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1 CDH1 AND FGFR2 GENE POLYMORPHISMS IN PATIENTS WITH BREAST CANCER MOHAMMAD-REZA JAVAN 1 MILAD ASADI 2 MEHDI MOHEBI 3 ASLANI SAEED 4 VENUS ZAFARI 5 HOMAYUN DOLATKHAH 6 1 Dept. of Biochemistry & Immunology, Faculty of Medicine, Zabol University of Medical Sciences, Zabol, Sistab & Baluchestan, Iran 2 Dept. of Medicine Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, East-Azerbaijan, Iran 3 Dept. of Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 4 Dept. of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 5 Dept. of Medicine Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, East-Azerbaijan, Iran 6 Dept. of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran ABSTRACT Through various studies, a strong heritable background has been related to breast cancer. Approximate cases who exhibit a family history of the disease are 15%. Mutations in genes starkly in BRCA1 and BRCA2 make people subject to an autosomal dominant inherited cancer possibility. Cadherin-1 also known as E-cadherin (CD324) is a protein that in humans is encoded by the CDH1 gene which is a tumor suppressor gene. Fibroblast growth factor receptor 2 (FGFR2) is a protein that is encoded by the FGFR2 gene, is a receptor for fibroblast growth factor. CDH1 and FGFR2 genes play a role in carcinogenesis. It has already been demonstrated that polymorphisms in CDH1 and FGFR2 genes is related to various malignant disorders. The aim of present study was to reveal the affection possibility towards breast cancer in relation to SNPs of CDH1 and FGFR2 genes. Peripheral blood of 100 breast cancer contracted women and 150 healthy controls were collected. Afterward, genomic DNA content of the PBMCs was extracted. PCR-RFLP was done and in order to detection of polymorphisms, AflIII and AciI restriction enzymes treated with PCR product of CDH1 and FGFR2 genes, respectively. Restriction enzymes could digest the DNA at selected SNPs. According to statistical analysis, SNPs were not significantly frequent in patients with breast cancer in comparison to healthy controls. Nucleotide changes comparison between patients and healthy controls depicted that there was not SNP frequency significantly in patients of North West of Iran. KEYWORDS: SNP, CDH1, FGFR2, Breast Cancer 114
2 INTRODUCTION Breast cancer is a heterogeneous group of malignancies originated from the ductal epithelium. It is the striking cause of cancer-associated mortality and the most common malignancy of women in all over the world (1). Nowadays, the incidence of breast cancer is increasing, and this increase is remarkably high in developing countries of Southeast Asia, Africa, and South America (2). Breast cancer incidence rates are also on the raise in all regions of India. It is the commonest cancer in urban Indian females and second commonest in rural Indian women. About 80,000 new cases of breast cancer in Indians are at the late stages III and IV. In some areas, about 70% of breast cancer cases are locally advanced in the diagnosis moment. It is because of the lack of organized screening programs, paucity of diagnostic aids, and a general ignorance toward the health of women in Indian Society (3). The etiology of breast cancer is the matter of controversy and multifactorial, consequence of the interactions between genetic, environmental, and lifestyle-related risk factors (4). Several mutations especially in BRCA1 and BRCA2 as well P53 and DNA repair-related gens such as ATM, ATM1, and PALB2 have been realized to be associated with breast cancer (5). It has been shown that geographical variation play a role in the incidence difference of breast cancer and can be attributed to exposure to various risk factors. Among these factors are early menarche, late menopause, late first full-term pregnancy, nulliparity, no breast feeding, and family history of breast cancer in two or more first-degree relatives have major attribution. To enumerate minor risk factors, obesity in postmenopausal women, hormone, replacement therapy (HRT), smoking, exposure to low-dose radiation, excessive alcohol intake, and so on (6, 7). CDH1 gene encodes E-cadherin which plays a fundamental role in the maintenance of cell differentiation and normal architecture of epithelial tissue (8). Mutations and diminished expression of CDH1 have been identified in a number of epithelial malignancies. Moreover somatic CDH1 mutations have been detected in lobular breast cancer with a frequency rate of 10 56% (9). FGFR2 gene- encoded protein is a member of fibroblast growth factors and has many functions in different cell processes. It is critical for morphogenesis and cell proliferation regulation (10). It has been shown that FGF receptors dysregulation plays an important role in cancer development and progression. These alterations have been proposed to occur through over expression, gene amplification or mutation (10). In the present study we aimed to realize if the polymorphisms of CDH1 and FGFR2 genes play a role in the increased risk of breast cancer. 115
3 Material and methods Patients and DNA extraction In this study which was carried out at Tabriz University of Medical Sciences, patients were chosen from Imam Reza Hospital, Tabriz, Iran. An ethics committee of Human Research of the hospital approved the study. Measurement of biochemical markers to realize the situation of patients is a routine procedure and all patients gave an informed consent when they referred to hospital. One hundred breast cancer affected patients and 150 healthy controls were assigned in our study and PBMCs were collected through Ficoll-Paque procedures. The individuals were age and sex matched. Cancer grade, lymph node metastasis, chemotherapy and radiotherapy-receiving context of the patients had been designated histologically. Characteristics of patients are listed at the Table 1 below with more details. Genomic DNA was extracted through Phenol-Chloroform method. Table 1. Properties of 100 patients with breast cancer Characteristic No (%) Total number 100 Mean age 51.4 Mean weight 67 Delivery times 0-4 Hormonal disorders 38 (76%) Menopausal status Premenopausal 18 (36%) Postmenopausal 32 (64%) Lymph nodes + 5 (10%) - 45 (90%) Tumor grade I 8 (16%) II 27 (54%) III 15 (30%) Chemotherapy Yes 2 (4%) No 48 (96%) Radiotherapy Yes 3 (6%) No 47 (94%) Metastasis Yes 0 (0%) No 100 (100%) RFLP-PCR procedures In order to continue the procedures, one polymorphism for each gene was selected on website: SNP-28 C/T (rs ) of promoter of 116
4 CDH1 gene and SNP-2447 C/G (rs ) of intron 2 of FGFR2 gene were chosen. The primers for CDH1 amplification were forward 5 -AGGTCTTAGTGAGCCACC-3 and reverse 5 -AACTGACTTCCGCAAG-3 and for FGFR2 amplification were forward 5 - CGTGAGCCAAGCCTCTACTT-3 and reverse 5 -TGTCTGGGGTAAGCTGAGCT-3. For accuracy and specificity all primers were blasted in NCBI website: Primers were produced by the custom oligonucleotide synthesis service, Metabion (Martinsried, Germany). Performing the StepOne RFLP-PCR (Thermo Fisher Scientific Inc.), each reaction mixture contained a total volume of 25μl (master mix 12.5μl, DNA 3μl, primer 3μl, and H 2 O 6.5μl). The PCR conditions were: 50 C for 2 minutes, 95 C for 10 minutes, then 30 cycles of 95 C for 30 seconds, and 60 C 30 seconds and 72 C for 30 seconds. After PCR, the reactions were centrifuged at 1000 x g for 2 minutes and the supernatants were collected for restriction digestion. The digestions were prepared directly in the supernatants with restriction Enzyme AflIII (0.4 U/μL) and AciI (0.4 U/μL) for amplified CDH1 and FGFR2 genes, respectively. Restriction enzyme processing and choosing procedures was done using website: AflIII can digest the DNA sequence 5 -CTTAAG-3 after C nucleotide. Also AciI are able to cut the DNA sequence 5 -CCGC-3 after first C. The 10 μl reactions were incubated for one hour at 37 C. The resulting fragments were analyzed on agarose gel and compared to untreated PCR products. Table 2. Chosen SNPs, primers used in PCR, and the melting temperature (Tm) of each reaction. Gene CDH1 FGFR2 Polymorphism marker 28 C/T (rs ) 2447 C/G (rs ) Primers 5 -AGGTCTTAGTGAGCCACC-3 F 5 -AACTGACTTCCGCAAG-3 R 5 -CGTGAGCCAAGCCTCTACTT-3 F 5 -TGTCTGGGGTAAGCTGAGCT-3 R Tm ( C) of Primers 58.5 Results Histological classification of breast cancer patients according to historical documents has shown that about 71.5 % were ductal carcinoma, and the remaining 28.5 % were lobular carcinoma. According the categorizations by TNM, about 30% of the patients were detected to be at stage III, and 54% of them were at the stage II. Also less than 16% of the cases were
5 at the stage I. Menopausal status of patients indicated that 36 % were premenopausal, and 64 % of them were in postmenopausal status. The axillary lymph node status of patients was found to be 95 % negative, and the remaining 5 % were positive with a higher risk of metastasis but lymph node metastasis stage analysis resulted that nobody had definite metastasis manifestation. Characteristics of the patients are illustrated at Table 1. A higher number of breast cancer cases had more than 40 years of age (p <0.001). According to their area of living, most of cases were found to be from rural population when compared to controls (p <0.001). However, in terms of occupation, there was no significant difference between the cases and controls. After treating PCR products with restricting enzymes and running on the gel electrophoresis, 91 (91%) out 100 patients had SNP-28 C for promoter of CDH1 as all of healthy controls had. Nine (9%) of patients had SNP-28 T on this region. Also 89 (89%) out of 100 patients had SNP-2447 G at the intron 2 of FGFR2 gene and 11 (11%) of cases had SNP-2447 C (Table 2). statistical analysis resulted that there were no significant correlation between SNPs of CDH1 and FGFR2 genes and contraction to Breast cancer (P-value >.05). Agarose gel electrophoresis results are depicted in Figure 1. Figure 1. Electrophoresis results of PCR products after treating with restriction enzyme for CDH1 (A) and FGFR2 (B) genes. 118
6 Table 3. Count and percentage of the each polymorphisms for CDH1 and FGFR2 genes. Polymorphism Count in patients Count in healthy controls CDH1 C -28 (rs ) 91 (91%) 150 (100%) T -28 (rs ) 9 (9%) 0 (0%) FGFR2 G (rs ) 89 (89%) 150 (100%) C (rs ) 11 (11%) 0 (0%) Discussion Breast cancer is one of the common cause of cancer death among women in both India and Western countries. The etiology of breast cancer is multifactorial, and several risk factors are associated with it. The environmental and lifestyle factors are important determinants of breast cancer risk. In women, the incidence of breast cancer increases with increasing age until 45 to 50 years. Chronological age could be a crude equivalent for breast tissue age, adding to the severity of the disease (11). According to Elizabeth B. claus et al. the onset age of the breast cancer was up to 35 years old. On the other hand in our study the onset age of the breast cancer was > 40 and the mean age of distribution of breast cancer patients was 51.4±7.44 years (12). Haixin LEI et al. reported that the mutations of CDH1 gene are correlated with increased risk of breast cancer. They also resulted that allelic variants of the CDH1 promoter did not show detectable increased risk for breast cancer (9). According to our study, CDH1, and also FGFR2, gene polymorphisms did not raise the risk of breast cancer. In the previous studies CDH1 gene polymorphisms had been associated with different cancer like bladder cancer (13). On the other side it has been shown that CDH1 promoter gene polymorphism was not associated with increased risk of stomach cancer (14). It s the matter of premature to conclude that promoter polymorphisms of the CDH1 do not increase the risk of breast cancer. Fibroblast growth factor receptor 2 (FGFR2) is involved in different cell processes as if cell proliferation, invasiveness, motility, and angiogenesis. Some genome-wide association studies (GWAS) have found FGFR2 as a breast cancer susceptibility gene in populations of European and Asian descent (15). In a meta-analysis study related polymorphisms of increasing breast cancer risk were identified to be rs , rs , and rs polymorphisms (16). Jie Liang et al. depicted that genetic variants in intron 2 of FGFR2 were significantly associated with increased risk of breast cancer in Chinese women, possibly associated with the status of hormone receptors and the reproductive history of women (17). 119
7 In ongoing study, we evaluated the association between breast cancer and rs polymorphism. This polymorphism on intron 2 of FGFR2 did not raise the risk of breast cancer in women of North-West of Iran. Considering all the facts, the study validates previous studies about no correlation between polymorphisms of CDH1 promoter and breast cancer risk. But interestingly FGFR2 gene polymorphism did not raise the risk of breast cancer in North-West of Iran. Acknowledgement We are thankful of the kind personnel of the Genetic department of Tabriz University of Medical Sciences and Imam Reza Hospital, Tabriz, Iran. References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, CA: a cancer journal for clinicians. 2008;58(2): Agarwal G, Ramakant P, Forgach ERS, Rendón JC, Chaparro JM, Basurto CS, et al. Breast cancer care in developing countries. World journal of surgery. 2009;33(10): Agarwal G, Ramakant P. Breast cancer care in India: the current scenario and the challenges for the future. Breast Care. 2008;3(1): Byrne C, Brinton LA, Haile RW, Schairer C. Heterogeneity of the effect of family history on breast cancer risk. Epidemiology. 1991: Easton DF, Pooley KA, Dunning AM, Pharoah PD, Thompson D, Ballinger DG, et al. Genome-wide association study identifies novel breast cancer susceptibility loci. Nature. 2007;447(7148): Pakseresht S, Ingle G, Bahadur A, Ramteke V, Singh M, Garg S, et al. Risk factors with breast cancer among women in Delhi. Indian journal of cancer. 2009;46(2). 7. Porter PL. Global trends in breast cancer incidence and mortality. salud pública de méxico. 2009;51:s141-s6. 8. Graziano F, Humar B, Guilford P. The role of the E-cadherin gene (CDH1) in diffuse gastric cancer susceptibility: from the laboratory to clinical practice. Annals of oncology. 2003;14(12): Lei H, SjöbergMargolin S, Salahshor S, Werelius B, JANDAkova E, Hemminki K, et al. CDH1 mutations are present in both ductal and lobular breast cancer, but promoter allelic variants show no detectable breast cancer risk. International journal of cancer. 2002;98(2): Eswarakumar V, Lax I, Schlessinger J. Cellular signaling by fibroblast growth factor receptors. Cytokine & growth factor reviews. 2005;16(2): McCawley LJ, Matrisian LM. Matrix metalloproteinases: multifunctional contributors to tumor progression. Molecular medicine today. 2000;6(4): Claus EB, Risch NJ, THOMPSON WD. Age at onset as an indicator of familial risk of breast cancer. American journal of epidemiology. 1990;131(6): Kiemeney LA, van Houwelingen KP, Bogaerts M, Witjes JA, Swinkels DW, Heijer Md, et al. Polymorphisms in the< i> E-cadherin</i>(< i> CDH1</i>) gene promoter and the risk of bladder cancer. European Journal of Cancer. 2006;42(18):
8 14. Pharoah PD, Oliveira C, Machado JC, Keller G, Vogelsang H, Laux H, et al. CDH1 c160a promotor polymorphism is not associated with risk of stomach cancer. International journal of cancer. 2002;101(2): Zheng W, Long J, Gao Y-T, Li C, Zheng Y, Xiang Y-B, et al. Genome-wide association study identifies a new breast cancer susceptibility locus at 6q Nature genetics. 2009;41(3): Jia C, Cai Y, Ma Y, Fu D. Quantitative assessment of the effect of FGFR2 gene polymorphism on the risk of breast cancer. Breast cancer research and treatment. 2010;124(2): Liang J, Chen P, Hu Z, Zhou X, Chen L, Li M, et al. Genetic variants in fibroblast growth factor receptor 2 (FGFR2) contribute to susceptibility of breast cancer in Chinese women. Carcinogenesis. 2008;29(12):
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