Received 21 September 2015; accepted 17 October 2015; published 20 October 2015

ABSTRACT

Worldwide, breast cancer (BC) is the most common invasive cancer in women. Fibroblast growth factor receptor 2 (FGFR2) is a tyrosine kinase receptor that is a member of the family of individually distinct fibroblast growth factor receptors involved in tumorigenesis. FGFR2 gene is amplified and over expressed in breast cancer (1 - 3). The aim of the study was to determine whether polymorphisms in rs2981582, rs2420946, rs17102287, rs1219648, rs2981578, and rs17542768 in FGFR2 gene are associated with breast cancer susceptibility in the population of Kazakhstan. The statistically significant associations between SNPs analyzed and breast cancer risk according χ² and p < 0.05 criterions were not evaluated. The information describing the association of SNPs in FGFR2 with BC risk in the world populations could not be unambiguously used for Kazakhstan population.

Keywords:

Fibroblast Growth Factor Receptor 2 (FGFR2), Single Nucleotide Polymorphism (SNP), Association, Breast Cancer (BC), Kazakhstan

1. Introduction

Worldwide, breast cancer (BC) is the most common invasive cancer in women. Fibroblast growth factor receptor 2 (FGFR2) is a tyrosine kinase receptor that is a member of the family of individually distinct fibroblast growth factor receptors involved in tumorigenesis. FGFR2 gene is amplified and overexpressed in breast cancer [1] -[3] .

А meta-analysis of 37 studies of rs2981582, rs2420946, rs17102287, rs1219648, rs2981578, and rs17542768 polymorphisms demonstrated that these FGFR2 SNPs are a risk factor associated with increased BC susceptibility, but these associations vary significantly in different racial and ethnic groups [4] .

Significant associations between breast cancer risk and SNPs in rs11200014, rs2981579, rs1219648, rs2420946 of FGFR2 (P_trend for all SNPs < 0.0001) were found in Jewish and Arab Israeli population [5] .

Kazakhstan is situated in the middle of Central Asia. The multinational population of Kazakhstan totaled 17.2 million in 2013 with the major ethnic groups represented by mongoloid Asian Kazakhs (65%) and Caucasian Russians (22%), according to the Agency of the Republic of Kazakhstan on Statistics data [6] .

The aim of the present work was to determine the association of individual SNPs in rs2981582, rs2420946, rs17102287, rs1219648, rs2981578, and rs17542768 sites of FGFR2 with BC in Kazakh and Russian ethnic groups of Kazakhstan.

In studies performed in Russia, the associations of FGFR2’s SNPs with BC risk were shown for rs1219648, especially in combination with polymorphisms in TP53 [7] ; rs2981582 in the population of West Siberia [8] ; and rs2981582, particularly in genetically-enriched BC patients versus elderly tumor-free women [9] . In Kazakhstan the presented research devoted to the evaluation of association of SNPs of FGFR2 with BC is performed at a first time.

2. Materials and Methods

2.1. Patients and Controls

Informed consent was received from all individuals prior to study inclusion. Ethical permissions were obtained from the ethical committees of the medical organizations listed below. Venous blood samples (5 ml) were collected from 495 women of Asian Kazakh (311 Kazakh) and Russian Caucasian (184 Russian) descent with diagnosed and histologically confirmed BC from the Kazakh Research Institute of Oncology and Radiology and Regional Oncological Dispensary (Almaty, Kazakhstan). Samples obtained from 190 healthy Kazakh and 170 Russian female blood donors (Almaty City Blood Center) without clinical symptoms or family history of cancer according to a questionnaire were used as a control. The average age of BC patients was 49.58 ± 8.70 (Kazakhs) and 53.40 ± 9.97 (Russians) years, while that of control donors was 49.84 ± 6.09 (Kazakhs) and 50.43 ± 6.56 (Russians) years old.

2.2. DNA Extraction and Genotyping

Genomic DNA was isolated from blood using commercially available DNA Blood & Tissue extraction kits (Qiagen, USA) according to the manufacturer’s instructions. Genotyping was performed by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) products. Deoxyribonucleoside triphosphates (dNTP), restriction endonucleases, and TaqDNA-polymerase were purchased from SibEnzyme (Russia).The PCR reaction mixture (10 μl total volume) contained 67 mM Tris-HCl, (pH 8.8), 16.6 mM (NH₄)₂SO₄, 2 mM MgCl₂, 0.01% Tween-20, 0.15 mg/ml bovine serum albumin, 2 pM primers, 0.25 mM dNTPs, 100 ng template DNA, and 1 unit of TaqDNA-polymerase using a Mastercycle gradient (Eppendorf, Germany). PCR-amplified products were separated by 8% polyacrylamide gel electrophoresis at 50 mA and 300 V for 2 - 3 h. RFLP products were visualized by 0.05% ethidium bromide staining and analyzed using GelDoc-Imager (BioRad, USA). Primers were designed by Primer3 (v. 0.4.0) [10] , and FGFR2 nucleotide sequences of interest were obtained from the Ensemble data base [11] . Amplification conditions and primers for each FGFR2 nucleotide sequence are presented in Table 1.

Each PCR product was digested with the appropriate restriction endonuclease according the manufacturer’s recommendations (SibEnzyme, Russia). PCR fragment and restriction product sizes and endonucleases used are presented in Table 2.

P―Fisher’s exact test p-value; OR―Odds Ratio; CI―Confidence Interval.

P―Fisher’s exact test p-value; OR―Odds Ratio; CI―Confidence Interval. ^*In the group of patients alleles frequencies did not corresponded to Hardy-Weinberg equilibrium.

2.3. Statistical Analysis

The Pearson χ² and Fisher’s exact tests were used to compare differences between allele frequencies and genotypes distribution between groups of BC patients and control. Cancer risk associated with genotype was calculated with odds ratios (ORs) and 95% confidence intervals (CI). All BC cases and controls were tested to be in Hardy-Weinberg equilibrium. Statistical analyses were performed using STATISTICA v. 5.0 software (StatSoft, USA). Fisher’s exact test and Chi-square criterion were estimated using Free Statistics Calculator v. 3.0 [12] .

3. Results and Discussion

To investigate whether SNPs in rs2981582, rs2420946, rs17102287, rs1219648, rs2981578, and rs17542768 of FGFR2 gene are associated with BC in Kazakhstan population, we performed PCR-RFLP based assay. The results of genotyping in Kazakh ethnic group (311 BC patients and 190 controls) are presented in Table 3 and the results of genotyping in Russian ethnic group (184 BC patients and 170 controls) are presented in Table 4. Allele frequencies in all groups corresponded to Hardy-Weinberg equilibrium with the exception of site rs1219648 in the group of BC patients. The differences neither in allele’s frequency nor in genotypes distribution were evaluated in both Kazakh and Russian ethnic groups.

The information describing the association of SNPs in FGFR2 with BC risk in the world populations could not be unambiguously used for Kazakhstan population.

Cite this paper

Timur S.Balmukhanov,Alexandra K.Khanseitova,Victoria G.Nigmatova,Alena S.Neupokoeva,Daria A.Sharafutdinova,Nagima A.Aitkhozhina, (2015) Lack of Association between Polymorphisms in rs2981582, rs2420946, rs17102287, rs1219648, rs2981578, and rs17542768 Sites of FGFR2 Gene with Breast Cancer in the Population of Kazakhstan. Advances in Breast Cancer Research,04,109-113. doi: 10.4236/abcr.2015.44012

References

NOTES

^*Corresponding author.