Estrogen receptor alpha (ESR1) gene polymorphism (rs2234693 and rs2046210) with breast cancer risk in pashtun population of Khyber Pakhtunkhwa

Breast cancer susceptibility is greatly influenced by single nucleotide polymorphisms (SNPs) both in penetrance and non-penetrance genes. The Estrogen Receptor Alfa (ESR1- rs2234693 and rs2046210) have been reported as risk factor of breast cancer in different ethnic groups with inconsistent results. In this study the association of ESR1 (rs2234693 and rs2046210) with breast cancer risk was investigated in patients of Khyber Pakhtunkhwa. A total of 312 females including 162 breast cancer patients and 150 healthy controls were enrolled in this study. The polymorphism was confirmed using T-ARMS-PCR. Our results revealed that ESR1-rs2234693 risk allele (C) (P = 0.21, OR = 1.27, CI = 0.87 to 1.87) and containing genotypes CC (P = 0.68, OR = 1.24, CI = 0.42 to 3.68) and TC (P = 0.23, OR = 1.32, CI = 0.83 to 2.13) were not associated with the risk of breast cancer. In case of rs2046210, the risk allele A (P < 0.0001, OR = 2.42, CI = 1.74 to 3.38) and corresponding genotypes GA (P = 0.0001, OR = 2.55, CI = 1.62 to 4.03) and AA (P = 0.02, OR = 2.20, CI = 1.12 to 4.34) were significantly associated with higher risk of breast cancer. Moreover, ESR1-rs2234693 was significantly (P < 0.05) associated with family history, stages, PR status, ER status and luminal B. The ESR1-rs2046210 showed significant (P ≤ 0.05) association with menstrual status, tumor grade and TNBC. Both the SNPs showed non-significant (P > 0.05) association with nulliparity, nodal status, HER2 status, metastasis, HER2 enriched subtype and luminal A. It is concluded that ESR1-rs2234693 is not associated with breast cancer, while rs2046210 is significantly associated with the risk of breast cancer in Khyber Pakhtunkhwa population. Further, to confirm the exact situation of ESR1 polymorphism, ESR1 existing and other SNPs need to be investigated in diverse data sets.


Introduction
Cancer (abnormal and/or rapid growth of cells) is one of the main causes of mortality worldwide. The deaths recorded by cancer in 2008 was 8 million and is estimated to reach 11 million by 2030 [1]. Among all types of cancers, breast cancer is the most leading cause of death in women globally [2]. Around 2.1 million cases were reported in 2018, and causing fatalities of more than 627,000 in the world. Breast cancer is caused by various factors, the most important are genetic make-up and hereditary, environmental and lifestyle [1]. Currently, early diagnosis, poor prognosis and drug resistance are the major challenges in breast cancer management [3]. Therefore, the attributes to decrease rate of breast cancer incidence and mortality, particularly in low-income countries, risk of genetic predisposition for breast cancer is necessary.
Breast cancer is a complicated molecular disorder in which several biological pathways are involved in cell development and propagation, like the MAPK, RB/E2F, P13K/AKT/mTOR, and TP53 pathways [4,5]. Estrogen receptor (ERα) signaling is known to be required for normal breast epithelial growth and differentiation and also the onset and advancement of estrogen-dependent breast cancer [6]. Numerous mechanisms are known to control ERα expression. Micro-ribonucleic acid (miRNA)-206 and miRNA-221/222 have been shown to negatively regulate ERα at post-transcription level [7]. These abnormalities, alone or in combination, change the role or levels of expression of ERα [8]. ERα silencing has been found in breast tumors, through an alteration inside the estrogen receptor alpha (ESR1) open reading frame and epigenetic alterations like DNA methylation of promoter-proximal CpG Island [9]. However, the ESR1 pathway is thought to be the most significant. At the time of diagnosis, more than two-thirds of breast tumors show estrogen receptor expression [10]. A transcription factor that can bind both estrogen and estrogen response element (ERE) is encoded by ESR1. Through their EREs, estrogen-activated ER dimers initiate transcription of downstream genes. Genetic alterations that modify the ER and its downstream signaling affect susceptibility to breast cancer, tumor progression, and response to treatment [11].
Research studies have been conducted on ESR1 polymorphism and risk of breast cancer in various population which displayed inconsistent results. In Pakistan, no such study has been conducted so far, particularly on association of ESR1 polymorphism (rs2234693 and rs2046210) and risk of breast cancer in the Pashtun population. Therefore, this study aimed to see the association of ESR1 polymorphisms with breast cancer risk in the population of Khyber Pakhtunkhwa, Pakistan.

Study design, ethical approval and sample collection
This case-control study was conducted at Institute of Biotechnology and Genetic Engineering (IBGE; Health Division), The University of Agriculture, Peshawar, Pakistan. Ethical approval was taken from IBGE (No: IBGE/2021/007), and written consent was taken from the patients while explaining the aim of the study. Based on inclusion (only breast cancer patients) and exclusion (patients who have a history of other complications such as diabetes etc.) criteria, a total of 162 breast cancer patients and similar age and gender matched 150 healthy controls data, including demographics, clinical, pathological, and blood samples (3 cc) were collected via venipuncture method from IRNUM hospital using a standard proforma.

Primer designing and DNA extraction
Specific primers were designed using NCBI PRIMER-BLAST with some parameter modification ( Table 1). Two sets of outer and inner primers (forward and reverse) were used to detect homozygous and heterozygous mutations. Extraction of genomic DNA was carried out using nonenzymatic/salting out methods already adopted in our lab [12,13].

Amplified refractory mutation system (ARMS) PCR and gel electrophoresis
Polymorphism in ESR1 was confirmed via ARMS-PCR protocol previously adopted in our lab [13][14][15] and validated elsewhere [16,17]. Briefly, the 25 µL PCR reaction mixture containing 12 µL of Master Mix, 1 µL of each forward and reverse primers, 3 µL of template DNA (~ 100ng/µL), and 8 µL of ddH 2 O was used. The PCR conditions were set at initial denaturation at 95ºC for 5 min, then 38 cycles of denaturation at 95ºC for 30 s, annealing at 58.5ºC for ESR1 (rs2234693), then extension at 72ºC for 30 s and final extension at 72ºC for 7 min. The PCR product was run and confirmed using 1% gel electrophoresis and a 100 bp DNA ladder (Thermo Fishier Scientific).

Statistical analysis
The data was statistically analyzed using SPSS software Version 26, to determine the association of ESR1 polymorphisms with breast cancer risk. Categorical data was represented by numbers and percentages and continuous data was shown as ± standard deviation. T-test was used to determine significance between variables. P-value equal or less than 0.05 was considered significant. The comparison of genotypes was analyzed via Chi-square test. Association was determined by Odd ratio and 95% CI by using the Medcalc Odd ratio Calculator.

ESR1 polymorphism (rs2234693 and rs2046210) association with breast cancer risk in pashtun population
ESR1 polymorphism (rs2234693 and rs2046210) was confirmed in 162 breast cancer patients and 150 healthy controls using ARMS-PCR protocol (Fig. 1 [19]. Other noticeable risk factors of breast cancer are nulliparity, delayed menopause, early menarche and early age at first pregnancy. Approximately 75% of breast cancer are associated with Estrogen receptor α (ER), which are then thus treated with drugs. However, most of the patients are relapsed and develop metastatic disease even after treatment. Multiple molecular mechanisms are involved in the proliferation and resistance of ER + targeted therapies [20]. Genetic mutation in ER could be one of the underlying mechanisms of treatment resistance, patient relapses and advance stages of breast cancer [21]. In the present study frequencies of ESR1 polymorphism (rs2234693 and rs2046210) were analyzed in 162 breast cancer patients and 60 healthy controls of Khyber Pakhtunkhwa population. Our results exhibited that rs2234693 risk allele and respective genotypes, both heterozygous and homozygous were not associated (P > 0.05) with breast cancer risk. However, rs2234693 showed significant association (P < 0.05) with family history, stage, PR status, ER status and luminal B subtype. Similar results were obtained by other studies conducted on Mexican [22] Chinese [23] Japanese and Japanese-Brazilians population [24], which revealed that rs2234693 did not showed significant association with breast cancer susceptibility. In contrast, a study conducted on Iranian population had shown significant association of rs2234693 with high risk of breast cancer [25].
Regarding rs2046210, our data set showed that the risk allele and its respective genotypes both heterozygous and homozygous genotypes displayed significant association (P < 0.05) with breast cancer risk. ESR1 (rs2046210) also

ESR1 polymorphism (rs2234693 and rs2046210) association with clinicopathological parameters of breast cancer
The clinicopathological parameters of breast cancer is necessary for disease management particularly to decide the available treatment options. The association of clinical and pathological parameters were checked with ESR1 polymorphism (rs2234693 and rs2046210) to see the relation of the selected polymorphism. Our data indicated that ESR1 rs2234693 was significantly associated (P > 0.05) with the family history, stages, PR status, ER status and luminal B (Table 3). Similarly, the ESR1 (rs2046210) display significant (P < 0.05) association with menstrual status, tumor grade and TNBC ( Table 3). Both the SNPs (rs2234693 and 2,046,210) showed non-significant (P > 0.05) association with nulliparity, nodal status, HER2 status, metastasis, HER2 enriched subtype and luminal A (Table 3).

Discussion
Breast cancer is the leading cause of death in women worldwide. There are several factors involved in the risk of breast cancer, of which genetic mutation particularly the single nucleotide polymorphism (SNP) is the major risk factor of breast cancer. Breast cancer risk are increases with the mutations in particular genes [18]. About 25% of breast cancer is because of hereditary mutation in highly penetrant genes like BRCA1 and BRCA2, and about 2-3% is because of the rare or moderate penetrant genes like ATM and PALB2 etc. However, genetic predisposition and their suggestive follow up recommendations reduce the risk of breast cancer. In addition to genetic predisposition, age of the patient, Pashtun ethnicity. Therefore, these contradictory results stress that there is an urgent need to understand the exact situation of ESR1 polymorphism in various ethnicities and associated complication in breast cancer. This will definitely revolutionize the breast cancer management with more precise approaches. displayed significant (P < 0.05) association with menstrual status, tumor grade and TNBC. Similar to our results, previous studies conducted on Chinese [26,27] and Vietnamese population [28], rs2046210 has displayed risk association with breast cancer. However, a study reported that rs2046210 is not associated with breast cancer in African and European population when compared with Asian population [29], which facilitate our study goal for focusing on

Conclusion
This study concluded that the ESR1 rs2234693 (P > 0.05) demonstrated non-significant association with breast cancer while rs2046210 exhibited significant association (P > 0.05) with breast cancer risk in Khyber Pakhtunkhwa population. Both ESR1 (rs2234693 and rs2046210) did not display association with nulliparity, nodal status, HER2 status, metastasis, HER2 enriched and luminal A subtype. rs2234693 displayed association with family history, stage, PR status, ER status and luminal B while rs2046210 was associated with menstrual status, grade and TNBC. However, more investigations will be required to investigate the exact situation of ESR1 existing and other associated SNPs in larger data sets.