br The e ects of estrogen in target tissues
The eﬀects of estrogen in target tissues are mediated through es-trogen receptors (ERs) (Anderson, 2002). ERs are members of the nu-clear receptor superfamily proteins that act as ligand-inducible tran-scription factors and modulate the transcription of target genes, and
Abbreviations: BC, breast cancer; BMI, body mass index; bp, AS1517499 pair(s); CI, confidence interval; df, degree of freedom; DNA, deoxyribonucleic acid; ER, estrogen receptor; ERα, estrogen receptor alpha; ESR1, gene encoding estrogen receptor alpha; ESR2, gene encoding estrogen receptor beta; HER2, human epidermal growth factor receptor 2; HWE, Hardy–Weinberg equilibrium; kb, kilobase; kg/m2, kilogram/meter2; M, metastasis; mRNA, messenger ribonucleic acid; N, lymph node; OR, odds ratio; PCR-RFLP, polymerase chain reaction - restriction fragment length polymorphism; PR, progesterone receptor; SNP, single nucleotide polymorphisms; T, tumor
Corresponding author at: Department of Molecular Biology, National Cancer Institute (NCI-UG), University of Gezira, Wad Medani, P.O. Box 20, Sudan. E-mail addresses: [email protected], [email protected] (A. Faggad).
A.T. Gebreslasie, et al.
membrane receptors (Razandi et al., 2004; Ribeiro et al., 1995). There are two main isoforms of estrogen receptors in human designated as estrogen receptor α (ERα) encoded by ESR1 (6q25.1) (Ponglikitmongkol et al., 1988), and estrogen receptor β (ERβ) encoded by ESR2 (14q22-24) (Enmark et al., 1997). The human ESR1 gene comprises eight exons spanning > 140 kb (Ponglikitmongkol et al., 1988). ERα is expressed mainly in classic estrogen target tissues such as uterus, mammary and ovary (Couse and Korach, 1999). The underlying mechanism linking estrogen exposure to development of breast cancer has been proposed to be the binding of estrogen to estrogen receptors in mammary tissue, which consequently stimulates the proliferation and diﬀerentiation of breast cells (Wang et al., 2007). The higher cellular proliferative activity make breast cells more susceptible to be trans-formed by environmental chemical carcinogens (Russo et al., 2000).
Currently, expression of the ER, progesterone receptor (Billings et al., 2012), and human epidermal growth factor receptor 2 (HER2), along with clinicopathologic variables are used to select treatment and predict disease prognosis (Vallejos et al., 2010). ERα is an important biomarker for determining breast cancer outcomes; most anti-breast cancer hormonal therapies act via their binding to the estrogen-binding domain of ERα and blocking the down-stream actions of estrogen. Pa-tients with ERα-positive breast cancer are seven to eight times more likely to benefit from endocrine therapy than ERα-negative patients (Ding et al., 2010).
Although some studies had reported the association of ESR1 SNPs with breast cancer risk, these genetic associations have arisen from research investigating patients of Caucasian and Asian ethnicity. Accordingly, the aim of this study was to investigate the possible as-sociation of two ESR1 SNPs (rs3020314 and rs1514348) with predis-position to breast cancer in Sudanese women, and to test the correlation of the SNP genotypes with breast cancer risk factors and clin-icopathologic features.
2. Materials and methods
2.1. Study design, area and subjects
This case-control study was carried-out at the National Cancer Institute-University of Gezira (NCI-UG), Sudan. An ethical approval was obtained from the Institution Ethics Committee, and all study subjects provided informed consent prior to enrollment into the study. A total of 144 unrelated ethnic Sudanese women were recruited into this study from January-2012 to January-2014, seventy-one of these subjects were patients diagnosed with breast cancer, and seventy-three were healthy community controls with no family history of breast cancer. A struc-tured questionnaire was completed for each participant to obtain de-tailed information on demographic data, reproductive history (age at menarche, age at first live birth, number of pregnancies, parity, age at menopause, hormone use e.g. oral contraceptives or hormonal re-placement therapies); in addition to, personal and family medical his-tory (a previous benign or malignant breast disease, past breast biopsy, family history of breast or related cancer). Anthropometric measure-ments were assessed, and clinicopathological characteristics were re-corded. Gene Reports 15 (2019) 100396
In selecting the ESR1 SNPs, we reviewed findings from GWAS and other independent studies, two SNPs rs3020314 and rs1514348 were selected after scrutinizing the frequency genotypes and frequency data available on the HapMap database http://hapmap.ncbi.nlm.nih.gov/ cgi-perl/gbrowse/hapmap3r2_B36 with a selection criterion of r2 > 0.8 and minor allele frequency (MAF) > 0.2 in Yoruba popula-tion. SNPs rs3020314 (+2464 C/T) and rs1514348 (−4576 A/C) are located in introns 4 and 2 of the ESR1 gene respectively. We used PCR-RFLP for genotyping ESR1 SNPs. Primers were designed based on the SNP flanking sequence (http://genewindow.nci.nih.gov/) and using OLIGO 7 primer analysis software, version: 7.57. Adequate restriction endonucleases for each SNP were selected using the online tool for restriction analysis (http://watcut.uwaterloo.ca/). A nearly perfect proxy SNP rs1643821 was used for the association analysis of rs1514348 with breast cancer risk.