PPAR-Gamma Pro 12 Ala Polymorphism and Gastric Cancer Risk in a Turkish Population

Gastric cancer (GC) is the fourth most common human malignant disease and second leading cause of cancer related death in both sexes worldwide (Ferlay et al., 2010). Recently, there has been intense interest in the search for common genetic variants as biomarkers for genetic susceptibility to GC development, namely single nucleotide polymorphisms (SNPs) (Milne et al., 2009; Yin et al., 2009). Beside associations between some of SNPs in genes and GC risk have been reported in previous studies, definition of susceptibility genes for GC risk remains to be determined. Peroxisome proliferator-activated receptor γ (PPAR γ) is a member of the nuclear hormone receptor superfamily that plays an important role in cellular differentiation and carcinogenesis as well as regulation of metabolism, glucose and lipid homeostasis, and intracellular insulinsignaling events (Michalik et al., 2004). PPAR γ exists as two isoforms, γ1 and γ2, generated by alternative promoters and differential splicing of at least three different


Introduction
Gastric cancer (GC) is the fourth most common human malignant disease and second leading cause of cancer related death in both sexes worldwide (Ferlay et al., 2010).Recently, there has been intense interest in the search for common genetic variants as biomarkers for RESEARCH ARTICLE

PPAR-Gamma Pro12Ala Polymorphism and Gastric Cancer Risk in a Turkish Population
Emel Canbay 1,2,3 *, Ozlem Kurnaz 4 , Bahar Canbay 5 , Dursun Bugra 6 , Bedia Cakmakoglu 4 , Turker Bulut 7 , Sumer Yamaner 7 , Necmettin Sokucu 7 , Yılmaz Buyukuncu 7 , Hulya Yilmaz-Aydogan 4 transcripts from the PPAR γ gene on chromosome 3p25.Several polymorphisms in PPAR γ2 have been identified so far and one of the common structural polymorphism in the PPAR γ2 gene was identified as CCA-to-GCA (Pro12Ala) missense mutation in codon 12 of exon B (Yen et al., 1997).This substitution possibly results in a conformational change in protein structure and reduced function of the PPAR γ2 gene.The role of Pro12Ala polymorphism of PPAR γ gene has been recently studied in cancers.Individuals with the Ala12 allele are found to have an increased risk of GC in Chinese (Liao et al., 2006), Indian (Prasad et al., 2008) and Japanese (Tahara et al., 2008) populations.As PPARγ Pro12Ala polymorphism is found to be associated with GC risk we examined the potential association between Pro12Ala polymorphism in the PPARγ gene and GC in Turkish population

Study Groups
Two hundred fifteen individuals included in this study.Sixty eight patients with GC at their initial staging admitted to Department of General Surgery, Istanbul Medical Faculty, Istanbul University were included in this study.One hundred twenty nine healthy controls were selected from individuals who came for routine health check during the same period of time.The patient and control groups were matched for age and sex.All participants signed an informed consent before enrollment and Institutional Ethical committee approval was obtained for the study.

Polymorphism Analysis
Blood samples from all study participants were collected in EDTA-containing tubes.Genomic DNA was extracted from peripheral whole blood according to salting-out technique (Miller et al., 1998).Genotyping was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism the procedures of PCR-RFLP are given in Table 1 (Liao et al., 2006).The appropriate primers were used to amplify the corresponding gene of the subjects by PCR and the reaction products were digested by using the appropriate enzyme at 60°C.The digested products were analyzed on 2.5% agarose gel stained with ethidium bromide and examined under transillumination.Each gel was read by two observers unaware of the subject's status.If there is any conflict, samples were repeated.The expected results after restriction for each gene were also given in Table 1.

Statistical Analysis
Statistical analyses were performed using the SPSS software package (revision 16.0 SPSS Inc., Chicago, IL, USA.).Data are expressed as means+SD.Differences in clinicopathological characteristics between patients and controls were tested by chi-square test for categorical data and Student's t-test for numerical data.Odds ratio (OR) and 95% confidence interval (CI) for the association between genotype and GC was computed.A two-sided p-value of less than 0.05 was considered statistically significant.

Characteristics of study group
Sixty eight patients with GC and 129 controls were studied.Characteristics of patients with GC and controls were given in Table 2.There was no significant difference in the baseline characteristics between patients and controls.

Discussion
In this study we investigated the association between PPAR γ Pro12Ala polymorphism and GC risk in Turkish population.
In our study we demonstrated that PPAR γ Pro12Ala gene polymorphism was associated with GC.Individuals carrying G (Ala 12) allele had increased risk for GC.Moreover, G allele carriers were higher in GC patients with tumor harboring poorly differentiation and metastatic process.
PPARs represent a family of nuclear receptors that are related to thyroid and retinoid receptors.PPARγ is the most extensively studied of three PPAR subtypes.Ligand binding to PPAR activates the transcription of PPAR responsive genes including cellular development, differentiation and carcinogenesis.PPARγ gene expression was identified in both gastric cancerous and normal gastric epithelium (Leung et al., 2004).It has also been shown activation of PPARγ inhibits the growth and induces apoptosis of gastric cancer cells (Leung et al., 2004).
Presence of the Ala12 polymorphism which is associated with reduced PPAR γ functional activity was thought to increase the risk of GC (Stuvoll and Haring, 2002;Meirhaeghe and Amouyel, 2004).In our study, Ala12 allele was frequently present in GC when compared to control group.We found that the patients with Ala12 allele carriers had 1.57-fold (95%CI: 1.103-2.238)increased risk of progression to GC. Regarding the association between Ala 12 polymorphism of PPAR γ gene and GC risk, Liao et al. (2006) reported 2.5-fold increased risk in Chinese, Prasad et al. (2008) reported 2.14-fold increased risk in Indian, and Tahara et al. (2008).reported 2.4-fold increased risk in Japanese population of progression to GC. Bazargani et al. (2010) investigated the association between PPAR γ polymorphism and noncardia GC risk in Iranian population.They have found that Ala 12 PPAR γ gene carriers had 3.28 fold increased risk for non-cardia GC development in the presence of H.pylori infection.It appears that the risk for development of GC related to Ala12 polymorphism of PPARγ gene in our Turkish population is similar with previous studies in other ethnic population.Furthermore, Xu et al. (2010) reported that Ala12 variant of PPARγ was associated with increased risk of GC (OR: 2.31, 95%CI: 0.95-1.23).When we analyzed the association of PPARγ gene polymorphism with patients' characteristics, the Ala12 carriers had higher in patients harboring poorly differentiation as well as with distant metastases.Our subgroup analyses support that PPARγ Ala12 polymorphism is associated with carcinogenesis and metastatic processes of GC in Turkish population.Functional role of this substitution is not clear but it possibly results in a conformational change in protein structure and reduced function of the PPAR γ2 gene.The functional role of Pro12Ala polymorphism of PPAR γ gene also needs to be studied with further studies.
Although the present study has a novel finding in Turkish population, it has some limitations such as small sample size and lack of tissue expression and association of PPAR γ polymorphism with presence of H.pylori infection in our patients.Indeed, GC risk was found to be increased in Ala12 allele carriers of PPARγ gene in the presence of H.pylori infection in previous studies (Liao et al., 2006;Prasad et al., 2008).Further analysis in GC patients with presence of H.pylori infection seems to be mandatory to clarify the association between H.pylori infection and PPARγ polymorphism in GC development.Beside this, the functional studies of this polymorphism will be clarified exact role of this polymorphism in GC.
In conclusion, this study suggests that PPARγ Pro12Ala polymorphism might be potential marker for genetic susceptibility to GC in Turkish population.We also report, PPARγ Pro12Ala polymorphism is associated with tumor characteristics suggesting the prognostic importance of this gene polymorphism in GC.Further studies on larger study group with expression of PPAR γ gene and determination of the presence of H.pylori infection and different ethnic groups are needed to confirm the association of this polymorphism with development and prognosis of GC.
patients (p: 0.01).Genotype frequencies of PPAR γ Pro12Ala among patients and controls are shown in Table

Table 3 . Frequencies of PPAR γ Genotype Distributions in Patients with GC and Controls
*p value less than 0.05 was considered as significant.