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Human Molecular Genetics Advance Access originally published online on July 13, 2005
Human Molecular Genetics 2005 14(16):2405-2413; doi:10.1093/hmg/ddi242
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Tobacco smoking, estrogen receptor {alpha} gene variation and small low density lipoprotein level

Amanda M. Shearman1,*, Serkalem Demissie2, L. Adrienne Cupples2, Inga Peter3, Christopher H. Schmid3, Jose M. Ordovas4, Michael E. Mendelsohn5 and David E. Housman1

1Center for Cancer Research, E17-536, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, 2The Department of Biostatistics, Boston University School of Public Health, Boston, MA 02118, USA, 3Biostatistics Research Center, Institute for Clinical Research and Health Policy Studies, Tufts-New England Medical Center, Boston, MA 02111, USA, 4Nutrition and Genomics Laboratory, USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, USA and 5The Molecular Cardiology Research Institute, Department of Medicine, New England Medical Center and Tufts University School of Medicine, Boston, MA 02111, USA

* To whom correspondence should be addressed. Tel: +617 2533015; Fax: +617 2535202; Email: shearman{at}mit.edu

Received February 26, 2005; Revised June 23, 2005; Accepted July 1, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
High levels of small low density lipoprotein (LDL) particles are a major risk factor for cardiovascular morbidity and mortality. Both estrogens and smoking, with known anti-estrogenic effects, alter the atherogenic lipid profile. We tested for a role of interaction between smoking and estrogen receptor {alpha} gene (ESR1) variation in association with plasma concentration of atherogenic small LDL particles and LDL particle size. We studied 1727 unrelated subjects, 854 women and 873 men, mean age 51 years (SD 10), from the population-based Framingham Heart Study. After covariate adjustment, women who smoked and had the common ESR1 c.454-397 TT genotype (in 30% of women, T was present on both chromosomes at position 397 prior to the start of exon 2) had >1.7-fold higher levels of small LDL particles than women with the alternative genotypes (P-value for smoking–genotype interaction was 0.001). Similar results were obtained for three other ESR1 variants including c.454–351A>G, in the same linkage disequilibrium block. A similar substantial gender-specific result was also evident with a fifth variant, in a separate linkage disequilibrium block, in exon 4 (P=0.003). Women who smoked and had specific, common ESR1 genotypes had a substantially higher plasma concentration of atherogenic small LDL particles. Significant results revealed a dose-dependent effect of smoking and were evident in both pre- and postmenopausal women. The reported association has the potential to explain the risks associated with estrogen use in certain women and a recent report of association between an ESR1 haplotype comprised of c.454–397 T and c.454–351 A alleles with increased myocardial infarction and ischaemic heart disease, independent of the standard, established cardiovascular risk factors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The mechanisms of action and effects of estrogen and hormone therapy are a topic of current interest and controversy, with important implications for clinical practice. Both estrogens and cigarette smoking have established effects on the atherogenic lipid and lipoprotein profile. Smoking causes increased lipid oxidation and alterations in estrogen metabolism. The effects of estrogen are mediated via activation of estrogen receptors, which are expressed in a wide range of tissues and regulate gene expression (1Go). Anti-estrogenic effects of cigarette smoking in women have been recognized for over a decade (2Go,3Go). More recently, evidence has shown that some of the compounds in tobacco smoke may act as non-steroidal estrogens (4Go) and activate estrogen receptor {alpha} (ESR1) (5Go).

We studied variation in the ESR1 gene for association with a comprehensive lipid profile in women and men from the Framingham Heart Study (5aGo). Consistent results were found in women, using four polymorphisms in a single linkage disequilibrium block (a TA repeat in the promoter, c.30T>C in exon 1 and c.454-397T>C and c.454-351A>G in intron 1), for correlated measures: small low density lipoprotein (LDL) particle concentration and LDL particle size. Our results also suggested modification of the association between ESR1 variation and small LDL particle concentration by menopausal status and hormone replacement therapy (HRT). In general, premenopausal women and postmenopausal women on HRT or not on HRT revealed similar patterns of effect, but the effect size was largest among postmenopausal women, particularly those on HRT. Between a third and a half of the variation in LDL particle size has been shown to be inherited (6Go), and a multilocus linkage study of LDL particle size in families with coronary artery disease confirmed reports of linkage to the LDL receptor and also found evidence of linkage to chromosome 6q25.3, near ESR1 (7Go). A recent genome-wide scan in familial hypertriglyceridaemia families found the only evidence for linkage to LDL particle size on 6q with the maximum LOD score ~1 cM away from D6S440 in the ESR1 gene (8Go). Results of this and other linkage and association studies of LDL particle size have been reviewed recently (9Go). Genotypes at several ESR1 variants have been associated with other estrogen-dependent disorders including an increase in cardiovascular disease risk and responses of lipid profile to HRT (10Go–14Go). One variant, ESR1 c.454-397T>C, has an allele that forms part of a potential binding site for the myb family of transcription factors, which are themselves estrogen activated (15Go,16Go). There is growing evidence of the importance of gene–environment interactions in human health. Several genetic association studies have reported a synergistic effect with smoking status, including genes involved in metabolism of the toxic constituents of cigarette smoke and genes that contain estrogen response elements (17Go,18Go), but such an interaction has not been explored for ESR1 variation and smoking status. Therefore, we tested the hypothesis that smoking status and genotypes at ESR1 polymorphisms might interact to affect small LDL particle concentration and LDL particle size in 1727 unrelated women and men from the Framingham Heart Study.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The characteristics of the unrelated study subjects, by smoking status, at the fourth examination cycle of the Framingham Heart Study offspring cohort are shown (Table 1). Smokers were significantly younger and had higher intakes of alcohol than non-smokers. In addition, participants who smoked had a more atherogenic profile. The differences were most significant among women and included higher LDL cholesterol, higher TG levels and lower HDL cholesterol levels, among women who smoked than in women who did not. With the exception of c.454-351A>G, genotype frequencies between smoking and non-smoking women were not significantly different (Table 2). The promoter, exon 1 and intron 1 polymorphisms were in strong linkage disequilibrium, whereas D6S440 and the exon 4 polymorphism were not in strong linkage disequilibrium with any of them, or with one another. The allele frequency distribution of the dinucleotide repeats is provided in Supplementary Material, Figure S1.


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  		Table 1. Characteristics of the study participants by gender and smoking status
 

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  		Table 2. Genotype and allele frequencies of ESR1 polymorphisms in women, by smoking status
 
Adjusted analyses of genotypes at three of the four polymorphisms in strong linkage disequilibrium provided evidence of interaction with smoking in association with small LDL in women (P=0.03, 0.001 and 0.003, respectively, for c.30TC, c.454-397T>C and c.454-351A>G), but not in men (Fig. 1 and Supplementary Material, Table S1). Of the other polymorphisms analysed for comparison, genotypes at the exon 4 variant (c.975C>G) also significantly interacted with smoking (P=0.003). Adjusted analyses of the six ESR1 polymorphisms, by smoking status, provided significant evidence of association of five polymorphisms in the smoking women (P-value from 0.009 to <0.001), but no evidence of association among the non-smoking women. Female smokers with the c.454-397TT genotype had a 75% higher mean level of small LDL particles than other women. The association with small LDL particles was present in two separate linkage disequilibrium blocks: first at TA, c.30T>C, c.454-397T>C and c.454-351A>G; second at c.975C>G. There appeared to be an allele dose effect in the second block, with c.975GG individuals having the highest level of small LDL, around 70% greater than the c.975CC individuals and 50% greater than the heterozygous individuals. There was a smoking dose–response relationship, for small LDL particle concentration by genotype, in both linkage disequilibrium blocks (Fig. 2). Analysis by menopausal and smoking status, run only for c.454-397T>C and c.975C>G, revealed that the association among smokers was only present in postmenopausal women for the former polymorphism, whereas it was present in both pre- and postmenopausal women for the latter (Fig. 3). Forty-four percent of women were premenopausal, 50% postmenopausal not on HRT and only 6% postmenopausal on HRT. Among the postmenopausal women on HRT, the non-smokers had lower small LDL particle concentrations than other postmenopausal women, whereas those who smoked had much higher concentrations than those who were not on HRT.



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  		Figure 1. Interaction of smoking status, and ESR1 genotypes, on LDL characteristics in 854 women. The five polymorphisms, TA repeat, c.30T>C, c.454-397T>C, c.454-351A>G and c.975C>G are ordered 5' to 3' along the ESR1 gene. The first four polymorphisms were in strong linkage disequilibrium with one another (D'>0.82), whereas the fifth, c.975C>G was in a separate linkage disequilibrium block, D' with each of the former markers <0.26. Analyses were adjusted for age, body mass index, diabetic status, alcohol intake, use of cholesterol lowering medications, use of beta-blockers, menopausal status and HRT. P-values are for interaction between smoking status and genotype in association with LDL characteristic: small LDL particle concentration in the upper panel and LDL particle size in the lower panel. Association analyses by smoking status were significant among smokers (P-values shown) and not among non-smokers (P-values provided in Supplementary Material, Tables S1 and S2). Interaction terms were also calculated in men, no significant results were obtained. Values are least squares mean±SE.

 


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  		Figure 2. Association of ESR1 c.454-397T>C and c.975C>G with small LDL particle concentration in never, formerly and currently smoking women. There were 250 never smokers, 365 former smokers, 124 current smokers of ≤20 cigarettes/day and 58 current smokers of >20 cigarettes/day. Analyses were adjusted for age, body mass index, diabetic status, alcohol intake, use of cholesterol lowering medications, use of beta-blockers, menopausal status and HRT. Values are least squares mean±SE.

 


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  		Figure 3. Association of ESR1 c.454-397T>C and c.975C>G with small LDL particle concentration, by smoking, menopausal and hormone replacement status. Analyses were adjusted for age, body mass index, diabetic status, alcohol intake, use of cholesterol lowering medications and use of beta-blockers. Values are least squares mean±SE. Only +SE has been depicted where –SE was less than zero. In the six menopausal/smoking status categories, n=289, 86, 45, 8, 328 and 96, respectively, in the order depicted.

 
Haplotype analysis of ESR1 c.454-397T>C and c.454-351A>G was performed for small LDL particle concentration. There were four observed haplotypes: c.454-397T-c.454-351A, c.454-397C-c.454-351G, c.454-397C-c.454-351A and c.454-397T-c.454-351G with frequencies of 54, 35, 11 and 0.2%, respectively. Using an additive model, when compared with the TA haplotype, the CG haplotype is associated with lower small LDL particle concentrations (P=0.001) among women who smoked.

Analysis of LDL size also revealed significant associations, for four of the six polymorphisms in smoking women, although only two markers provided data that reached statistical significance for the interaction (Fig. 1 and Supplementary Material, Table S2). The genotypes that previously had the highest level of small LDL tended to correlate with lower LDL sizes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
We observed significant interaction between smoking status and genotype at each of five ESR1 polymorphisms, in association with small LDL particle concentration in women. ESR1 c.454-397T>C and c.454-351A>G, in intron 1, were both in the same linkage disequilibrium block as the studied TA repeat in the ESR1 promoter and c.30T>C in exon 1. Women who smoked and were homozygous for the c.454-397T or c.454-351A allele had over 70% higher small LDL particle levels than other women. Both of these were the more common alleles and there was strong linkage disequilibrium between them. An exon 4 polymorphism over 100 kb away in a separate linkage disequilibrium block provided equally significant evidence for a smoking status–genotype interaction in association with small LDL phenotypes. The pairwise D' values between these markers were consistent with previous studies (12Go). The significant interaction between smoking status and ESR1 genotype for small LDL particle concentration and similar findings for LDL particle size were independent of a number of covariates and potentially confounding factors. The fact that there was a dose–response relationship with smoking status, the largest effect being observed among current smokers of more than 20 cigarettes per day reinforces our findings. The effect was also observed with varying menopausal and HRT status.

In postmenopausal women, participants from the previous Framingham Study generation to those reported here, significantly increased rates of myocardial infarction were observed, particularly among estrogen users who smoked cigarettes (19Go). Haplotypes of the two intron 1 polymorphisms, associated with high risk lipid levels in women, were recently associated with increased risk of myocardial infarction and ischaemic heart disease in women from the Rotterdam Study (20Go). In contrast, in men, including those who were studied and gave no significant results here, the alternative, rarer c.454-397CC homozygote has been associated with myocardial infarction (10Go,11Go). This gender difference for cardiovascular disease risk is consistent with previous reports where estrogen receptor variation was associated with opposite effects in males and females (21Go,22Go). Although this may potentially result from gender differences in circulating estrogen levels, the results we obtained in women revealed similar direction of effect regardless of menopausal and HRT status. One limitation of the current study is the absence of data for circulating estrogen levels, a factor that should be considered in future studies. The fact that our positive results were specific to women provides support that ESR1 variation may contribute to cardiovascular risk in men and women via separate, gender-specific mechanisms. Although we did not obtain significant evidence of association among non-smokers, there did appear to be some trend towards an opposite effect to that among smokers. Our findings are compatible with the results of complex segregation analyses of small dense LDL phenotypes, which have included support for recessive inheritance, causative allele frequencies ranging from 19 to 42%, reduced penetrance in young men and an effect of menopausal status (23Go).

It is commonly believed that LDL cholesterol is functionally deleterious; however, this may just be a surrogate for the biologically functional factor represented by lipoprotein particle concentration. Cells regulate their cholesterol content by a combination of de novo synthesis and uptake via LDL receptors. Small dense LDL particles have decreased receptor-mediated uptake, have greater propensity for uptake by the arterial tissue and are more readily oxidized than larger LDL particles. The menopausal transition is associated with an increase in small dense LDL levels. Higher concentrations of these particles are associated with hypertension, the metabolic syndrome (central obesity, insulin resistance and dyslipidaemia) and cardiovascular disease and are inversely associated with extreme longevity (6Go,24Go).

Here, as in the previous Framingham Study reports, smokers had enhanced atherogenic lipid profiles (25Go). Smoking has smaller effects on lipid profile in men than in women. In addition to smoking status, differences in lipid profiles may be due to differences in lifestyle and behaviour or comorbidity between smokers and non-smokers. The ESR1 TA repeat polymorphism has also been associated with anxiety (26Go,27Go) and other aspects of personality (28Go) that may be related to propensity to tobacco smoking or other factors, such as diet that can affect lipid profile. In women, the TA repeat alleles associated with high anxiety (27Go) were in disequilibrium with genotypes (10Go,11Go) associated with higher small LDL concentrations. Cigarette smoking has been associated with anxiety, and anxiety may be a risk factor for CHD (29Go–31Go). Future studies will be required to determine the effects of some of these potential confounders. Adjustment for physical activity (data not shown) made little difference to our findings. When individuals who had never smoked, formerly smoked, smoked 20 or fewer cigarettes or more than 20 cigarettes per day were analysed separately, there was a dose–response relationship and the largest effect was observed among current smokers of more than 20 cigarettes per day. Although there have been reports that components of tobacco smoke may act as non-steroidal estrogens (4Go) and activate ESR1 (5Go), there is a more general belief that smoking results in reduced levels of natural estrogens (2Go). Sources of evidence that tend to support this idea include early menopause and smaller increases in bone mineral density in response to HRT, in smokers (32Go).

There are limitations inherent in all association studies. Results may overestimate the true size of effect (33Go) or identify spurious associations due to population stratification. The risk of population stratification may, however, have been overestimated (34Go) and the gender-specific nature of our findings may be taken as evidence against extreme stratification, which would be expected to affect both genders equally in a population such as Framingham. Our smallest P-value for interaction was 0.001, which for a single test would represent a false positive in one out of a thousand occurrences. Our results are particularly robust due to similar findings from polymorphisms on two separate linkage disequilibrium blocks. A standard correction to adjust for multiple testing by increasing the threshold for significance may be used, but relies on the assumption that all statistical tests are independent. We have not made such an adjustment because many of the markers were in strong linkage disequilibrium with one another and the lipid measures were also correlated. Our findings may be due to a direct effect of some of the genotyped polymorphisms or to other markers in linkage disequilibrium with them.

The fact that we obtained positive results in two separate ESR1 linkage disequilibrium blocks provides support for multiple functional alterations in the ESR1 gene. It is likely that the range of polymorphisms and spectrum of disease severity observed in the studies of genes causing Mendelian disorders will also apply to genes contributing to complex traits (35Go). A male non-smoker has been described with LDL-, HDL- and total cholesterol levels below the 10th percentile for men of his age, because of homozygosity for an ESR1 mutation that resulted in an absence of functional ESR1 protein (36Go,37Go). Genotype-specific alterations in ESR1 function may also have critical effects among female smokers. The ESR1 c.454-397C allele, but not the T allele, forms part of a potential binding site for the myb family of transcription factors that are themselves activated by hormone (15Go,16Go). Several groups have independently reported that presence of the T allele resulted in a lower ESR1 transcription level (16Go,20Go). In women, Schuit et al. (20Go) also point out that presence of the c.454-397T allele has been associated with multiple phenotypes of estrogen depletion, consistent with it leading to lower estrogen action. Thus, in the c.454-397T homozygous women who smoked, the higher levels of small LDL particles might result from the effects of lower numbers of receptors combined with lower levels of estrogens, which may result from smoking. It is noteworthy that even without consideration of interaction with smoking status, ESR1 c.454-397TT genotype was also associated with a much larger increase in small LDL particles among postmenopausal women on HRT, than among other women (5aGo). Here, the results suggest that this is confined to women who smoke.

Linkage analyses and previous association studies of candidate genes with small dense LDL (reviewed in 9) provide evidence of positive hits that are in general not consistent across all populations and genetic backgrounds, but in addition to evidence to support a locus including ESR1 on 6q25.3 estrogen receptor function and hormone replacement clearly can affect lipid metabolism. LDL receptor expression can be stimulated via an ESR1/Sp1 complex through which estrogen and related hormone therapies lower plasma LDL concentrations (38Go). Both hepatic lipase, in which gene polymorphisms have been associated with LDL particle size (39Go), and lipoprotein lipase, which can increase production of smaller, denser LDL from larger more buoyant precursors, have known estrogen response elements (40Go,41Go). Expression of apolipoprotein E, which is associated with small dense LDL phenotypes, is influenced by estrogen in an apolipoprotein E allele-dependent fashion, which affects estrogen receptor binding (42Go).

In summary, we have observed a significant interaction between smoking status and common ESR1 genotypes, in association with small LDL concentration in women. It is now important to have these analyses replicated in other populations and ethnicities, including studies with both detailed lipid data and large enough sample size to provide statistical power to study cardiovascular disease risk. The underlying mechanism and the effect of a comprehensive profile of ESR1 variation are promising areas for future investigation. Finally, further analysis of ESR1 variation and smoking status may illuminate other estrogen-related characteristics of complex etiology, particularly the increased early morbidity among women receiving HRT in recent clinical trials.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Study sample
The Framingham Heart Study began in 1948 with enrolment of men and women from Framingham, MA, USA. In 1971, 5124 subjects who were offspring of original cohort members and the spouses of the offspring were recruited. After their initial evaluation, these individuals have undergone repeat examinations approximately every 4 years. Offspring cohort members, including many unrelated individuals (n=2933), provided blood samples for DNA extraction at the sixth examination cycle. A subset (n=1811, 907 men and 904 women) of biologically unrelated individuals was selected for inclusion in a panel of DNAs that would be suitable for multiple studies. These subjects had been selected to include approximately equal numbers of men and women and to have other characteristics not significantly different from the unstudied offspring cohort members.

Plasma lipid subclasses and particle sizes in the current study were only measured at the fourth examination, during 1988–1991, and thus subject characteristics are taken from that examination. Both phenotypic and ESR1 genotypic information were available for 1727 individuals (873 men and 854 women) who were described in the current study.

All subjects gave written informed consent (including for DNA analysis) at each clinic visit. The study protocol was approved by the Review board for Human Subjects Research, Boston Medical Center, and the Human Investigation Research Committee, Tufts University New England Medical Center.

Lipid, smoking status and covariate measurements
Fasting venous blood samples were collected and plasma was separated from blood cells by centrifugation and immediately used for the measurement of lipids. Plasma total cholesterol, HDL cholesterol and TG concentrations were measured, and when plasma TG concentration did not exceed 400 mg/dl, LDL cholesterol concentrations were estimated with the Friedewald equation. Plasma lipoprotein concentrations and subclass distributions were determined by proton nuclear magnetic resonance (NMR) spectroscopy (43Go). Concentrations of three LDL subclasses (large, 21.3–27.0 nm; intermediate, 19.8–21.2 nm; and small, 18.3–19.7 nm) are provided, in units of cholesterol (mg/dl). Weighted-average particle diameter (nm) of LDL is calculated from the subclasses concentrations. NMR measures of LDL subclass distributions and particle sizes are highly correlated with measures obtained using gradient gel electrophoresis and density gradient centrifugation (44Go,45Go). Small LDL particle levels are highly correlated with and nearly equivalent to ‘small dense LDL particles’ (6Go). The coefficient of variation or reproducibility of LDL size is <0.5% (46Go).

Potential confounders and covariates included in the analyses were age, body mass index, diabetes status, smoking status, alcohol intake, use of cholesterol lowering medications, use of beta-blockers and menopausal status and HRT use in woman (11Go,47Go). Current cigarette smoking status was self-reported. Non-smokers included both never and former smokers. Current smokers were subdivided according to whether they did or did not smoke >20 cigarettes per day. Physical activity index was calculated from self-report of daily physical activity (48Go).

DNA extraction and genotyping
Genomic DNA was extracted from peripheral blood leukocytes using standard methods. Genotyping was blinded to participant characteristics, and allele calling carried out independently by two separate investigators. The single nucleotide polymorphisms, described at http://www.ncbi.nlm.nih.gov/SNP, were genotyped as described previously (11Go). The two dinucleotide repeats were amplified by PCR using standard methods and an annealing temperature of 58°C. For the TA repeat in the promoter, 5' to 3' primer sequences were: 6FAM-GAC GCA TGA TAT ACT TCA CC and GCA GAA TCA AAT ATC CAG ATG; for D6S440: 6FAM-GCT AAG GAT AAC TTT CTG GTA GAC and TTC TTC ATT TTA CGG ATG G. Fragments were separated on an ABI 3700 and detected using Genescan and Genotyper software (Applied Biosystems, Foster City, CA, USA).

Statistical analysis
Single nucleotide polymorphism genotype frequencies were analysed using the {chi}2 test, to confirm that they conformed to the expectations of Hardy–Weinberg equilibrium (P>0.05). The dinucleotide repeats had multiple alleles (Supplementary Material, Fig. S1) that were coded as ‘long’ (L) if the number of repeats exceeded the median allele number or ‘short’ (S) otherwise; genotypes were denoted LL, LS and SS. For the TA repeat, both the observed bimodal allele distribution and analytical strategy were similar to that in several previous studies (49Go,50Go). For each pair of markers, linkage disequilibrium was evaluated by Lewontin's D' calculated from haplotype frequencies estimated by the expectation–maximization algorithm (snphap, http://www-gene.cimr.cam.ac.uk/clayton/software/). A linkage disequilibrium block was defined as a group of markers with pairwise values of |D'|>0.70. Descriptive statistics, values of mean±SD for continuous variables, or proportions for categorical variables were computed separately by smoking status in men and women. The distribution of these variables was compared between smokers and non-smokers using t-tests for continuous variables and {chi}2-tests for categorical variables.

To evaluate the relationships between genotype, smoking and plasma lipid or lipoprotein concentrations, we used analysis of covariance (ANCOVA) techniques with adjustment for covariates. Lipid concentrations were used as dependent variables, and genotype indicator variables and smoking status as primary independent variables. To test for interaction between cigarette smoking and ESR1 variants, product terms of ESR1 genotypes and smoking status were included in ANCOVA models, together with the main effects of genotypes and smoking status and covariates. The simple main effects of ESR1 genotypes were evaluated by comparing the adjusted mean values between ESR1 genotypes within smokers and within non-smokers. Statistical analyses were performed using SAS software version 8.2 (SAS Institute Inc., Cary, NC, USA). The threshold for significance was set at {alpha}=0.05. To allow comparison with results of the previous study (20Go), haplotype analysis of ESR1 c.454-397T>C and c.454-351A>G was performed for small LDL particle concentration, using HAPLO.STAT software (http://www.mayo.edu/statgen/software).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We are indebted to all those who participated in the Framingham Study and thank Kristen M. Gruenthal and Shanie Coven for genotyping of DNA samples. Support was given by a Specialized Center of Research in Ischaemic Heart Disease from the National Heart, Lung, and Blood Institute (NHLBI, P50 HL63494) to D.E.H. and M.E.M.; an NIH/NHLBI grant (HL54776) and US Department of Agriculture Research Service contracts (53-K06-5-10 and 58-1950-9-001) to J.M.O. and NHLBI funding for the Framingham Heart study (Contract N01-HC-25195). This manuscript has been reviewed by Boston University and the NHLBI for scientific content and consistency of data interpretation with previous Framingham publications.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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