Association of 
Breast cancer is a type of cancer that originates in the cells of the breast . It usually begins as a small confined tumor that can spread to other parts of the body if not treated early . It encompasses various types of carcinomas including ductal and lobular carcinomas. Common symptoms of breast cancer include a lump or mass of the tumor in the breast area, changes in breast size or shape, skin changes (dimpling), nipple changes (inversion or discharge), and persistent breast discomfort . According to Globocan Cancer Data Statistics 2022, breast cancer is the second most diagnosed cancer worldwide, accounting for one in every four cancer cases and one in every six cancer deaths. The registered number of breast cancer statistics in global cancer data for 2022 is around 2,308,897 cases and 656,84 mortalities worldwide . Several risk factors contribute to the development of breast cancer, including environmental factors, hormonal influences, genetic factors, and lifestyle choices (alcohol consumption, obesity, and lack of physical activity) . Genetic mutations play a crucial role in the etiology of breast cancer. Some common genetic mutations associated with breast cancer include BRCA1 , BRCA2 , TP53 , PALB2 , and ATM . Alongside these extensively studied genes, recent studies have associated genetic mutations within ESR1 , HER1 , and HER2 genes in breast cancer . The estrogen receptor1 ( ESR1 ) gene encodes estrogen receptor α (ERα), a key protein expressed in most breast cancers and serves as an important biomarker in tumors . ERα regulates breast cancer risk by affecting estrogen metabolic pathways, and is essential for normal breast epithelial growth and differentiation. Variations in ESR1 , particularly single nucleotide polymorphisms (SNPs), have been studied exclusively to understand their association with breast cancer . The two most studied polymorphisms in the ESR1 gene are rs2234693 and rs2046210 . The rs2234693 polymorphism, located in intron one, is a well-characterized SNP associated with an increased risk of breast cancer and other estrogen-related diseases . This variant may affect breast cancer risk by altering transcription factor-binding sites and affecting alternative splicing of the ESR1 gene, thereby altering gene expression . ESR1 and rs2046210 are located at the 6q25.1 locus on chromosome 6, approximately 29 kb upstream of ESR1 . Its proximity suggests a potential role in modulating ESR1 expression, possibly influencing the proliferation of breast epithelial cells, and contributing to breast tumorigenesis . HER1 , also known as ErbB1 or EGFR, is a transmembrane receptor protein involved in important cellular processes such as cell division, growth promotion, apoptosis inhibition, and angiogenesis stimulation . Activation occurs when ligands, such as transforming growth factor alpha (TGF alpha), bind to its extracellular domain, leading to autophosphorylation of tyrosine kinase residues in the intracellular domain. This activation initiates a cascade of downstream signals, including the MAPK and PI3K pathways, which regulate cell proliferation and survival . SNP rs11543848, also known as HER 1 R497K, involves a single nucleotide substitution (142285G > A) at codon 521 of the extracellular subdomain IV of the EGFR gene, leading to an arginine (R) to lysine (K) change . This genetic variation may affect EGFR-mediated signaling pathways, which may further affect cancer susceptibility and treatment responses, including implications for breast cancer research . HER2 is a proto-oncogene located on chromosome 17q12 that encodes a 185 kDa transmembrane glycoprotein involved in regulating cell growth and differentiation . Unlike other members of this family, HER2 is constitutively active and independent of ligand binding . Dimerization of HER3 or HER4 triggers transphosphorylation of the intracellular tyrosine kinase domain, thereby initiating pathways such as MAPK and PI3K . Overexpression or amplification of HER2 occurs in 20–25% of breast cancers, correlating with aggressive tumor behavior, increased metastasis, and therapy insensitivity . The rs1136201 SNP, known as I655V, causes a valine-to-isoleucine substitution in the transmembrane domain of the HER2 protein . This polymorphism in the transmembrane region of HER2 is associated with a higher risk of breast cancer . This amino acid change promotes enhanced dimerization, tyrosine kinase activity, and autophosphorylation, thereby initiating cell transformation . This study aimed to investigate the association of ESR1 (rs2234693 and rs2046210), HER1 (rs11543848), and HER2 (rs1136201) polymorphisms with breast cancer risk in the Pashtun population. Previous studies have identified genetic markers for this demographic . However, the purpose of this research was to provide strong validation and deep insight through a comprehensive analysis of larger datasets. 

The study was conducted from January to December 2023 at the Institute of Biotechnology and Genetic Engineering (IBGE), University of Agriculture, Peshawar, Pakistan. The study cohort included 528 patients with breast cancer and 530 age- and sex-matched healthy controls. Patients with breast cancer were enrolled at the Institute of Radiology and Nuclear Medicine (IRNUM) Hospital in Peshawar. Ethical approval for the study was obtained from the IBGE and IRNUM Hospital Peshawar. Informed written consent was obtained from all participants after explaining the study objectives. Inclusion Exclusion Criteria and Blood Collection Blood samples were collected from patients with breast cancer based on the inclusion and exclusion criteria. Patients with other significant comorbidities, such as diabetes or other chronic conditions that could potentially affect the study outcomes, were excluded from the study. Additionally, individuals who did not meet the age- and gender-matching criteria with the breast cancer patient group were also excluded from the health control group. Furthermore, demographic and clinicopathological data alongside a 3mL blood sample, were collected from breast cancer patients. DNA Extraction, Genotyping and Gel Electrophoresis DNA extraction was performed using non-enzymatic or salting-out method [ – ]. Genotyping was performed by T-ARMS PCR. Specific sequenced matched primers were designed using the NCBI primer BLAST software (Table ). The inner and outer primers were designed in two sets (reverse and forward each) to detect heterozygous and homozygous dominant and recessive alleles, respectively. The conditions used for the genotyping are presented in Table . The PCR results were confirmed by 1.5% gel electrophoresis. Statistical Analysis The collected data were analyzed statistically using the MedCalc odd ratio calculator and Statistical Package for Social Sciences (SPSS) version 25 to assess the association between polymorphisms and breast cancer risk. Statistical significance was set at P < 0.05. Clinicopathological and genetic variant associations were determined using the chi-squared test. Odds ratios and 95% confidence intervals (CI) were calculated to determine risk factors associated with the selected polymorphisms. 

Blood samples were collected from patients with breast cancer based on the inclusion and exclusion criteria. Patients with other significant comorbidities, such as diabetes or other chronic conditions that could potentially affect the study outcomes, were excluded from the study. Additionally, individuals who did not meet the age- and gender-matching criteria with the breast cancer patient group were also excluded from the health control group. Furthermore, demographic and clinicopathological data alongside a 3mL blood sample, were collected from breast cancer patients. 

DNA extraction was performed using non-enzymatic or salting-out method [ – ]. Genotyping was performed by T-ARMS PCR. Specific sequenced matched primers were designed using the NCBI primer BLAST software (Table ). The inner and outer primers were designed in two sets (reverse and forward each) to detect heterozygous and homozygous dominant and recessive alleles, respectively. The conditions used for the genotyping are presented in Table . The PCR results were confirmed by 1.5% gel electrophoresis. 

The collected data were analyzed statistically using the MedCalc odd ratio calculator and Statistical Package for Social Sciences (SPSS) version 25 to assess the association between polymorphisms and breast cancer risk. Statistical significance was set at P < 0.05. Clinicopathological and genetic variant associations were determined using the chi-squared test. Odds ratios and 95% confidence intervals (CI) were calculated to determine risk factors associated with the selected polymorphisms. 

Association of ESR1 (rs2234693 and rs2046210), HER1 (rs11543848) and HER2 (rs1136201) Polymorphism with Breast Cancer Patients Genotyping was performed for 528 patients with breast cancer and 530 healthy controls using T-ARMS-PCR, followed by gel electrophoresis (Fig. ). Genotyping results were statistically analyzed, which yielded the following results (Table ). The ESR1 (rs2234693) analysis indicated that risk allele C (OR = 1.18,95% CI = 0.967– 1.445, P = 0.102) and both heterozygous TC (OR = 1.23, 95% CI = 0.9597– 1.5986, P = 0.1002) and homozygous CC genotype (OR = 1.26, 95% CI = 0.7355– 2.1645, P = 0.398) indicated a higher risk but non-significant association with breast cancer. The risk allele A (OR = 1.55, 95% CI = 1.3085– 1.8481, P = 0.001) of rs2046210 and both heterozygous GA (OR = 2.33, 95% CI = 1.7423– 3.1239, P = 0.001) and homozygous AA genotypes (OR = 2.62, 95% CI = 1.7703– 3.8989, P = 0.001) were significantly associated with breast cancer. Moreover, both the selected SNPs of HER1 and HER2 were significantly associated with breast cancer patients. The risk allele A (OR = 1.65, 95% CI = 1.3951– 1.9702, P = 0.001) of HER 1 (11543848) and both the heterozygous GA (OR = 3.31, 95% CI = 2.4323– 4.5272, P = 0.001) and homozygous AA (OR = 3.19, 95% CI = 2.1208– 4.8152, P = 0.001) genotypes showed a higher risk and significant association. The analysis for HER2 (rs1136201) indicated that the risk allele G (OR = 1.37, 95% CI = 1.1498– 1.6361, P = 0.004) and both heterozygous AG (OR = 2.00, 95% CI = 1.5208– 2.6411, P = 0.001) and homozygous GG (OR = 1.96, 95% CI = 1.2424– 3.1220, P = 0.003) genotypes were significantly associated with breast cancer. Association of ESR1 (rs2234693 and rs2046210), HER1 (rs11543848) and HER2 (rs1136201) Polymorphisms with Receptor Status of Breast Cancer The association of the selected SNPs with receptor status was statistically determined using SPSS and MedCalc, an odd ratio calculator. The results indicated a significant association between ESR1 (rs2234693) polymorphism and PR Status ( P = 0.001). Similarly, both HER1 (rs11543848) and HER2 (rs1136201) variants are significantly associated with HER2 receptors in patients with breast cancer. Moreover, all selected variants showed a non-significant ( P < 0.05) association with ER and TNBC. 

ESR1 (rs2234693 and rs2046210), HER1 (rs11543848) and HER2 (rs1136201) Polymorphism with Breast Cancer Patients Genotyping was performed for 528 patients with breast cancer and 530 healthy controls using T-ARMS-PCR, followed by gel electrophoresis (Fig. ). Genotyping results were statistically analyzed, which yielded the following results (Table ). The ESR1 (rs2234693) analysis indicated that risk allele C (OR = 1.18,95% CI = 0.967– 1.445, P = 0.102) and both heterozygous TC (OR = 1.23, 95% CI = 0.9597– 1.5986, P = 0.1002) and homozygous CC genotype (OR = 1.26, 95% CI = 0.7355– 2.1645, P = 0.398) indicated a higher risk but non-significant association with breast cancer. The risk allele A (OR = 1.55, 95% CI = 1.3085– 1.8481, P = 0.001) of rs2046210 and both heterozygous GA (OR = 2.33, 95% CI = 1.7423– 3.1239, P = 0.001) and homozygous AA genotypes (OR = 2.62, 95% CI = 1.7703– 3.8989, P = 0.001) were significantly associated with breast cancer. Moreover, both the selected SNPs of HER1 and HER2 were significantly associated with breast cancer patients. The risk allele A (OR = 1.65, 95% CI = 1.3951– 1.9702, P = 0.001) of HER 1 (11543848) and both the heterozygous GA (OR = 3.31, 95% CI = 2.4323– 4.5272, P = 0.001) and homozygous AA (OR = 3.19, 95% CI = 2.1208– 4.8152, P = 0.001) genotypes showed a higher risk and significant association. The analysis for HER2 (rs1136201) indicated that the risk allele G (OR = 1.37, 95% CI = 1.1498– 1.6361, P = 0.004) and both heterozygous AG (OR = 2.00, 95% CI = 1.5208– 2.6411, P = 0.001) and homozygous GG (OR = 1.96, 95% CI = 1.2424– 3.1220, P = 0.003) genotypes were significantly associated with breast cancer. 

ESR1 (rs2234693 and rs2046210), HER1 (rs11543848) and HER2 (rs1136201) Polymorphisms with Receptor Status of Breast Cancer The association of the selected SNPs with receptor status was statistically determined using SPSS and MedCalc, an odd ratio calculator. The results indicated a significant association between ESR1 (rs2234693) polymorphism and PR Status ( P = 0.001). Similarly, both HER1 (rs11543848) and HER2 (rs1136201) variants are significantly associated with HER2 receptors in patients with breast cancer. Moreover, all selected variants showed a non-significant ( P < 0.05) association with ER and TNBC. 

Breast cancer is a complex multifactorial disease with mechanisms that have not been fully explored . Genome-wide association studies (GWAS) have demonstrated that both genetic and environmental factors contribute to breast cancer pathogenesis . Additionally, specific nucleotide polymorphisms within susceptibility genes may play a significant role in breast cancer development . Numerous clinical studies have investigated SNPs within ESR1 , HER1 , and HER2 with contradictory results across several populations [ , , ]. This study aimed to explore the role of ESR1 (rs2234693 and rs2046210), HER1 (rs11543848), and HER2 (rs1136201) polymorphisms in breast cancer risk in the Pashtun population using a larger dataset. The study cohort included 528 patients with BC and 530 healthy controls. The research methodology included DNA extraction using a non-enzymatic method and genotyping using ARMS-PCR. The results were statistically analyzed. The analyzed results indicated a non-significant association between ESR1 (rs2234693) risk allele C ( P = 0.102) and both heterozygous TC ( P = 0.1002) and homozygous CC ( P = 0.398). Additionally, ESR1 (rs2234693) was significantly associated with the PR status in breast cancer. In contrast, ESR1 (rs2046210) risk allele A ( P = 0.001) and both heterozygous GA ( P = 0.001) and homozygous AA ( P = 0.001) were significantly associated with breast cancer risk. Moreover, ESR1 (rs2046210) failed to exhibit a significant association with the receptor status of breast cancer. Our results are consistent with those of a previous study conducted in Mexican  and Chinese  populations, where rs2234693 did not show any association with breast cancer risk. Conversely, a study on an Iranian population showed a significant association between rs2234693 and breast cancer risk . Furthermore, for ESR1 (rs2046210), previous studies in Chinese  and Vietnamese populations  also demonstrated a risk association with breast cancer, supporting our results. Additionally, another study conducted in the Pashtun population also aligns with our results, further confirming our results. However, the results diverged in terms of receptor status, where rs2046210 was significantly associated with TNBC, whereas rs2234693 was significantly associated with ER status . Moreover, both the selected SNPs of HER1 and HER2 were significantly associated with breast cancer risk. For HER1 (11543848), the risk allele A ( P = 0.001), heterozygous GA ( P = 0.001), and homozygous AA ( P = 0.001) genotypes indicated a higher risk and a significant association. For HER2 (rs1136201), the risk allele G ( P = 0.004), heterozygous AG ( P = 0.001), and homozygous GG ( P = 0.003) genotypes were significantly associated with breast cancer. Furthermore, both the selected variants exhibited a significant association with HER2 receptor status. A previous study conducted in the Egyptian population supported our results, where both HER1 (11543848) and HER2 (rs1136201) polymorphisms were significantly associated with breast cancer . In contrast, a study of Tunisian patients found no significant association between HER1 (11543848) and breast cancer risk . Furthermore, the research study conducted in the Pashtun population also supported our results, except for both homozygous mutant genotypes, which indicated a non-significant association with breast cancer risk . 

In conclusion, this study confirmed the role of ESR1 (rs2234693 and rs2046210), HER1 (rs11543848), and HER2 (rs1136201) in breast cancer risk in the Pashtun population using a larger dataset. Despite the utilization of larger datasets, it is necessary to acknowledge certain limitations inherent to this study. This study focused on only a few SNPs within the entire gene, which may not fully capture the genetic variability that could contribute to breast cancer risk. Future studies should further consider whole-gene sequencing to explore other potential SNPs within the same or other associated genes. 

