The VEGF -634G>C promoter polymorphism is associated with risk of gastric cancer

Background Both TGF-β1 and VEGF play a critic role in the multiple-step process of tumorgenesis of gastric cancer. Single nucleotide polymorphisms (SNPs) of the TGFB1 and VEGF genes have been associated with risk and progression of many cancers. In this study, we investigated the association between potentially functional SNPs of these two genes and risk of gastric cancer in a US population. Methods The risk associated with genotypes and haplotypes of four TGFB1 SNPs and four VEGF SNPs were determined by multivariate logistic regression analysis in 171 patients with gastric cancer and 353 cancer-free controls frequency-matched by age, sex and ethnicity. Results Compared with the VEGF-634GG genotype, the -634CG genotype and the combined -634CG+CC genotypes were associated with a significantly elevated risk of gastric cancer (adjusted OR = 1.88, 95% CI = 1.24-2.86 and adjusted OR = 1.56, 95% CI = 1.07-2.27, respectively). However, none of other TGFB1 and VEGF SNPs was associated with risk of gastric cancer. Conclusion Our data suggested that the VEGF-634G>C SNP may be a marker for susceptibility to gastric cancer, and this finding needs to be validated in larger studies.


Background
Gastric cancer is one of the major causes of cancer-related deaths worldwide, although both incidence and mortality of gastric cancer have been declining in recent years [1]. In 2008, there were 21,500 new gastric cancer patients, and 10,880 patients died of this disease in the United States [2]. Epidemiological studies have identified many risk factors for gastric cancer, including older age, being a man, Helicobacter pylori (H. pylori) bacteria infection, diets high in smoked foods, salted fish and meats, and pickled vegetables, tobacco use and obesity [2]. Although about two-thirds of gastric cancer could be prevented by changing lifestyle and diet habits [3], the fact that some individuals develop gastric cancer while others do not when having similar exposures suggests that genetic factors may also play an important role in the etiology of gastric cancer.
It has been reported that both TGF-β1 and VEGF played an important role in the oncogenesis of gastric cancer [4,5]. Both TGFB1 and VEGF genes are highly polymorphic, reportedly having 168 and 140 variants, respectively, but few are within the promoters or coding regions that may be potentially functional http:// www.ncbi.nlm.nih.gov/SNP/. Of these variants, several common single nucleotide polymorphisms (SNPs) have been described as important [6][7][8] and reported to be involved in the etiology of various cancers [9][10][11][12]. For example, some TGFB1 and VEGF variants are associated with protein functions, which may contribute to an individual's susceptibility to cancer [13]. Several studies have investigated the association between TGFB1 and VEGF SNPs and risk of cancers, including breast cancer [14][15][16], lung cancer [17,18], and gastric cancer [19][20][21][22], but the results were inconsistent.
To investigate the role of TGFB1 and VEGF SNPs in the etiology of gastric cancer, we conducted a case-control study to evaluate the association between three TGFB1 SNPs (one promoter SNP -509C>T and two exon 1 SNPs +869T>C and +915G>C) and three VEGF SNPs (one promoter SNP -1498T>C, one 5'-untranslated region SNP -634G>C and one 3'-untranslated region SNP +936C>T) and gastric cancer risk in a US population.

Study population
The case-control study consisted of 171 patients with newly diagnosed and histologically confirmed gastric cancer at The University of Texas MD Anderson Cancer Center (Houston, TX) between February 1990 and July 2008. The 353 cancer-free controls subjects were selected from an ongoing molecular epidemiology study of the head and neck cancer since 1995 [23] by frequency matching by age, sex and ethnicity. All cancer-free control subjects were recruited from persons who were not hospital patients, nor seeking health care but accompanying the patients visiting the clinics, and who were genetically unrelated to the cases. The study protocol and informed consent form used in this study were approved by our institutional review board.

Genotyping
Genomic DNA was extracted from the buffy coat fraction of each blood sample by using a Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. DNA purity and concentrations were determined by spectrophotometric measurement of absorbance at 260 and 280 nm by UV spectrophotometer.
The selected three TGFB1 SNPs [-509C>T (rs1800469) in the promoter, +869T>C (rs1800470) and +915G>C (rs1800471) in the exon 1] and three promoter VEGF SNPs [-1498T>C (rs833061) in the promoter and -634G>C (rs2010963) in the 5'-untranslated region, and VEGF +936C>T (rs3025039) in the 3'-untranslated region] were genotyped using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method. These six SNPs are relatively common (a minor allele frequency > 0.05) in Caucasian populations. Genotypes of TGFB1 SNPs were determined as previously described [24]. Assays on the VEGF -1498T>C (rs833061), -634G>C (rs2010963), and +936C>T (rs3025039) SNPs were previously reported [17] For the PCR-RFLP assay, the genotyping was performed without knowing the subjects' case and control status, and almost the same number of cases and controls were assayed in each 96-well PCR plate. Two research assistants independently read the gel pictures and performed the repeated assays, if they did not reach a consensus on the tested genotype. In addition, 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant.

Statistical Analysis
The χ 2 tests were used to compare the distribution of demographic variables and selected risk factors between cases and controls. The Hardy-Weinberg equilibrium (p 2 +2pq+q 2 = 1), where p is the frequency of the variant allele and q = 1-p, was tested by a goodness-of-fit χ 2 test to compare the observed genotype frequencies with the expected genotype frequencies in cancer-free controls. Unconditional logistical regression analysis was used to calculate odds ratios (ORs) and their 95% confidence intervals (CIs) with and without adjustment for age, sex, ethnicity, smoking status and alcohol use. Age was also dichotomized according to the median age (57 years) of the controls. The logistic regression model was also used for the trend test of the genotypes with different number of the minor alleles. The 2-linkage disequilibrium (LD) program and Proc ALLELE in SAS/Genetics software were used to detect the LD of any pair of SNPs. Proc HAPLO-TYPE in SAS/Genetics software using the expectation-maximization (EM) algorithm was applied to generate maximum likelihood estimates of haplotype frequencies.
Haplotype were combined when their frequency was <5%. The joint effects of the TGFB1 and VEGF SNPs and their interactions with smoking and drinking on gastric cancer risk were also evaluated. Finally, we calculated the false-positive report probability (FPRP) to test for falsepositive associations, using prior probabilities of 0.0001, 0.001, 0.01, 0.1, and 0.25. The OR was set close to the observed value obtained in our study, and a probability of <0.2 was considered a noteworthy association. All statistical tests were two-sided, with a P value of 0.05 considered significant, by using SAS software (version 9.1; SAS Institute, Cary, NC).

Characteristics of the study population
This analysis included 171 gastric cancer cases and 353 cancer-free controls. Because we used frequency matching, there were no significant difference in distributions of age, sex and ethnicity between the cases and controls (P = 0.135, 0.444 and 0.486, respectively). For the tumor types, more than 50% of the cases had intestinal type, about 125 had difuse type, and the remaining was mixed. There were more current and former smokers in cases (19.9% and 39.2%, respectively) than in controls (13.6% and 38.5%, respectively), but the difference was not statistically significant (P = 0.126). However, there were significantly more alcohol drinkers among cases than among controls (P = 0.037) ( Table 1). These variables were further adjusted for in multivariate logistic regression models to control for any residual effect of their confounding on the main effect of selected SNPs.

TGFB1 and VEGF genotypes and risk of gastric cancer
The genotype and allele distributions of the three TGFB1 SNPs and three VEGF SNPs between the cases and controls are summarized in Table 2. The observed genotype frequencies of these SNPs were all in agreement with the Hardy-Weinberg equilibrium in the control subjects, except for TGFB1 -509C>T and VEGF -634G>C, indicating some possible selection bias in the hospital-based controls. Although none of the minor alleles had a different frequency distribution between the cases and the controls, the VEGF-634G>C genotype frequencies were statistically significantly between the cases (40.4% for GG, 42.1% for CG, and 17.5% for CC) and the controls (51.0% for GG, 28.0% for CG and 21.0% for CC) (P = 0.003) ( Table 2).
Further logistic regression analysis revealed that, of the six SNPs examined, only the VEGF -634CG heterozygous and the combined -634CG+CC variant genotypes were associated with a significantly elevated risk of gastric cancer (adjusted OR = 1.88, 95% CI = 1.24-2.86 and adjusted OR = 1.56, 95% CI = 1.07-2.27, respectively). In further stratified analysis by age, sex, ethnicity, alcohol use and smoking status, the risk associated with the combined -634CG+CC variant genotypes was more evident in subgroups of ever smokers (adjusted OR = 1.75, 95% CI = 1.06-2.90), never drinker (adjusted OR = 2.00, 95% CI = 1.14-3.54) and non-white subjects (adjusted OR = 2.39, 95% CI = 1.15-4.98) ( Table 3). This kind of significant association was not found for the three TGFB1 SNPs and other two VEGF SNPs (Table 2).

TGFB1 and VEGF haplotypes and risk of gastric cancer
To further analyze possible combined effects of the TGFB1 and VEGF SNPs, we estimated the haplotypes on the basis of the observed genotypes. As shown in Table 4, the four common TGFB1 haplotypes (CTG, CCG, TCG and CCC) represented 85.6% and 89.9% of the chromosomes of the controls and cases, respectively. For the VEGF haplotypes, the common alleles included CGC, TCC, CCC and CGT. However, the frequency distributions of these haplotypes between the cases and controls was not significantly different for both TGFB1 (P = 0.759) and VEGF (P = 0.808), nor were they associated with risk of gastric cancer.
Finally, because the sample size of this study was small, we calculated FPRP for the positive findings. We found that under the assumption of a prior probability of 0.

Discussion
Gastric cancer is a genetic disease developing from a multifactorial, multigenetic and multistage process [25,26]. It was widely accepted that both genetic and environmental factors may be involved in the etiology of gastric cancer  [27]. During the multistage carcinogenesis, TGFB1 may be involved in multiple important cellular processes, play a biphasic role in carcinogenesis, and inhibit proliferation of tumors in early stages of cancers, whereas it may also promote tumor growth and metastasis in later stages of cancers [28,29]. Several molecular epidemiological studies have shown an association of TGFB1 SNPs with risk of various cancers, including breast [14,15,30,31], prostate [32,33] and hepatocellular carcinoma [34,35] and gastric cancer [4,19].
The roles of -509C>T, +869T>C and +915G>C SNPs in TGFB1 have been implicated in the etiology of various human cancers. For example, in high-risk Chinese populations, TGFB1 -509C>T was associated with a reduced risk of gastric cancer [19] and the -509T variant genotypes were found to be associated with a decreased risk of among stage I+II cases [20]. In the present study, we did not observe a significant association between TGFB1 SNPs and gastric cancer risk, partly because our study size was small. Another possible explanation is that there may be an ethnicity difference in the etiology of gastric cancer. For example, the H. pylori infection was common in Asians but not in Caucasians [36], which may interact with genetic factors. Furthermore, cancer is a disease involving multiple genes/SNPs, and therefore the combined analysis of multiple gene or haplotype-based approach might be more powerful than the analysis of single allele or locus effect [19], which requires studies with much large sample sizes. It is also likely that our results could be biased owing to possible selection biases in hospitalbased case-control studies. Therefore, larger studies are warranted to further test whether the TGFB1 variants are associated with gastric cancer risk.
Among other genetic factors, VEGF, the key mediator of angiogenesis, also plays an important role in the development of different kind of tumors, including gastric cancer. Several studies have investigated the associations between   [16,17,21,22,37], However, the results from published studies remain inconsistent rather than conclusive. Recently, Ke Q et al [21] found that none of four potentially functional SNPs (-2578C>A, -1498T>C, -634G>C, and +936C>T) of the VEGF gene or their haplotypes achieved a significant difference in their distributions between gastric cancer cases and controls in a Chinese population, whereas in a Greek study of the association between these four potentially functional SNPs in VEGF and gastric cancer risk in 100 cases and 100 controls [38], it was found that the -634CC variant genotype was associated with an increased the risk of gastric cancer with a marginal significance. However, the -634CC was shown to be associated with a significantly decreased risk of gastric cancer in a Korean population [22]. Our current study indicated that the heterozygous -634CG and the combined -634CG+CC carriers had an increased risk of gastric cancer when compared with the -634GG genotype. Again, these studies demonstrated some possible ethnic difference in the etiology of gastric cancer [19].
An obvious limitation in the present study was the lack of information on the H. pylori infection status of the subjects. Because H. pylori infection was relatively rare in the US compared with Asian countries, these patients were not tested for H. pylori infection upon their visit to the hospital. This defect of the study has to be corrected in future well-planned studies. Also, we observed that genetic effects were more profound in never drinkers in this study population, a phenomenon we often observed in our association studies, in which the risk associated with adverse genotypes appears to be higher in nonexposed or less exposed subjects, suggesting that the related susceptibility to cancer may be high in such subgroups. Alternatively, there may be some unknown risk factors that were not identified in these subgroups.

Conclusion
In this hospital-based case-control study of sporadic gastric cancer, our finding supports the notion that variant genotypes of the VEGF-634C>G polymorphism, but not other tested SNPs, may contribute to gastric cancer risk. However, because we used a hospital-based case-control study design, the observed associations may have been biased or simply due to chances. Therefore, larger, prospective studies, particularly with the information about H. pylori infection, are needed to verify our findings.