The estrogen receptor beta is an obvious candidate gene to harbor allelic variants which predispose to breast cancer risk along the sex steroid hormone synthesis, metabolism, and signaling pathway. However, it is not the only candidate along this pathway, and many other genes are currently under study to examine associations between common variants and breast cancer risk. At the present time, no clear consensus in the field has been reached with regards to studying the effect of variants in large numbers of genes simultaneously on disease risk. Therefore, we have chosen to present results from the ESR2 gene independently of other genes.
Given that the global-test for association between ESR2 haplotypes and breast cancer risk was of borderline significance (p=0.04), with only one (CCAC) of the six common haplotypes showing a statistically significant increase in risk (p=0.0007) we used permutation testing as an additional multiple comparisons correction procedure. After correction for multiple comparisons (at the gene level) using permutation testing, the CCAC haplotype remains nominally statistically significantly associated with breast cancer risk (corrected p-value = 0.002), though not at the stringent threshold (10−4) that has been proposed for candidate gene studies.
The low magnitude of risk limits the power to detect interactions with non-genetic risk factors. Nevertheless, we did find some intriguing results upon stratification by age at diagnosis () and estrone levels (). The stratified analyses by age suggest that the CCAC haplotype is a risk factor only in younger women. We have chosen to dichotomize at age 63, as this is the median age at diagnosis across all cohorts, and is similar to the median age at diagnosis in the SEER data (61 years)22
. While breast cancer incidence rates increase dramatically after menopause, they continue to increase well into the seventh decade. In fact, risk factors for breast cancer, particularly body mass index, have been shown to vary in their effect on premenopausal or postmenopausal diagnosis of breast cancer. Therefore, the most likely interpretation of the interaction between the CCAC haplotype and age at diagnosis on breast cancer risk is related to overall lifetime risk, as opposed to risk relative to some specific life event, such as menopause. Among women with lower estrone levels, women carrying the CCAC haplotype had a further reduction in breast cancer risk. This could imply that a variant on this haplotype reduces the ability of cells to respond to estrogen signaling by altering the function of the ESR2
gene. These stratified analyses, particularly with respect to estrone levels where the number of samples available leads to very unstable risk estimates (as evidenced by the very wide confidence intervals) must be interpreted very cautiously however, and further replication is necessary before making definitive conclusions.
Examining the other polymorphisms genotyped in the screen for htSNPs does not yield any a priori candidate causal SNPs (ie non-synonymous or splice site SNPs) on this haplotype. However, a putatively causal polymorphism, either part of the screen or not, could be incompletely tagged by this haplotype, either due to incomplete linkage, different allele frequency, or both. Given that no obviously functional polymorphisms have been described on this haplotype, we can not rule out that the association we observe between the CCAC haplotype and breast cancer risk is due to chance.
The Breast and Prostate Cancer Cohort Consortium (BPC3) was established to overcome the sample size limitation of many studies which examine genetic variants for association with breast and prostate cancer. Given the sample size in this study (5,789 cases, 7,761 controls), we have >90% power with type I error rate of 10−4 to detect a 0.2 frequency allele with per-allele risk of 1.2. As such, the results we present here confidently exclude common variation of ESR2 from being associated with moderate or greater breast cancer risk. However, one less common variant (the CCAC haplotype, 8% of control chromosomes) is found to be associated with a modest increase in breast cancer risk. Even with the large sample size of the current study, roughly 12,000 cases and controls would be needed for 80% power to detect a similar association (per-allele OR 1.17) at type I error rate of 10−4. For this reason, we should be cautious when interpreting the association between the CCAC haplotype and breast cancer risk. Similarly, the population studied here is predominantly post-menopausal Caucasian women, and the htSNPs selected tag haplotypes most efficiently among Caucasians. Therefore, we can not make conclusions about the association between variants of ESR2 and breast cancer risk in other populations, nor should these htSNPs be assumed to tag variants in non-Caucasian populations.
In conclusion, we have performed an exhaustive scan of SNPs in the ESR2 gene, selected htSNPs based on this scan, and evaluated the association between these htSNPs and breast cancer risk. One haplotype of ESR2 is significantly associated with a seventeen percent increase in breast cancer risk per copy of the haplotype carried among Caucasian women.