential ligand activation of estrogen receptors ERalphaand ERbeta at AP1 sites. Science 1997;277:1508–1510.

37. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J, NilssonS. Differential response of estrogen receptorα and estrogen receptorβ to partial estrogen agonists/antagonists. Mol Pharmacol 1998;54:105–112.

38. Hodges YK, Tung L, Yan XD, Graham JD, Horwitz KB, Horwitz LD.Estrogen receptorsα andβ: prevalence of estrogen receptorβ mRNAin human vascular smooth muscle and transcriptional effects. Circula-tion 2000;101:1792–1798.