Reference
Complete the bibliographic reference for the article according to AJE format.

Shearman AM, Cupples LA, Demissie S, Peter I, Schmid CH, Karas RH, Mendelsohn ME, Housman DE , Levy D. Association between estrogen receptor alpha gene variation and cardiovascular disease. JAMA 2003;290(17):2263-70.

Category of HuGE information
Specify the types of information (from the list below) available in the article:

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

1. Prevalence of gene variant
2. Gene-disease association

Study hypotheses or purpose
The authors study hypotheses or main purpose for conducting the study.

Gene(s)
Identification of the following:

1. Gene name
2. Chromosome location
3. Gene product/function
4. Alleles
5. OMIM #

1. Gene name: ESR1, Estrogen receptor alpha
2. Chromosome location: 6q25.1
3. Gene product/function: ESR1is a ligand-activated transcription factor with multiple promoters encoded in its sequence (1). It is known to mediate the effects of estrogen on blood vessel walls including accelerated re-endothelialization, altered endothelial nitric oxide production, and inhibited vascular injury response (2). ESR1 binds phosphatidylinositol-3-hydroxy kinase (PI3K) in a ligand-dependent manner. Estrogen stimulation increases ESR1-associated PI3K activity, leading to the activation of protein kinase B/AKT and endothelial nitric oxide synthase (eNOS). Mice treated with estrogen showed increased eNOS activity and decreased vascular leukocyte accumulation after ischemia and reperfusion injury. This vascular protective effect of estrogen was abolished in the presence of PI3K or eNOS inhibitors. Interestingly, ESR1 knockout mice are obese and exhibit hyperplastic and hypertrophied adipocytes, insulin resistance and glucose intolerance. ESR1 is found in the eye, bone, ovary, and the uterus (1). In addition to cardiovascular tissue, its targets also include the uterus, breast, pituitary, bone, and central nervous system (3). In humans, ESR1 variants have been associated with gynecologic (4,5), bone density (6,7), autoimmune diseases (8-10), Alzheimer’s disease (11,12), and coronary artery disease (13,14).
4. Alleles: At least 19 somatic mutations are known including basepair insertions, transitions, deletions, and alternative splice variants (1). At least 14 polymorphisms (15) have been reported including the 4 SNPs examined in this paper: c. 454-397T>C, c.30T>C, c.454-351A>G and c.975C>G. The c. 454-397C allele forms part of a potential binding site for the myb family of transcription factors, whose expression is activated by estrogen (2).
5. OMIM #: 133430

Environmental factor(s)
Identification of the major environmental factors studied (infectious, chemical, physical, nutritional, and behavioral)

N/A

Health outcome(s)
Identification of the major health outcome(s) studied

1. Cardiovascular disease

1. Case-control
2. Cohort 
3. Cross-sectional
4. Descriptive or case series
5. Clinical trial
6. Population screening

1. Disease case definition
2. Exclusion criteria
3. Gender
4. Race/ethnicity
5. Age
6. Time period
7. Geographic location
8. Number of participants

Please see cohort definition below.

1. Disease case definition: CVD diagnosed based on consistent clinical symptoms, electrocardiographic changes, diagnostic elevation of biomarkers, and supportive imaging studies for strokes. A panel of 3 physicians reviewed available medical records according to standardized Framingham Heart Study criteria. 3 major outcomes: 1. total CVD [recognized and unrecognized myocardial infarction (MI), angina pectoris, coronary insufficiency, intermittent claudication, coronary heart disease death, or atherothrombotic stroke]; 2. major CVD [acute MI, coronary insufficiency, coronary heart disease death, or atherothrombotic stroke]; 3. recognized acute MI
2. Exclusion criteria: Unavailable blood samples during the 1995-1998 examination cycle (6 th), related to other participants, absence of data available for ESR1 c. 454-397T>C polymorphism.
3. Gender: 1. total CVD group: 122 M vs. 56 F, 2. major CVD group: 69 M vs. 14 F, 3. recognized acute MI group: 54 M vs. 5 F
4. Race/ethnicity: Not specified, but population is presumably predominantly Caucasian because of its source.
5. Age: mean 60 years
6. Time period: 1971-1998 at 4-year intervals
7. Geographic location: Framingham , MA
8. Number of participants: 178 (max: total CVD group)

1. Control selection criteria
2. Matching variables
3. Exclusion criteria
4. Gender
5. Race/ethnicity
6. Age
7. Time period
8. Geographic location
9. Number of participants

1. Control selection criteria: Both cases and controls came from the same cohort and differ only by the presence of CVD
2. Matching variables: frequency matched by gender
3. Exclusion criteria: Unavailable blood samples during the 1995-1998 examination cycle (6 th), related to other participants, absence of ESR1 c. 454-397T>C polymorphism.
4. Gender: about matched for M and F (753 vs. 808)
5. Race/ethnicity: Not specified, but population is presumably predominantly Caucasian because of its source.
6. Age: mean 60 years
7. Time period: 1971-1998
8. Geographic location: Framingham , MA
9. Number of participants: 1561

Cohort definition
For study designs 2, 3, and 6, define the following if available:

1. Cohort selection criteria
2. Exclusion criteria
3. Gender
4. Race/ethnicity
5. Age
6. Time period
7. Geographic location
8. Number of participants

1. Cohort selection criteria: Offspring and corresponding spouses of original Framingham Heart Cohort participants
2. Exclusion criteria:
3. Gender: M and F
4. Race/ethnicity: Caucasian
5. Age: mean age at baseline (1971): 36 years
6. Time period: 1971-1998
7. Geographic location: Framingham , MA
8. Number of participants: 5124 recruited in 1971 (this study’s participants were those available for genotyping 27 years later)

1. Environmental factor
2. Exposure assessment
3. Exposure definition
4. Number of participants with exposure data (% of total eligible)

No data regarding gene-environment interaction was provided, precluding analysis. However, information regarding exposure to alcohol and smoking obtained in the time interval from 1971-1998 was provided according to genotype.

1. Alcohol consumption (mean) in oz/week by self report
   1. (3.6) for 4.2% of those with TT genotype (n=525)
   2. (3.5) for 4.4 % of those with CT genotype (n=862)
   3. (3.4) for 4.1% of those with CC genotype (n=352)
2. Smoking either currently smoking cigarettes or quitting within 1 year prior to the clinic visit by self report
   1. 333 or 63% of those with TT genotype
   2. 516 or 60% of those with CT genotype
   3. 225 or 64% of those with CC genotype

1. Gene
2. DNA source
3. Methodology
4. Number of participants genotyped (% of total eligible) 

1. Gene: ESR1
   The 4 SNPs examined were: c. 454-397T>C, c.30T>C, c.454-351A>G and c.975C>G. The c. 454-397C allele forms part of a potential binding site for the myb family of transcription factors, whose expression is activated by estrogen.
2. DNA source: peripheral blood
3. Methodology: Restriction fragment length analysis and polymerase chain reaction amplification with allele calling by 2 independent investigators
4. Number of participants genotyped: 1739 analyzed out of 1811 participant DNA samples

1. Prevalence of gene variant
2. Gene-disease association
3. Gene-environment interaction
4. Gene-gene interaction
5. Genetic test evaluation/monitoring

1. Prevalence of gene variants (attached as Tables 1-5)
   For c. 454-397T>C , the genotype frequencies in this study comprised of 30% for TT (others: 19-36%), 50% for CT (others: 49-53%), and 20% for CC (others: 15-28%) are comparable to those reported by others for Caucasian and Japanese subjects. For c.454-351A>G, the genotype frequencies in this study were similar to those reported for other Caucasian groups (40% for AA, 48% for AG, 16% for GG) but differed from those reported for a Japanese cohort (67% for AA, 25% for AG, and 5% for GG). For c.30T>C and c.975C>G, this study’s genotype frequencies were similar to those reported in another Caucasian cohort. For c.30T>C, genotype frequencies for TT were 40% for TT, 48% for CT, and 16% for CC while for c.975C>G, they were 58% for CC, 35% for CG, and 5% for GG in the latter.
   
   
2. Gene-disease association (attached as Tables 6-7)
   CVD risk was associated with the c. 454-397 CC genotype in the acute MI group with OR=2.1 (1.1-4.0). Attributable fraction for the acute MI group due to this specific genotype was 0.2. Absolute risk for acute MI due to this genotype could not be calculated because no data was provided regarding the cumulative incidence of MI in this study cohort.

• Table 1
• Table 2
• Table 3
• Table 4
• Table 5
• Table 6
• Table 7

The authors assert that their study shows an association between the ESR1 c.454-397CC genotype and increased odds of myocardial infarction which persists after adjustment for traditional CVD risk factors. They caution that while their results support the importance of estrogen receptors in CVD susceptibility for men, further studies are needed to determine if these results can be generalized to other genetically distinct populations and to women. Larger studies would also be needed to assess gene-gene and gene-environment interactions.

1. There may be population selection bias since participants were selected 27 years after initial enrollment and had to survive this time period and be able to give a blood sample.
2. Since the number of MI cases was small (n=59), it would be important to see if this association can be replicated in further studies. In addition, since there were only 5 women with MI, additional studies with larger numbers of women with MI will be helpful. Since estrogen is thought to protect women from CVD, it would be interesting to evaluate this potential association in post-menopausal women on and off hormone replacement therapy. Finally, as asserted by the authors, studies in other ethnic populations are also needed.
3. Strengths of this study included: large population-based sample size, well-characterized participants with consistent and fairly complete ascertainment, small p-value, and biologic plausibility.
4. Although the authors and I report similar crude OR ratios for association of the ESR1 c.454-397T>C genotype with both the total CVD and acute MI groups listed in Table 4, we have differing values for the crude OR ratios reported for both the CT and CC genotypes associated with the major CVD group. They report 0.9 (0.5-1.5) for the CT genotype and 1.7 (1.0-2.8) for the CC genotype while I calculated 0.4 (0.13-1.2) and 1.2 (0.39- 3.7).
5. Based on this study, the attributable fraction of MI due to the ESR1 c.454-397CC genotype is 20%. Because of the significant morbidity and mortality associated with CVD, this finding is interesting. However, current prevention and medical management such as smoking cessation; treatment of hypertension and hyperlipidemia through diet, exercise, and medications; and management of diabetes which are known to exert significant reduction in risk of cardiovascular events for everyone, regardless of ethnicity and gender, would still apply to individuals with the CC genotype. Moreover, with smoking cessation and good control of diabetes during pregnancy, adverse fetal conditions which some investigators hypothesize may predispose the fetus for development of cardiovascular disease and diabetes in adulthood are diminished (Barker Hypothesis). Thus, there is currently no public health justification for population-based screening. Interestingly, the authors have filed a provisional patent application for medical uses related to the ESR1c.454-397T>C polymorphism after this article went to press.

References

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