Effect of SMAD4 gene polymorphism on breast cancer risk in Bangladeshi women

Breast cancer, one of the most prevalent cancer types among women worldwide as well as in Bangladesh, is the leading cause of cancer death in women throughout the globe. The risk of breast cancer development was found to be associated with genetic polymorphism according to several studies. As a convenient prognostic marker, a biomarker helps to identify disease progression, can lead to an effective therapeutic strategy, development of prognostic marker is very important for any cancer to initiate treatment strategy early to increase the possibility of the success rate of the treatment along with reduction of the treatment cost. This study aims to establish the correlation between polymorphism of SMAD4 rs10502913 and risk of breast cancer development in Bangladeshi women. This study was conducted on 70 breast cancer patients and 60 healthy volunteers through blood sample collection followed by DNA separation between the intervals of August 2019–October 2019. The collected DNA sample was arranged for the RFLP analysis of a PCR amplified fragments followed by gel electrophoresis. The obtained data was analyzed by structured multinomial logistic regression model. Obtained different fragment size after gel electrophoresis indicated different genotypes in this experiment. Our findings demonstrated that mutant homozygous A/A genotype, plays a significant role in breast cancer development among Bangladeshi women (P = 0.006, OR = 4.9626, 95% CI = 1.9980–12.3261) compared to the reference homozygous G/G genotype. Moreover, heterozygous G/A genotype was also found to be significantly associated with the risk of breast cancer development (P = 0.0252, OR = 2.6574, CI = 1.1295–6.2525). Considering the A/A genotype and G/A genotype combined, it also indicates a strong association of breast cancer development in Bangladeshi women (P = 0.008, OR = 3.5630, CI = 1.6907–7.5068). Our study indicated a novel association between SMAD4 (rs10502913) polymorphism and increased risk of breast cancer development in Bangladeshi women.


Background
Cancer is a disease in which a group of untypical cells grows uncontrollably without adhering to the standard rules of cell division [1]. Regular healthy cells are constantly subject to signals that decide whether the cell should divide, differentiate into another cell or die. But the division of cancer cells does not correspond to these signals, resulting in uncontrolled growth and proliferation. When this proliferation is allowed to continue and spread, cancer develops. Almost 90% of cancer-related deaths are due to tumor spreading-a process called metastasis [2]. Cancer genetics can predict the networks and pathways that contribute to tumor development and the mechanism of cancer genes promoting tumor evolution [3]. One of the most common invasive cancers in females worldwide is breast cancer [4]. In the beginning, breast cancer starts with a lump with or without other manifestations [5]. As with other types of cancer, breast tumors can be benign or malignant, the benign type is not life-threatening, can usually be removed, and does not spread to other parts of the body [6]. However, malignant breast tumors are life-threatening and can invade surrounding tissues. It can metastasize to other parts of the body via the lymphatic system (lymphatic vessels and lymph nodes) such as the liver and bone [7].
According to the estimation of web-based global cancer statistics platform, GLOBOCAN, in 2020, worldwide female breast cancer has overtaken lung cancer and become the most commonly diagnosed cancer with an estimation of 2.3 million new cases [8]. At the end of 2020, breast cancer was the world's most prevalent cancer as 7.8 million breast cancer survived women were found in the past 5 years [9]. Breast cancer is responsible for the most frequent cause of death in different regions of the world [4]. The peak age of breast cancer patients in Asia as well as in Bangladesh is around 40-50 years while in western countries the peak age is around 60-70 years [10,11]. According to GLOBOCAN estimation of 2020, in Bangladesh, 13,028 new breast cancer cases have been diagnosed in 2020, the most prevalent cancer type among females with an occurrence rate of 19% of the total female cancers [12].
Variation at single position of DNA is termed as Single nucleotide polymorphicm (SNP). According to multiple studies, SNPs are associated with breast density and the risk of breast cancer development [13,14]. SMAD4, a gene located on chromosome 18q21.1, is a downstream mediator of transforming growth factor-beta (TGF-β) and plays a role in regulating cellular function such as proliferation, differentiation, etc. [15]. Both alleles of the gene are inactivated in pancreatic carcinomas, but their role in the tumorigenesis of other cancer is still unknown [16]. A study suggested that SMAD4 might be a potential prognostic marker for the early detection of breast cancer [17]. An earlier study indicated that SMAD4 has a role to play in the regulation of the initiation, progression and prognostic outcome of breast cancer [18]. TGFβ, an inhibitory signaling molecule, generally binds with TGF-β receptor and via SMAD signaling pathway inhibits cell growth [19]. SMAD4 is a candidate for the tumorsuppressor gene [20]. In estrogen receptor-α positive breast cancer cells, SMAD4 can induce programmed cell death by TGF-β-mediated inhibition of ERα estrogenic transcription activity in tumor samples [21]. In recent years, studies on SNPs have increased due to their use to predict certain disease susceptibility. Gene transcription and translation as well as necessary protein synthesis can be altered due to SNPs [22].
Till date, according to our knowledge, no study has been conducted to assess the risk of SMAD4 gene polymorphism and breast cancer risk in Bangladeshi women. The aim of this study is to investigate the potential involvement of the SMAD4 gene polymorphism at position rs10502913 in Bangladeshi women to detect breast cancer in the early stage so that patients can be unclogged in terms of cure and cost.

Study population and sample collection
The case-control study was conducted on seventy breast cancer patients (case) and sixty healthy female volunteers (control). Two groups, case and control, were matched by means of age. The healthy volunteers were subjected to physical examination to mark them as control group. Breast cancer patients have been chosen from the National Institute of Cancer Research and Hospital (NICRH), Dhaka, Bangladesh, between the intervals of August 2019-October 2019. Ethical approval was taken before approaching to patients from Ethical Approval Committee of Medical Oncology Department of NICRH, Dhaka, Bangladesh. Details of ethical approval procedure is outlined in Supplementary Information (Additional file 1). Patients had been histologically diagnosed with breast cancer according to the TNM staging device accustomed by American Joint Committee of Cancer (AJCC). Demographic traits and lifestyle factors were acquired through interviews by means of skilled nurses in presence of professional physicians. The study was performed in accordance with the statement of Helsinki and its subsequent revisions [23]. Breast cancer patients or their respective caregivers filled out a consent form after being informed about the study protocol. The blood sample (3 mL) was collected from the patient and control using the EDTA tubes and was stored at − 80 °C until further use and the freeze-thaw cycle was avoided.

DNA extraction and genotyping
DNA was extracted by chemical method mentioned by Daly et al. [24]. DNA was isolated from the blood samples of the patients and the healthy volunteers and kept at − 20 °C temperature till analysis was done. Amplification of the SNP of interest for this study was performed by Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique to genotype the SMAD4 rs10502913 polymorphism [25][26][27]. Thermostable Taq DNA polymerase, isolated enzyme from Thermus aquaticus, was used to automatize the specific DNA sequence amplification repetitively during the PCR technique. The forward primer and reverse primer were designed by following the rule of the National Center for Biotechnology Information (NCBI). Primer details are given in Table 1.
The produced fragment was isolated and kept in a thermostat chamber overnight at 37 °C then digested by the restriction enzyme HpyCH4IV (source: Helicobacter pylori). Gel electrophoresis (with 2% agarose) was performed to visualize extracted DNA fragments after the digestion and ethidium bromide was used to stain the fragments. The restriction enzyme, Taq DNA polymerase and the primers were procured from New England Bio-Labs Inc., USA. Table 2 represents the patient group and control group with clinicopathological features.

Statistics
A statistical multinomial logistic regression model was used to evaluate the association between SMAD4 polymorphism and breast cancer risk in the studied Bangladeshi women. p value less than 0.05 was considered statistically significant with a 95% confidence interval. The statistical analysis was performed by the statistical software SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA).

Results
This population-based case-control study was conducted to demonstrate the prevalence of SMAD4 gene polymorphism in the Bangladeshi population of breast cancer patients and normal healthy controls. We conducted the study through 70 breast cancer patients with a number of 60 controls.
The splitting of the normal functioning allele G provided 46 bp and 206 bp sized fragments for SMAD4 rs10502913 polymorphism. The cleaving of the polymorphic allele A provided 46 bp, 206 bp and 252 bp sized fragments (Fig. 1). Different sized DNA fragments got after digestion with the restriction enzyme and it has been presented in Table 3.
The result has been depicted through classified the genotype of patient and control group into four; the heterozygous genotype, mutant homogygous genotype and combination of both were scrutinized against the reference genotype. In the case of SMAD4 rs10502913 the G/G homozygous genotype showed statistically lower frequency in breast cancer patients if it is compared to healthy controls volunteers. In this analysis, we set this GG genotype as a reference value to compare other data. The percentage frequency of G/A heterozygous genotype in the studied breast cancer cases was found in 34.28% with a p value of 0.0252. The G/A genotype showed an odds ratio of 2.6574 and the 95% confidence Interval was in the range between 1.1295 and 6.2525. Again, in the case of mutant homozygous A/A genotype the patients were found to have a frequency of 41.24%. With an odds ratio of 4.9626. The p value was found 0.0006 with a 1.9980-12.3261 range of 95% confidence interval. Moreover, considering both genotypes G/A and A/A which  are deviated from normal genotype the odds ratio was found to be 3.5630 with p value of 0.0008 and the confidence Interval was in the range between 1.6907 to 7.5068 (Table 4).

Discussions
This study shows that the carriers of the G/A genotype population were found to have 2.6574 times more risk of developing breast cancer compared to the normal genotype. The carriers of the A/A genotype population have 4.9626 times more risk and G/A and A/A genotypes combined has 3.4630 times more risk to develop breast cancer. Heterozygous (G/A), mutant homozygous (A/A) along combination both of these genotypes were found to be significantly associated with breast cancer development in the studied population group of Bangladeshi women. The shortcomings of the current study is that the function of gene of interest related to breast cancer development has not been evaluated in vivo and in vitro. Science is blending on regular basis successfully to set up the innate and nuclear reason of various complex disorders corresponding to certain sorts of carcinomas. In  explicit, SNPs have been productive in genome-based contemplates perceiving the genetic defenselessness of malignant growths [28]. The foundation of positive connection between polymorphism in genomic DNA and the possibility of tumor development has empowered various cover in looking for after development consideration in disease science but such associations can vary considerably between various ethnic populations [29]. A study suggested that breast cancer is associated with lower expression of the SMAD4 gene [30]. As earlier studies suggested that SMAD4 rs10502913 was associated with the development of colon cancer and gastric cancer, we targeted the alleles for its previous association with cancer [31,32].
Worldwide, there was no study conducted to find out the relationship between mutation at position rs10502913 of SMAD4 gene and risk of development of breast cancer. SNPs can be positioned in the promoter region, exon or intron. SMAD4 rs10502913 is an intronic variant functional SNP [33]. Intronic SNPs are responsible for splicing and change the long noncoding RNAs in terms of function and binding [34]. A study found that an intronic SNP was related to the risk of breast cancer development [35]. As an intronic variant SNP, SMAD4 rs10502913 might be associated with the risk of breast cancer development. SMAD4 rs10502913 polymorphic variant was found to be responsible for colorectal cancer and missense mutation was observed in the MH2 domain of SMAD protein [16,32]. Mostly Arg361 region and Pro356 region were found to be affected due to this mutation [36]. Missense mutation generally renders the protein to be less effective. Earlier studies found that missense mutation is associated with the risk of breast cancer development [37,38]. Upon binding with the DNA, SMAD4 protein regulates particular gene that is responsible for cellular proliferation [39]. So disruption of the function of this protein might be associated with the risk of breast cancer development.
A study with colorectal cancer in the Polish population found that in colorectal cancer patients the SMAD4 rs10502913 G/G genotype (60%) was predominant, followed by heterozygous GA (35%) and mutant homozygous A/A (5%) [40]. But in the case of samples from breast cancer patients in Bangladesh from this study, it was found that SMAD4 rs10502913 A/A mutant homozygous genotype is predominant with an occurrence of 41.42%, followed by heterozygous G/A genotype (34.28%). In case of SMAD4 rs10502913 G/G genotype, it was found only 24.28% occurrence. Previous studies have suggested that different ethnic backgrounds and different geographic populations may act as factor of incidence of cancer as well as treatment efficacy and prognosis [41,42]. Furthermore, race factor was found to be a major differentiator of risk of breast cancer development [43]. Due to differences in cancer nature and geographical location, the genotypes involved in cancer development were found to be different though same the SNP was found to be involved.
As there was no previous study for Bangladeshi women regarding breast cancer development associated with the mutated SMAD4 gene, this study can help us to find a novel correlation. It has been observed that, if breast cancer can be diagnosed at an earlier stage, the treatment cost would be much lower compared to detection in the late stage [44]. So developing a potential early prognostic marker will help us to treat the patient at the right time with a significant reduction in the cost. In our study, we have illustrated the impact of a distinctive SNP on the development of breast cancer. The primary SNP of interest was SMAD4 gene rs10502913. Polymorphism in this SNP was found to have a significant correlation with breast cancer risk in Bangladeshi women.

Conclusion
SMAD4 gene rs10502913 allele polymorphism identification can be a possible promising diagnostic marker for breast cancer in Bangladeshi women. Further studies are needed to corroborate the findings of the research.

Bp
Base pair GLOBOCAN Global cancer observatory SNP Single nucleotide polymorphism PCR-RFLP Polymerase chain reaction-restriction fragment length polymorphism TGF-β Transforming growth factor beta EDTA Ethylene diamine tetra acetic acid MH2 MAD homology 2