Link between the ESR2 (rs3020449) polymorphism and breast cancer risk in Bangladeshi women

Abstract

Worldwide, breast carcinoma represents the single most widespread disease among women as well as the second most prevalent malignancy overall. This condition is heterogeneous, comprising multiple distinguished subgroups characterized by unique gene transcription variations and varying mortality rates. Multiple diverse studies suggest that estrogen receptor gene polymorphisms might impact BC susceptibility and formation, which may alter receptor function, tumor expansion, and sensitivity to hormonal therapies. These genes regulate estrogen signaling pathways, affecting breast cancer (BC) formation. The linkage of various polymorphisms of ESR2 gene on BC development is not comprehended. Hence, our objective was to scrutinize the correlation among the ESR2 (rs3020449) polymorphism and BC risk in Bangladeshi women. A case-control genetic association study was undertaken on 209 clinically diagnosed BC female patients against 201 healthy women of Bangladeshi ethnicity using the amplification refractory mutation system polymerase chain reaction (ARMS-PCR). We evaluated p-value, odds ratio (OR) and 95% confidence interval (95% CI) for comprehending the level of risk association of rs3020449. This SNP showed reduced risk in AG vs. AA (heterozygous/additive) model where, OR = 0.58, 95% CI = 0.38 to 0.90, p = 0.015. Moreover, rs3020449 indicated a lower tumor aggressiveness in tumor grade (II vs. I) with the statistics of OR = 0.3138, 95% CI = 0.10 to 0.96, p = 0.043 and in tumor staging, both stage-II (OR = 0.37, 95% CI = 0.15 to 0.90, p = 0.028) and stage-IV (OR = 0.16, 95% CI = 0.05 to 0.53, p = 0.002) against Stage-I. The findings of the research suggested that ESR2 (rs3020449) polymorphism may decrease the risk of BC formation and progression in Bangladeshi women.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-12843-5.

Introduction

Cancer is frequently defined as a condition of malignancy that is characterized by the unregulated and uncontrolled proliferation of altered cells which are prone to natural selection-driven evolution¹. Globally, over 19.96 million new cases of cancer and 9.74 million deaths attributable to cancer were projected in 2022. Considering the anticipated shifts in demographic trends and the aging population, and presuming that cancer incidence rates stay stable, projections indicate that the number will exceed 35 million additional cases of cancer by the year 2050, representing a 77% rise from the 20 million cases projected for 2022^2–4. BC consists of a diverse range of tumors that show significant differences in clinical appearance, morphological and cellular characteristics, physiological behavior, and treatment response. Conversely, benign lesions usually appear with a lucent center and exhibit a more consistent shape, in contrast to the erratic characteristics of malignant lesions^5,6. Accordingly, the American Cancer Society states that yearly, around 175 thousand women and over 1,300 men get newly diagnosed with BC, leading to the deaths of 43,300 women and 400 men. BC is a hundred times more prevalent in women in comparison to men^5–8.

Tumor differentiation, comprising tubule growth, nuclear pleomorphism, and cellular activity, is reflected in grade, which indicates the invasiveness of the tumor⁹. The predominant forms include invasive ductal and invasive lobular BC. Collectively, they represent more than 70% of the total incidents¹⁰. Among the most widespread forms of cancer is invasive lobular carcinoma. Nearly 90% of in-situ carcinomas that do not spread to other parts of the body typically are ductal, whereas 10% are lobular¹¹. Inflammatory and metaplastic BC are less common but deadly forms of BC. Other rare types are Paget disease, tubular, papillary, mucinous, and Phyllodes tumors^9,11. Based on genetic investigations, there are five biological categories of BC: luminal A, luminal B, basal-like (triple-negative), HER2-positive non-luminal, and special histological classification¹². Excessive body mass index (BMI), previous chest radiation, delayed menopause, postmenopausal hormone treatment, early menstruation, and a genetic background of cancer are additional concerns. Individuals with a greater risk include women on hormone treatment who fall under a healthy BMI range but have heavy breasts^11,13. Nearly 30% of BC occurrences include evidence of a detectable tumor in the region of the breast. When a condition progresses, ulcers develop. Fibroadenomas, cysts, and papillomas are examples of benign causes. Ultrasound and screening mammography are necessary for any noticeable lump^11,14. When no tumor is seen in the axillary lymph nodes, sentinel lymph node biopsy is chosen instead of lumpectomy or bilateral mastectomy as a surgical alternative. Chemotherapy, hormonal inhibitors, and monoclonal antibodies targeting HER2 are a part of the adjuvant treatment. To mitigate adverse effects, patients should adhere to particular hygiene protocols throughout radiation therapy^11,15–19.

Genetic polymorphism refers to the inheritance of an attribute determined by a single genetic region with two distinct alleles, where a less prevalent allele possesses an incidence of approximately 1% or higher^20,21. Single nucleotide polymorphisms (SNPs), copy number variations (CNVs), and structural variations (SVs) represent types of variation in genes that may possess substantial consequences on the standard of life²². SNPs and mutations are essential genetic variations that may affect disease progression. SNPs represent alterations of the sequence at a single position of the nucleotide in the DNA and are prevalent across both coding and noncoding regions of the genome²³. Human ERβ gene (ESR2) has been identified on chromosomes 14q23.2 and spans approximately 61.2 kb. An ERβ protein is generated through eight exons comprising 530 amino acids and having a predicted molecular dimension of 59.2 kDa, the complete human ERβ protein may refer to ERβ1²⁴²⁵,. Estrogen receptor beta (ERβ), encoded by ESR2, has been implicated in breast cancer as a potential tumor suppressor. Functional SNPs in the ESR2 promoter region such as rs2987983, rs3020450, and rs3020449 could influence transcription factor binding and gene expression, thereby affecting ERβ-mediated regulation of cell proliferation and apoptosis²⁶. The investigation of estrogen’s effects on BC and other targeted tissues has been prompted by the discovery of the second estrogen receptor gene ESR2, and its byproduct Erβ^27,28. ESR2 polymorphisms such as rs 3,020,449, rs 3,020,450, rs 2,987,983, rs 1,271,572, rs1887994, and rs 4,986,938 are associated with a distinctive likelihood of developing BC levels²⁹. SNP investigated in this study is rs3020449, a single nucleotide polymorphism located in the promoter region of the ESR2 gene, which encodes estrogen receptor beta (ERβ). This receptor plays a crucial role in estrogen signaling pathways involved in various physiological processes, including neurodevelopment and immune function. Variants may influence gene transcription and have been implicated in modulating susceptibility to several hormone-related disorders³⁰. Previous studies in European cohorts have shown that promoter SNPs like rs2987983 are associated with altered breast cancer risk. However, data from South Asian populations, especially Bangladeshi women, are lacking. Investigating these variants in our cohort offers critical insight into genetic risk factors in this underrepresented group²⁶. We, therefore, aimed to identify the the association of ESR2 (rs3020449) polymorphism in BC formation and progression in Bangladeshi ethnic population for the very first time.

Methods

Ethical clearance

The study design and the protocol were reviewed and approved by the research ethical committee of the University of Asia Pacific (Ref: UAP/REC/2024/204) 74/A Green Road, Dhaka-1205, Bangladesh. Prior to the analysis, signed consent was obtained from each enlisted BC patient along with healthy subjects after they were informed of the goals of the project along with the experimental methods. Subjects who refused to participate in the research (either as cases or controls) and were unwilling to provide information were excluded from this investigation.

Research population

The research involved 209 female BC patients coming from various regions of Bangladesh, who were confirmed by histopathology and were hereditarily unrelated. The subject matter was subsequently chosen from the National Institute of Cancer Research and Hospital (NICRH), Mohakhali, Dhaka, Bangladesh, from mid to late 2024. Following a comprehensive individual discussion and diagnostic technique, every medical record of the patient, including tumor stage, histological type, tumor grade, and lymph node characteristics, alongside the physical attributes (age and BMI), socio-demographic profile (region, number of children, profession, marital status etc.) were obtained by a qualified nurse under the supervision of a physician. 201 Bengali ethnic women without genetic history were recruited from various parts of Bangladesh as control population, matched by age with the cases and the demographic properties were recorded in structured questionnaires. The investigation was conducted per the Helsinki Declaration and its subsequent amendments³¹. The research study was conducted in the Biotechnology Laboratory of the Pharmacy Department, University of Asia Pacific.

Genotyping

Genomic DNA was extracted utilizing DNA extraction mini kit (Favorgen, Taiwan) according to the protocol from the whole blood samples which were collected from all the recruited cases and healthy volunteers (approximately 5 ml peripheral blood were taken into sterile EDTA-Na₂ vacutainer tubes by venipuncture). Amplification of the DNA was carried out employing the predesigned set of forward and reverse primers and standardized PCR conditions (Table 1). For this experiment of ESR2 (rs3020449) polymorphism, we used ARMS-PCR method. DNA fragments were confirmed and visualized in ethidium bromide blemished 1.7% agarose gel electrophoresis (Fig. 1).

Table 1.

SNP, primers, PCR condition and expected fragment size for ARMS-PCR.

SNP	Primers	PCR Condition	Expected Fragment Size (bp)
ESR2 (rs3020449)	FI: 5’-GCATTGTCCTTTTTACATATTGTTAGGGTA-3’ RI: 5’-AATTCTCAAGGAAATTTTAGCAAAGCC-3’ FO: 5’-ATTAGGAACGGATGTTAGATTTTGTCAA-3’ RO: 5’-CCAAATGATTAAGGAGAAATAACAGCAG-3’	95º C 15 min 95º C 20 s 56º C 50 s 72º C 1 min 72º C 5 min 95º C 15 min	AA: 231, 382 AG: 208, 231, 382 GG: 208, 382

Fig. 1.

Fragments of ESR2 (rs3020449) polymorphism (Lane-1 contained 100 bp DNA ladder and rest of the lanes represented the ARMS-PCR product. The expected fragments were- A/A: 231 bp, 382 bp; A/G: 208 bp, 231 bp, 382 bp; G/G: 208 bp, 382 bp).

Statistical analysis

Statistical analysis was carried out with the SPSS software package 17.0 (SPSS Inc., Chicago, IL). To understand the risk association of each variant, the odds ratio (OR) and 95% confidence interval (95% CI) were assessed using MedCalc (version 23.1.6) software. Hardy–Weinberg equilibrium (HWE) was tested with a goodness of fit chi-square test (χ²) to compare the observed genotype frequencies among the subjects. To addressed multiple variables testing corrections we used Bonferroni correlation. For the sample size determination, we used sample size calculator (version 2025) online software.

Results

Socio-demographic distribution of the patients and the healthy volunteers

In this case-control study, 209 BC female patients and 201 healthy female volunteers were chosen. The clinical, physical and demographical information is listed in Table 2.

Table 2.

Demographic and clinicopathological data of BC patients and healthy controls.

Variables	Case (%) N = 209	Control (%) N = 201	p-value
Age (years)
< 45	81 (38.8%)	116 (57.7%)	< 0.0001
≥ 45	128 (61.2%)	85 (42.3%)
Region
Rural	170 (81.3%)	41 (20.4%	< 0.0001
Urban	39 (18.7%)	160 (79.6%)
BMI
Underweight	7 (3.3%)	27 (13.4%)	< 0.0001
Healthy weight	87 (41.6%)	110 (54.7%)
Overweight	69 (33.0%)	53 (26.4%)
Obese (class- I, II, III)	46 (22%)	11 (5.5%)
Marital status
Married	208 (99.5%)	155 (77.1%)	< 0.0001
Unmarried	1 (0.5%)	46 (22.9%)
Occupation
Housewife	200 (95.7%)	64 (31.8%)	< 0.0001
Student	1 (0.5%)	41 (20.4%)
Working	8 (3.8%)	96 (47.8%)
Number of children
0	9 (4.3%)	69 (34.3%)	< 0.0001
1	15 (7.2%)	35 (17.4%)
2	75 (35.9%)	46 (22.9%)
More than 2	110 (52.6%)	51 (25.4%)
Breastfeeding status
No	9 (4.3%)	76 (37.8%)	< 0.0001
Yes	200 (95.7%)	125 (62.2%)
Taking contraceptive pill
No	78 (37.3%)	105 (52.2%)	0.005
Yes	131 (62.7%)	96 (47.8%)
Menstrual cycle stopped
No	41 (19.6%)	164 (81.6%)	< 0.0001
Yes	168 (80.4%)	37 (18.4%)
Genetic history of cancer (Family members)
No	162 (77.5%)	174 (86.6%)	0.037
Yes	47 (22.5%)	27 (13.4%)

p < 0.05 was considered statistically significant; p < 0.01 highly significant; p < 0.001 very highly significant (indicated with bold).

Correlation of rs3020449 SNP in breast cancer

The association between the ESR2 (rs3020449) and the formation of BC was assessed in this study (Table 3). From Table 3, it was evident that rs3020449 is significantly associated (p < 0.05) with lowering the risk of BC development in the heterozygous model against the normal homozygous model, AG vs. AA (additive model). In this particular model, OR = 0.58, 95% CI = 0.38 to 0.90, p = 0.015. However other genotype models, GG vs. AA (OR = 1.36, 95% CI = 0.75 to 2.50, p = 0.313), AG + GG vs. AA (OR = 0.75, 95% CI = 0.51 to 1.11, p = 0.147), G vs. A (OR = 0.97, 95% CI = 0.72 to 1.31, p = 0.834) revealed no association for BC formation. The genotype distributions across both groups conform to Hardy-Weinberg Equilibrium (cases: χ2 = 29.382 and p = 0.505; controls: χ2 = 1.083 and p = 0.298).

Table 3.

Statistical presentation of genotypes of ESR2 (rs3020449) polymorphism and their comparative role in breast cancer risk.

Polymorphism	Genotype	BC Cases (N = 209) (%)	Controls (N = 201) (%)	OR (95% CI)	p-value
ESR2 (rs3020449)	AA	121 (57.89%)	102 (50.75%)	1
	AG	54 (25.83%)	78 (38.81%)	0.58 (0.38–0.90)	0.015
	GG	34 (16.27%)	21 (10.45%)	1.36 (0.75–2.50)	0.313
	AG + GG	88 (42.11%)	99 (49.25%)	0.75 (0.51–1.11)	0.147
	A	296 (70.81%)	282 (70.15%)	1
	G	122 (29.19%)	120 (29.85%)	0.97 (0.72–1.31)	0.834

p < 0.05 was considered statistically significant (indicated with bold).

Effect of genetic variations regarding the clinicopathological parameters of the BC patients

Multiple clinicopathological factors, for instance, age, tumor grade, nodal status, cancer histology, stages in the patients were evaluated by comparing the data of the rs3020449 variations carrier (HE + MH) and non-carrier (NH) in Table 4.

Table 4.

Correlation of ESR2 (rs3020449) polymorphism with clinicopathological properties of the cases.

Category	Cases (N = 209)	ESR 2 (rs3020449)
		HE + MH	NH	OR (95% CI)	p-value
Age (years)
< 45	81	32	49	Ref.
≥ 45	128	54	74	1.12 (0.6338-1.97)	0.701
Grade
I	15	10	5	Ref.
II	153	59	94	0.3138 (0.10–0.96)	0.043
III	41	17	24	0.35 (0.1025-1.22)	0.101
Nodal status
Negative	91	41	50	Ref.
Positive	118	45	73	0.75 (0.43–1.31)	0.314
Stages
I	27	17	10	Ref.
II	93	36	57	0.37 (0.15–0.90)	0.028
III	61	27	34	0.47 (0.18–1.18)	0.109
IV	28	6	22	0.16 (0.05–0.53)	0.002
Cancer histology
Invasive ductal carcinoma	99	44	55	Ref.
Ductal carcinoma in-situ	47	13	34	0.48 (0.23–1.01)	0.054
Invasive lobular carcinoma	13	8	5	2.00 (0.61–6.55)	0.252
Infiltrating ductal carcinoma	26	8	18	0.56 (0.22–1.40)	0.212
Metastatic ductal carcinoma	8	4	4	1.25 (0.30–5.29)	0.762
Others	16	9	7	1.61 (0.55- 4.66)	0.382

p < 0.05 was considered statistically significant (indicated with bold). HE = Heterozygote; MH = Mutant homozygote; NH = Normal homozygote.

A statistically significant correlation was found for the clinicopathological attributes of the tumor stage and grade Table 4. Variant carriers of rs3020449 showed decreased association (p < 0.05) in tumor grade (II) against the tumor grade (I) where OR = 0.3138, 95% CI = 0.10 to 0.96 and p = 0.043. In case of tumor staging, both stage-II (OR = 0.37, 95% CI = 0.15 to 0.90, p = 0.028) and stage-IV (OR = 0.16, 95% CI = 0.05 to 0.53, p = 0.002) revealed lower risk association comparing to stage-I for HE + MH carriers. Regarding the remaining clinicopathological features, no statistically important association was noticed for any genotypes Table 4. In the present study correction for multiple comparisons, Bonferroni correlation was conducted and found adjusted α: 0.02500 (for Table 3) and adjusted α: 0.01000 (for Table 4), which reflects the present study affirmative.

Discussion

The variation in genetics serves as a significant risk associated with cancer³². Single nucleotide polymorphism (SNP) represents among the most common variability found within a genome. A genome-wide association study uncovered a variety of susceptibility regions for BC within genes associated with tumors³³. Several correlational studies have been conducted to explore the association among polymorphic locations in the ESR2 gene and the risk of developing BC; nevertheless, the findings remain inconsistent³⁴. The SNP rs3020449 is found within the promoter domain of the ESR2 gene³⁵. The rs3020449 SNP is located in the promoter region of the ESR2 gene, a non-coding regulatory region that does not result in an amino acid change. However, this variant may affect ESR2 transcriptional activity by altering transcription factor binding sites, thereby influencing estrogen receptor beta expression. Previous studies have associated rs3020449 with breast cancer risk and other estrogen-related conditions, supporting its potential functional relevance through regulatory mechanisms^30,36. According to the analysis, decreased risk was found regarding BC progression suggesting a protective effect in heterozygous model. A comparative overview of the previous studies and the current study of rs3020449 in Table 5 is presented^26,37,38.

Table 5.

Comparison study of ESR2 (rs3020449) polymorphism between the previous studies and the current study.

Author	Year of Publication	Number of Cases	Number of Controls	Association Status	Population
Treeck et al.	2009	318	Undefined	Association (X)	Caucasian women
Chen et al.	2013	645	700	Association (X)	Southwestern Chinese women
Dai et al.	2019	459	549	Association (↑)	Chinese Han women
Current study		209	201	Association (↓)	Bangladeshi women
(X) No association; (↑) Increased risk; (↓) Decreased risk.

The research study of rs3020449 was carried out on Caucasian women for the very first time involving 318 BC patients alongside various controls from comparable age cohorts. The study indicated no association between BC progression and Caucasian women²⁶. ESR2 (rs3020449) had also been studied for BC risk assessment in western Chinese population with 645 cases and 700 controls in which no statistically significant correlation was identified either for the particular SNP³⁷.

Promoter polymorphisms like rs3020449 may impact gene expression by modifying transcription factor binding affinity, altering epigenetic marks, or affecting chromatin structure. In the case of ESR2, reduced expression of estrogen receptor beta (ERβ) has been associated with enhanced tumor proliferation, decreased apoptosis, and greater invasiveness in breast and other hormone-related cancers. Given that ERβ is generally considered to have tumor-suppressive properties, a SNP that reduces ESR2 transcriptional activity could biologically contribute to tumor progression. Functional studies have also suggested rs3020449 may influence ESR2 expression levels, though further investigation is required to confirm this regulatory effect^39,40.

In the present study of rs3020449 on Bangladeshi ethnicity, AG vs. AA model detected as statistically significant in lowering the risk of BC development (OR = 0.58, 95% CI = 0.38 to 0.90, p = 0.015). A/G allele frequency for the patients and the controls were 25.83% and 38.81% whereas the A/A allele frequency for the cases and the healthy volunteers were 57.89% and 50.75% respectively (Table 3). In case-control studies, the same SNP may demonstrate varying outcomes in disease progression among different populations due to multiple factors. Population stratification, which includes differences in allele frequencies among groups, can lead to discrepancies. Gene-environment interactions are significant, as environmental exposures can differentially influence the SNP across various populations. The sample size and statistical power may differ among populations, influencing the reliability of associations. Eventually, variations in the characterization of cases and controls can result in differing outcomes among populations. From the analysis we might state that this study was more likely region and ethnicity specific.

The current study identified a significant association between the ESR2 promoter polymorphism rs3020449 and breast cancer susceptibility. Global evidence supports the regulatory influence of ESR2 variants on gene expression and cancer risk. For example, studies in Iranian and Chinese populations have linked rs3020449 with increased breast and endometrial cancer risk, respectively, possibly due to altered ERβ transcriptional activity^39–42. Similarly, rs3020449 has been associated with prostate cancer progression in European cohorts suggesting a broader oncogenic relevance across cancer types. The functional role of this SNP, being located in a promoter region, may lie in modulating transcription factor binding and gene expression, thereby influencing tumor development. These findings reinforce the need to explore ESR2 polymorphisms as potential biomarkers for cancer risk stratification across populations^41–45. Although this study focused on the rs3020449 polymorphism of the ESR2 gene, it is important to note that other SNPs within the ESR2 gene such as rs1256049, rs4986938, and rs1271572; have also been implicated in the susceptibility to various cancers, including breast, endometrial, and prostate cancers. Incorporating multiple SNPs in future investigations would provide a more comprehensive understanding of the gene’s role in cancer risk. A multi-locus analysis could also help clarify gene-gene and gene-environment interactions affecting ESR2 expression and function in breast cancer pathogenesis^41,44–46.

The functional implications of the rs3020449 polymorphism, located in the promoter region of the ESR2 gene, remain inconclusive across studies. While promoter polymorphisms may affect transcription factor binding sites and subsequently gene expression, evidence regarding the specific influence of rs3020449 is still evolving. Some studies suggest that variants in the ESR2 promoter region may modulate receptor expression and influence susceptibility to hormone-related cancers, including breast cancer. However, functional validation of rs3020449 remains limited, and further in vitro and in vivo studies are needed to confirm its regulatory role and downstream biological effects^44,45.

This current study has certain strength for instance; this is the very first significant investigation of genetic association study of ESR2 (rs3020449) variation on Bangladeshi women concerning the BC development. An ARMS-PCR method for this SNP was developed and validated simultaneously through this study. There were also a few shortcomings of this investigation including, small size of subject population which tends to limited statistical evidence, lacking age-matched patients and controls due to hospital-based recruitment, subjects selected from a specific subset of the population such as a hospital setting may not be representative of the overall population. It is always advantageous to validate the genotyping results by DNA sequencing, which is a limitation in the present study.

Conclusion

The result of this research indicated that ESR2 (rs3020449) polymorphism may lower the risk of formation of breast cancer among Bangladeshi women. Moreover, this study showed that the reduced tumor invasiveness and cancer staging progression in Bangladeshi female BC patients. Nevertheless, it can be projected that this study may contribute to future studies and provide further persuasive data, as it demonstrated the correlation between the variations of the ESR2 gene and BC. Lastly, ESR2 (rs3020449) could function as a genetic indicator for BC, possibly contributing to the conception and designing of tailored medications and advancing targeted therapies for enhanced treatment outcomes using the gene’s protective effect in BC progression.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Tasnim Ahmed Shayla: Writing & performing the procedures. Samiha Rahman Arthi: Writing & data curation. Sanjida Chowdhury Ivy: Writing and interpretation of the data, review & editing. Mohammad Safiqul Islam: Review & conceptualization. Mohammad Shahriar: Writing and interpretation of the data, review & editing, visualization, planning & supervision.

Data availability

The data supporting the present study findings are obtainable from corresponding authors upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.