br Fig PCR RFLP analysis of
Fig. 1. PCR-RFLP analysis of the two SNPs: Lane M, 1 kb ladder DNA; Lanes 1–7, DNA samples from study subjects:
SNP Genotype frequencies
Genotype Cases Controls OR (95%CI) p value Allele Cases Controls OR (95%CI) p value
ref., reference; CI, confidence interval; OR, odds ratio; χ2, chi square; df, degree of freedom.
the homozygous (TT) variant Tamoxifen signifying the presence of re-striction sites at the point of the SNP, and three fragments were pro-duced (346, 170 and 107 bp). The homozygous wt genotype (CC) was digested into two fragments (453 and 170 bp), and hence, the hetero-zygous genotype produced four fragments (453, 346, 170 and 107 bp). The results of PCR-RFLP analysis of the two SNPs are shown in Fig. 1.
3.3. Single-marker analysis
Hardy–Weinberg equilibrium (HWE) for the distribution of each SNP was evaluated using Haploview 4.2. There was a slight deviation from expected HWE (HWp = 0.001) in the genotype frequency of rs3020314 in the control group, and in the genotype frequency of SNP-rs1643821 in breast cancer patients (HWp = 0.003).
We analyzed established risk factors of breast cancer, such as body mass index (BMI), family history of cancer, and reproductive variables with respect to the genotype frequencies of the two SNPs. There were significant associations in the subgroups with increased BMI and post-menopausal state. For rs3020314, a significantly increased risk of breast cancer in patients with the heterozygous genotype CT in the subgroup of patients having BMI ≥25 kg/m2 (OR: 7.14, CI: 2.18–23.45; p = 0.001). Similarly, in the postmenopausal patient subgroup, in-dividuals with the heterozygous genotype CT were found to have 6-times increased risk of breast cancer (OR: 5.95, CI: 1.22–28.96; p = 0.021) as presented in Table 3. Regarding the SNP rs1643821, the heterozygous genotype CT had a decreased risk of breast cancer with a trend towards significance (OR: 0.41, CI: 0.15–1.13; p = 0.086) as shown in Table 3. Testing association of the two SNPs with other risk factors such as age at menarche and parity, revealed no statistically significant associations. However, increased risks of breast cancer were observed in the homozygous variant TT and heterozygous genotype CT for rs3020314; whereas reduced risk of breast cancer was seen in the subgroup with heterozygous genotype CT of rs1643821.
Furthermore, despite the small sample size, we explored stratifica-tion of the patient group by established factors such as expression of hormone receptors (ER and PR) and clinicopathologic characteristics (tumor size, lymph node involvement, tumor grade). Investigating the
association between subgroups of these parameters and the genotype frequencies of the two SNPs, no statistically significant diﬀerence was found between the distribution of genotype frequencies of the two SNPs and the clinicopathologic features. Patients with the TT variant were more likely to have a T3 and T4 tumor size, compared to the CC or CT variant in the rs1643821 polymorphism. The same was true for rs3020314 in patients with estrogen receptor negative cancers, the TT variant had higher odds ratio than the wild type CC. It should be mentioned that, for tumor histology, the genotype frequencies were not evenly distributed within the histologic types, for instance there were only four patients with ductal carcinoma, whereas the majority of the cases (63) had lobular carcinoma, the most common morphology, therefore, this might obscure the result.
3.4. Haplotype analysis
Haplotype blocks for SNPs rs3020314 and rs164382 of the ESR1 are shown in Table 4. The pairwise haplotype associations for the two SNPs were estimated using the expectation-maximization algorithm by Plink software. The two SNPs are separated by 87.1 kb, no significant dif-ference was observed between each haplotype frequency in cases and controls. In addition, the haplotypes for the combined cases and con-trols groups were not significantly diﬀerent compared to the reference haplotype (TT) (chi-square = 0.93, df = 3, p = 0.82).The D′ and r2 were calculated for the pairs of SNPs using Plink and Haploview soft-wares, the LD was weak (D′ = 0.11, R-sq = 0.008).
This study aimed to assess the association of two ESR1 poly-morphisms (rs3020314 and rs1514348) with breast cancer suscept-ibility, risk factors, pathological and clinical characteristics. The es-trogen receptor-α, encoded by ESR1 gene, is an important mediator of the hormonal response in estrogen-sensitive tissues, so the genetic polymorphisms on the ESR1 that could alter the ESRα and its down-stream signaling are expected to aﬀect predisposition to breast cancer, tumor progression and response to treatment (Dunning et al., 2009).