Association of Fibroblast Growth Factor Receptor Gene (FGFR2) Polymorphism (rs2981582) and its Expression with Breast Cancer

The association between Fibroblast Growth Factor Receptor 2 (FGFR2) gene polymorphism and expression with breast cancer varies with ethnicity and geographical distribution. Several studies have been conducted with different genotypes of this particular gene polymorphism among various ethnicities throughout the world. The main purpose of this study was to evaluate the relationship between FGFR2 (rs2981582) gene polymorphism and FGFR2 gene expression with breast cancer in Indian women and also to establish the relative gene expression with different genotypes of the polymorphism, if there is any. Method: A total of 75 cases were selected, out of which 25 were malignant cases,25 healthy controls and 25 benign cases. Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP PCR) was used to determine FGFR2 Single Nucleotide Polymorphism and Real Time Quantitative Polymerase Chain Reaction (RT qPCR) for FGFR2 gene expression. The strength of association was estimated using Odds Ratios (ORs) and 95% Confidence Intervals (CIs). RESULTS: Our results showed that FGFR2 (rs2981582) polymorphism is not significantly associated with breast cancer in Indian women. However, FGFR2 gene expression is significantly increased in breast cancer but the differential gene expression associated with different genotypes (TT / TC / CC) is not significant in both malignant and benign cases. Therefore, we conclude from this study that FGFR2 rs2981582 SNP does not influence the expression of FGFR2 in this population.


Introduction
Breast Cancer (BC) is one of the most common malignant tumors among women all over the world and has the highest mortality rate amongst the cancers afflicting women [1]. In recent years, its incidence has increased among young women with increasing tendency of chemo-resistance and recurrence [2,3]. Genetic factors such as BRCA1, BRCA2, HER2, CyclinD1, MAP3K1, FGFR2, TOX3, etc play a major role in etiopathogenesis of breast cancer The association between Fibroblast Growth Factor Receptor 2 (FGFR2) gene polymorphism and expression with breast cancer varies with ethnicity and geographical distribution. Several studies have been conducted with different genotypes of this particular gene polymorphism among various ethnicities throughout the world. The main purpose of this study was to evaluate the relationship between FGFR2 (rs2981582) gene polymorphism and FGFR2 gene expression with breast cancer in Indian women and also to establish the relative gene expression with different genotypes of the polymorphism, if there is any. Method: A total of 75 cases were selected, out of which 25 were malignant cases,25 healthy controls and 25 benign cases. Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP PCR) was used to determine FGFR2 Single Nucleotide Polymorphism and Real Time Quantitative Polymerase Chain Reaction (RT qPCR) for FGFR2 gene expression. The strength of association was estimated using Odds Ratios (ORs) and 95% Confidence Intervals (CIs). RESULTS: Our results showed that FGFR2 (rs2981582) polymorphism is not significantly associated with breast cancer in Indian women. However, FGFR2 gene expression is significantly increased in breast cancer but the differential gene expression associated with different genotypes (TT / TC / CC) is not significant in both malignant and benign cases. Therefore, we conclude from this study that FGFR2 rs2981582 SNP does not influence the expression of FGFR2 in this population.
FGFR2 polymorphism and BC risk [5][6][7]. FGFRs mediate signaling of fibroblast growth factors (FGFs) and have numerous important functions [8]. FGFR2 gene is located at chromosome 10q26 and acts through multiple downstream signaling pathways that play vital roles in cell proliferation, survival and differentiation [9]. The role of FGFR pathway as a predictive/prognostic marker has been investigated through various studies. According to these studies, aberrant FGFR2 expression is associated with an increased risk of BC and correlates with poor prognosis [10,11]. According to Gene Wide Association Study (GWAS), five single nucleotide polymorphisms (SNPS) (rs7895676, rs2912781, rs10736303, rs2912778 and rs2981582) in the non-coding region of FGFR2 were found to have a significant association with breast cancer. FGFR2 (rs2981582) in intron 2 has been identified as the most significant breast cancer risk locus by the Breast Cancer Association Consortium genome wide association study [12]. Hunter et.al (2007) also identified four other SNPs, rs11200014, rs2420946, rs1219648 and rs2981579 in intron 2 of FGFR2 that are also associated with breast cancer [13]. Therefore, it becomes very important to understand the FGFR pathway on how it leads to BC and how it can be targeted. Several trials are being done with multiple agents that target FGFR pathway components [14][15][16]. According to these trials, identification of patients with FGFR pathway amplified tumors who may respond to this treatment may be possible. Large number of studies are available on relevance between FGFR2 single nucleotide gene polymorphisms (rs2981582 and rs2981579) and breast cancer, but the results vary with geographical differences [17]. There is hardly any study exploring the association of rs2981582 FGFR2 polymorphism in patients of breast cancer with Indian ethnicity.
Therefore, we developed an interest to elucidate the frequency of this polymorphism and its influence on FGFR2 expression in Indian patients with breast cancer.

Study Site and Patient Recruitment
The present study was conducted at the Department of  Table 1. The restriction digestion fragments were resolved and analyzed by 3% to 3.5% agarose gel electrophoresis using EtBr as staining dye.

RNA Isolation and cDNA Synthesis
Total RNA was extracted from all the subjects using Geneaid The ingredients of the eppendorf tube were then transferred to columns, provided with the kit, and centrifuged at high speed (14000 RPM) for 2minutes. The temperature of the centrifuge machine was set at 4°C. The columns were then washed with 75% alcohol and RNA was eluted down in RNAse free water (100µL).

RNA integrity was determined by agarose gel electrophoresis (2%)
and concentration was determined by measuring absorbance at 260 nm as discussed above.
The RNA was converted to complementary DNA (cDNA) using cDNA synthesis kit (Thermo Fisher Scientific, USA) by following manufacturer's instructions as provided with minor modifications. I brief, total RNA (100 ng), 2 µL oligo (dT) primer, and 9 µL water were mixed and incubated at 70°C for 5 minutes in a thermal cycler. This reaction mixture was then mixed with 5X RT buffer reverse transcriptase enzyme in a total volume of 20 µL. This mix was incubated at 42°C for 1 h and then heated to 95°C to stop the reaction. The cDNA was stored at −80°C until used for expression studies.

Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
Quantitative RT-PCR was used to carry out relative expression of FGFR2 gene on a Rotor-Gene Instrument (QIAGEN; Skelton House, Lloyd, Manchester, UK). β-actin gene was used as reference gene for normalizing. The primers used were as follows:

Statistical Analysis
Chi-square test was performed for comparison of genotypic frequency distribution between:

I)
All patients and healthy controls,

III) Malignant patient's vs healthy controls and IV) benign vs malignant patients. Odds ratio along with corresponding 95%
confidence intervals (CIs) were used for estimation of individual genotypic risk for breast cancer by employing codominant model of inheritance. Continuous data was analysed by student t-test and

Results
The clinicopathological and demographic characteristics of patients and healthy controls are represented in Tables 1 & 2. In contrast to healthy controls (mean ±SD=34.28±9.3), the breast cancer patients tended to be younger (mean ± SD=32.52+11.13) taken together was more. Further, when the breast cancer patients were subdivided, benign patients were comparatively found to be younger (mean ±SD=30.76±12.65) followed by healthy subjects (mean ±SD=34.28±9.3) and then by malignant patients (mean ±SD=46.2±15.54). Principally, we did not find any significant differences in basic demographic characteristics among the healthy subjects and breast cancer patients when taken together except that breast cancer patients significantly tend to be multiparous and married. However, significant differences were seen in basic characteristics including age at menarchy, parous status, menopausal status, breast feeding and marital status, but feeding habits, between subtypes. All the basic characteristics of healthy and breast cancer subjects, along with statistical analysis, have been presented in Tables 2 & 3.

FGFR2 mRNA Expression and Polymorphism (rs2981582) in Concert with Breast Cancer
We analysed the mRNA expression of FGFR2 gene and compared it among healthy controls and breast cancer patients.
It was observed that RGFR2 mRNA expression varied significantly among healthy controls and breast cancer patients. Breast cancer patients exhibited an increased FGFR2 mRNA expression compared to healthy control subjects (mean fold ± SD=4.33+4.20, Table 3).
We further compared FGFR2 mRNA expression among benign and malignant breast cancer patients and observed that FGFR2 mRNA expression was significantly raised by 2.95-fold in malignant subjects compared to benign tumor ones (mean fold ±SD=2.49+1.21 vs 7.35+5.88; p=0.0002 respectively, Table 4 and Figure 1).  Figure 2). Similarly, FGFR2 mRNA expression in concert with FGFR2 (rs2981582) polymorphism genotype was analysed in patients of benign and malignant tumour separately (Table 5). However, we could not reach any meaningful inference as the FGFR2 mRNA expression did not follow a meaningful pattern.
The results of the analysis are depicted in Table 4 below.  p-value of 0.0002 (Fig.1). This finding is correlating with the previous studies on overexpression of FGFR2 in breast cancer [19]. So we infer that altered expression of FGFR2 might have a role in pathogenesis/malignant transformation of breast tumor / cancer. However, on comparison of FGFR2 expression among benign patients with different genotypes (rs2981582 T>C) the differential gene expression associated with different genotypes of the polymorphism (TT/TC/CC) was not found to be statistically significant ( Figure 2). On comparing the same among malignant patients, FGFR2 gene expression among different genotypes in malignant cases was also statistically insignificant ( Figure 3). So, we hypothesize from this study that rs2981582 SNP does not influence the expression of FGFR2 in this population.