Significant increase in risk of gastroesophageal cancer is associated with interaction between promoter polymorphisms in thymidylate synthase and serum folate status

Wen Tan, Xiaoping Miao, Li Wang 1, Chunyuan Yu, Ping Xiong, Gang Liang, Tong Sun, Yifeng Zhou, Xuemei Zhang, Hui Li 1 and Dongxin Lin *

Department of Etiology and Carcinogenesis, Cancer Institute and 1 Department of Epidemiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

* To whom correspondence should be addressed. Tel: +86 10 87788491; Fax: +86 10 677 22460; Email: dlin{at}public.bta.net.cn


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thymidylate synthase (TS) catalyzes the 5,10-methylene-tetrahydrofolate-mediated conversion of deoxyuridine monophosphate to deoxythymydine monophosphate, a nucleotide required for DNA synthesis and repair. The impaired TS expression has been shown to be related to 28 bp tandem repeats and a G->C SNP in the 5'-UTR of TS. Folate deficiency has been demonstrated to play a role in gastroesophageal carcinogenesis. This case–control study was to examine the hypothesis that the TS polymorphisms, alone or in combination with serum folate status, may confer susceptibility of the hosts to gastroesophageal cancer. We analyzed TS genotype and serum folate concentration in 324 patients with esophageal squamous cell carcinoma (ESCC), 231 patients with gastric cardia adenocarcinoma (GCA) and 492 controls. It was found that compared with the normal expression TS genotype, the low expression TS genotype alone was significantly associated with increased risk of ESCC [adjusted odds ratio (OR) 1.47; 95% confidence interval (CI) 1.03–2.10] but not GCA (OR = 0.98, 95% CI = 0.68–1.40). More importantly, a significant interaction between the TS polymorphisms and serum folate status in risk of ESCC and GCA was observed. Among subjects with low serum folate concentration (<3 ng/ml), the ORs of ESCC and GCA for the low expression genotype were 22.63 (95% CI = 10.44–49.05) and 4.08 (95% CI = 1.94–8.59), which were greater than respective 9.97 (95% CI = 5.67–17.53) and 1.88 (95% CI = 1.18–3.24) for the normal expression genotype (P = 0.002 and 0.029). These results suggest an important role for folate deficiency and impaired TS activity in the etiology of ESCC and GCA.

Abbreviations: CI, confidence interval; ESCC, esophageal squamous cell carcinoma; GCA, gastric cardia adenocarcinoma; MTHFR, methylenetetrahydrofolate reductase; MTRR, methionine synthase reductase; OR, odds ratio; SHMT, serine hydroxymethyltransferase; SNP, single nucleotide polymorphism; THF, tetrahydrofolate; TS, thymidylate synthase; UTR, untranslated region


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), a critical reaction in maintaining a balanced supply of deoxynucleotides required for DNA synthesis and DNA repair (1). This reductive methylation reaction requires 5,10-methylene-tetrahydrofolate (THF) as methyl donor, which is derived from the metabolism of folate, an important nutrient whose deficiency has been associated with increased risk of certain cancers including gastroesophageal cancer (25). The balance of deoxynucleotide pools is an important mechanism in maintaining DNA integrity and preventing DNA damage from excess incorporation of uracil in replication (6,7). Accordingly, impaired TS activity and/or folate deficiency have been shown to be associated with increased chromosome damage and fragile site induction (710), which is believed to play an important role in tumorigenesis (11).

The 5'-UTR of human TS gene contains a number of cis-acting regulatory elements, in which a variable number of 28 bp tandem repeats, mainly 2R and 3R, has been identified, and this tandem repeat polymorphism contributes to the efficiency of expression of the gene (12). Individuals homozygous for the 3R/3R genotype have 3.6-fold elevated TS mRNA levels compared with those homozygous for the 2R/2R genotype (13). Recently, a G->C SNP within the second repeat of the 3R allele has been found and this base substitution disrupts an USF-1 binding consensus element and consequently down-regulates TS expression (14,15). Several previous studies have shown that the TS tandem repeat polymorphism may be a risk modifier of the occurrence and clinical outcome of certain malignant diseases such as colorectal cancer (16,17), lymphocytic leukemia (18), and malignant lymphoma (19), and it may also be a useful predictor of response to 5-FU-based chemotherapy due to the enzyme being a target of 5-FU (13,20,21).

Esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA) are two common cancers in the world and the mortality rates of these cancers are well known to be particularly high in certain parts of northern China. Interestingly, GCA appears to be more prevalent in areas of high-risk of ESCC in China. For example, in Linxian, a well-known high-risk area for ESCC, one-third of the gastroesophageal cancers occurred in the gastric cardia (22), suggesting that GCA may share common risk factors with ESCC for carcinogenesis. Folate deficiency resulting from low consumption of vegetables and fruits has been associated with increased risk of ESCC and GCA in at high-risk population in China (23,24). We and other investigators have previously shown that genetic polymorphisms in folate metabolizing enzymes including methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) as well substantially influence risk of ESCC and GCA in this population (2528). These data strongly suggest that folate deficiency and/or impaired folate metabolism play an important role in the etiology of these cancers.

Because TS is a unique enzyme catalyzing cellular conversion of dUMP to dTMP, a critical and rate-limiting step in maintaining a balanced deoxynucleotide supply required for faithful DNA synthesis and repair, we hypothesized that genetic polymorphism resulting in impaired TS expression or activity might also confer the hosts more susceptible to ESCC and GCA. This report described a case–control study that aimed to test this hypothesis. In this study, we evaluated the contribution of the tandem repeats and the G->C polymorphism in the TS promoter, alone and in interaction with serum folate status, to risk of the cancers.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
This study consisted of 324 patients with ESCC, 231 patients with GCA and 492 healthy controls and all subjects were ethnic Han Chinese. Patients were consecutively recruited from October 2000 to July 2002, at the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing), with a response rate of 94%. All patients were newly diagnosed incident cases and histopathologically confirmed ESCC or GCA. In this study, ESCC was defined as squamous cell carcinoma confined entirely to the esophageal subsite whereas GCA was defined as tumors arising at the gastric cardia and/or gastroesophageal junction with or without involvement of other esophageal and/or gastric subsite. Population controls were cancer-free individuals living in the same region and recruited during the same time period as the cases were recruited; they were selected from a community cancer screening program for early detection of cancer as described (26). Briefly, these controls were randomly selected from a pool of 2800 individuals based on a physical examination, and the response rate was 96%. They had no history of cancer and were frequency matched to ESCC cases on sex and age (±5 years). At recruitment, informed consent was obtained from each subject and each participant was then interviewed to collect detailed information on demographic characteristics and lifetime history of tobacco use. Five ml peripheral blood sample was drawn from each subject when physical examination was performed for both patients and controls. This study was approved by the Institutional Review Board of the Chinese Academy of Medical Sciences, Cancer Institute.

TS genotyping
Genomic DNA was isolated from peripheral blood of the subjects. TS genotyping was accomplished by PCR–RFLP analysis essentially as described by Mandola et al. (14). The primers used to amplify a 210 bp fragment for 2R allele and 238 bp fragment for 3R allele of the TS promoter containing G/C polymorphism were: TSF5'-CGT GGC TCC TGC GTT TCC C and TSR5'-GAG CCG GCC ACA GGC AT. PCR was carried out with a 25 µl reaction mixture containing ~100 ng DNA, 1.0 µM each primer, 0.2 mM dNTP, 1.25 mM MgCl2, 1.0 U Taq DNA polymerase with 1x reaction buffer (Promega, Madison, WI) and 10% dimethylsulfoxide. The PCR profile consisted of an initial melting step of 5 min at 95°C; followed by 35 cycles of 30 s at 95°C, 30 s at 63°C and 1 min at 72°C; and a final elongation step of 7 min at 72°C. PCR product was then electrophoresed to distinguish the tandem repeats (2R and 3R alleles). The PCR product containing the 3R allele was additionally digested with HaeIII restriction enzyme (New England BioLabs, Beverly, MA) to determine the G->C polymorphism. The digested and undigested PCR products from each patient were loaded into adjacent lanes on a 3% agarose gel (Figure 1). Due to loss of HaeIII restriction site, the 3Rc allele produces five fragments of 94, 47, 46, 44 and 7 bp, whereas the 3Rg allele generates 66, 47, 46, 44, 28 and 7 bp bands; the 3Rg/3Rc heterozygote had all 94, 66, 47, 46, 44, 28 and 7 bp fragments (Figure 1).



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Fig. 1. Representative gel picture showing PCR–RFLP analysis of the TS tandem repeats and G->C polymorphism. The PCR product from each subject was electrophoresed before HaeIII digestion to distinguish the 2R or 3R allele. A part of PCR product containing the 3R allele was additionally digested with HaeIII to determine the G->C genotype. M, DNA size markers; subjects 1, 2R/2R genotype; subject 2, 3Rg/3Rg genotype; subject 3, 3Rg/3Rc genotype; subject 4, 3Rc/3Rc genotype; subject 5, 2R/3Rg genotype; and subject 6, 2R/3Rc genotype.

 
Genotyping was performed without knowledge of case–control status, and a 10% random sample set of cases and controls was genotyped twice by different investigators, and the reproducibility was 100%.

Serum folate analysis
Blood sample collected from each subject was immediately refrigerated at 4°C and serum sample was isolated by centrifugation and stored at –80°C before analysis. Serum folate concentration was determined by radioisotope dilution assay with a commercially available SimulTRAC-SNB Radioassay Kit (ICN Pharmaceuticals, Orangeburg, NY) according to the manufacturer's instruction. All samples were assayed in duplicate and the average value for the duplicate determination was reported as the concentration for the sample. Below-normal concentration for serum folate was defined as <3 ng/ml (29).

Statistical analysis
{chi}2 test or t-test was used to examine differences in demographic variables, smoking, serum folate concentration, and distributions of genotypes between cases and controls. The associations between the TS polymorphism and risk of ESCC and GCA were estimated by odds ratios (ORs) and their 95% confidence intervals (CIs), which were calculated by unconditional logistic regression. The ORs were all adjusted for age, sex, smoking, or serum folate status where it was appropriate. Test for interaction between TS polymorphism and serum folate concentration were performed by using the likelihood ratio test. All of the statistical analysis was carried out using Statistical Analysis System software (version 6.12; SAS Institute, Cary, NC).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subject characteristics and serum folate concentration
The relevant characteristics of the study subjects are shown in Table I. The distributions of mean age and sex between ESCC cases and controls were not statistically different, suggesting that the frequency matching was adequate. However, although an effort was made simultaneously to obtain a frequency match on age and sex between GCA cases and controls, more males and more old subjects were presented in the GCA group (P < 0.0001, respectively). In terms of cigarette smoking, more smokers were presented in ESCC cases compared with controls (57.7 versus 44.7%; P < 0.0001), but this difference was not significant between GCA cases and controls. We observed a significant difference in serum folate concentration between ESCC or GCA cases and controls. The mean ± SD of serum folate concentration was 4.35 ± 1.59 ng/ml in controls, which was significantly higher than those in ESCC cases (1.97 ± 1.03 ng/ml; P < 0.0001) and GCA cases (3.56 ± 1.98 ng/ml; P < 0.0001). Seventy-three percent of ESCC cases and 32% GCA cases had serum folate <3 ng/ml; these proportions were much higher than that of controls (16.9%; P < 0.0001, respectively). The adjusted ORs of ESCC and GCA were 13.73 (95% CI = 9.61–19.60) and 2.43 (95% CI = 1.64–3.60), respectively, for low serum folate status (<3 ng/ml) compared with high serum folate status (≥3 ng/ml). In addition, we detected a significantly lower level of serum folate among smokers compared with non-smokers (4.04 ± 1.56 ng/ml versus 4.57 ± 1.60 ng/ml; P = 0.008) in controls. However, in patient groups, this difference between smokers and non-smokers was not significant (1.88 ± 1.97 ng/ml versus 2.05 ± 1.81 ng/ml in ESCC patients, P = 0.235; 3.54 ± 2.05 ng/ml versus 3.73 ± 1.91 ng/ml in GCA patients, P = 0.373). In order to examine influences of TS genotype on serum levels of folate, ANOVA was used to calculate age-adjusted means among the control individuals with the different TS genotypes, compared with a trend test. No effects of the TS polymorphisms on serum folate levels were found after adjusting for age, sex, and smoking status (data not shown). We also observed a concomitant lower serum vitamin B12 concentration among ESCC or GCA cases compared with controls (data not shown).


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Table I. Distributions of select characteristics by case–control status

 
The association between TS genotype and risk of ESCC and GCA
Genotyping results (Table II) showed that the allele frequencies for the TS 2R, 3Rc and 3Rg were 17.6, 40.3 and 42.1%, among controls, compared with 20.5, 41.4 and 38.1% among ESCC cases and 19.1, 39.3 and 41.6% among GCA cases, respectively. The frequencies of six TS genotypes, i.e. 3Rg/3Rg, 3Rg/3Rc, 3Rc/3Rc, 3Rg/2R, 3Rc/2R and 2R/2R, were 16.1, 38.0, 14.4, 14.0, 13.8 and 3.7%, respectively, among controls, which fit the Hardy–Weinberg equilibrium ({chi}2 = 2.47, P = 0.78). The respective TS genotype frequencies among ESCC cases were 14.2, 35.2, 14.5, 12.7, 18.5 and 4.9%, and among GCA cases were 16.5, 37.6, 13.8, 12.6, 13.4 and 6.1%, both of which did not differ significantly from those among controls. We also observed 4 controls, 1 ESCC case and 2 GCA cases carrying the 3R/5R genotype, and 2 GCA cases carrying the 2R/5R genotype. Because these genotypes were extremely rare, they were excluded from this study. Multivariate logistic regression analysis showed that the tandem repeat polymorphism of TS was not associated with risk of ESCC or GCA. However, when the G/C SNP within the second repeat of the 3R allele was considered in the analysis, subjects having the 2R/2R genotype had a >3-fold increased risk for ESCC compared with those having the 3Rg/3Rg genotype (Table III). Risk of GCA related to the 2R/2R genotype was also increased (OR = 1.74, 95% CI = 0.69–4.36), although it was not statistically significant. Because the 3Rc and 2R alleles are related to impaired TS expression (1215), we therefore combined these alleles for further estimation of the risk. It was found that the combined 3Rc/3Rc, 3Rc/2R and 2R/2R genotype was also associated with an increased risk of ESCC compared with the combined 3Rg/3Rg, 3Rg/3Rc and 3Rg/2R genotype (adjusted OR = 1.47, 95% CI = 1.03–2.10), although it was not the case for GCA (adjusted OR = 0.98, 95% CI = 0.68–1.40).


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Table II. Distribution of TS 5'-UTR tandem repeat and the G->C polymorphism in the 3R allele among controls and cases with ESCC or GCA

 

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Table III. Risk of ESCC and GCA associated with TS genotypes

 
Interaction between TS genotype and serum folate concentration
The association between TS genotype and risk of ESCC or GCA by serum folate concentration was further analyzed. The likelihood ratio test indicated a significant interaction between the TS polymorphism and serum folate status on risk of ESCC or GCA (both P = 0.001). As shown in Table IV, the low expression genotype (3Rc/3Rc, 3Rc/2R or 2R/2R) did not significantly increase ESCC risk among subjects whose serum folate concentration was ≥3 ng/ml (OR = 1.35, 95% CI = 0.85–2.14). However, among subjects whose serum folate concentration was <3 ng/ml, the OR of ESCC for the low expression genotype increased to 22.63 (95% CI = 10.44–49.05), which was significantly >9.97 (95% CI = 5.67–17.53) for the normal expression genotype (3Rg/3Rg, 3Rg/3Rc and 3Rg/2R; P = 0.002, test for homogeneity). Similar results were also observed in GCA, with the OR being 4.08 (95% CI = 1.94–8.59) among subjects having both low expression genotype and low serum folate compared with 1.88 (95% CI = 1.18–3.24) among subjects having the normal expression genotype but low serum folate status (P = 0.029, test for homogeneity).


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Table IV. Risk of ESCC and GCA related to TS genotype by serum folate status

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies shown by us and other groups have demonstrated that impaired folate metabolism resulting from genetic polymorphisms in folate-metabolizing enzymes such as MTHFR and MTRR are associated with increased risk of gastroesophageal cancer in a Chinese population (2528). In the present study, we extend the results to genetic polymorphism in TS, another key enzyme involved in the metabolism of folate. We observed that the low expression alleles (2R and 3Rc) were associated with increased risk of ESCC and GCA. Moreover, we demonstrated a statistically significant interaction between the TS polymorphism and serum folate concentration in intensifying risk for the development of these two cancers. These results are in agreement with our prior hypothesis that genetic polymorphism resulting in impaired TS expression or activity may confer the hosts susceptible to ESCC and GCA, and further support epidemiological observations that folate deficiency may play an important role in risk of gastroesophageal carcinogenesis (25,23,24,30).

Our findings showing an association between the TS polymorphisms and increased risk of ESCC and GCA may reflect impaired function of TS in the conversion of dUMP to dTMP. The investigated polymorphisms in the TS 5'-UTR (2R and 3Rc alleles) have been demonstrated to down-regulate the expression of TS (12,14,15). Suppressed TS expression and/or folate deficiency may cause an increase in intracellular dUMP, thus leading to uracil misincorporation into DNA (6,7,10,11). This misincorporation is believed to particularly occur in some tissues such as gastroesophageal epithelia, where the rate of DNA replication is high due to rapid turnover of the cell. Accumulation of high level uracil in DNA may consequently generate mutagenic mispairs and abasic sites and cause the degradation of newly synthesized DNA due to excision repair by uracil DNA glycosylase, which tends to cause double-strand DNA breaks (10,3133). DNA strand breaks could contribute to chromosome instability, translocations and aberrations, which have been observed in folate-deficiency cells and TS-negative mutant cells (8,10,34) and are common events in gastroesophageal cancer (35,36). On the other hand, the increased risk of ESCC and GCA among subjects with the low expression TS genotype may also result from impaired DNA repair. It has been documented that folate deficiency significantly reduces DNA repair capacity both in experimental animals and in humans (37,38). Therefore, impaired TS activity resulting from the genetic polymorphisms, which can be considered to further impair folate status, may suppress DNA repair capacity by reduced thymidylate bioavailability or other unrecognized mechanisms. In this context, it would be expected to observe a significant association between the functional polymorphisms in TS and elevated risk of ESCC and GCA.

In the present study, we observed a significant gene–nutrient interaction between the TS polymorphisms and serum folate status, and subjects having the low expression TS genotype and low serum folate concentration were at highest risk for both ESCC and GCA. This statistical interaction is biologically plausible, because TS catalyzed dUMP to dTMP conversion requires 5,10-methylene-THF as methyl donor, which is a central metabolite in folate metabolism. In order to keep balances of nucleotide pools, it is necessary for individuals to have both normal TS activity and abundant folate provision. Theoretically, either of TS defect or short of folate provision may cause imbalances in thymidylate synthesis and concomitant TS defect and low folate intake may exacerbate the problem and are thus expected to have the greatest risk of the cancers. However, it is interesting to note that although the TS polymorphisms and low serum folate status together had a greater than multiplicative interaction, risk of ESCC related to low serum folate concentration appeared to be higher than that related to the low expression TS genotype (Table IV). These findings may reflect the fact that the polymorphisms of TS confer only less but not complete loss of TS expression and suggest that a proper intake or supplementation of folate may overcome the effects of genetically determined reduction in TS activity. This conception might warrant folate chemoprevention of gastroesophageal cancer in at-risk population of individuals carrying the low expression TS genotype. Furthermore, we observed a significantly lower serum folate level among smokers compared with non-smokers in control subjects, although this difference was not significant in the patient groups. This finding is consistent with previous reports showing that smoking is inversely associated with plasma folate concentrations (3941). The null correlation between serum folate levels and smoking in ESCC and GCA groups might be attributed to quitting smoking of the patient smokers at the time of blood donation because of cancer diagnosis. Smoking has been shown to be a risk factor for ESCC and GCA (4244). The effect of smoking on folate may eventually contribute to folate deficiency in smokers and subsequently aggravate the hazard effect by genetic polymorphisms in folate-metabolizing enzymes such as TS.

Several case–control studies on the relationship between TS polymorphism and risk of other cancers have been published. Consistent with our findings, Skibola et al. (18) reported an increased risk of adult acute lymphocytic leukemia associated with the 2R allele and the 3R allele had a strong protective effect. Furthermore, the 2R allele appeared to interact with the T allele of SHMT1, another enzyme that plays an important role in providing one-carbon units for purine and thymidylate synthesis, to further increase the risk. Hishida et al. (19) showed similar results in their study on malignant lymphoma, indicating the 2R allele as risk allele and a combined effect of the TS 2R and SHMT1 T alleles. However, Ulrich et al. (17) and Chen et al. (16) observed a non-significantly lower risk of colorectal adenoma and carcinoma for the TS 2R allele. In addition, it appeared that among the 2R homozygotes, high folate intake was associated with a 1.5-fold increased risk of colorectal adenoma (17). Recently, Shi et al. (45) have shown that an increased lung cancer risk was not associated with the TS 5'-UTR polymorphism but was associated with the 3'-UTR 6-bp insertion polymorphism. In contrast, Zhang et al. (46) observed a decreased risk of ESCC and GCA associated with the TS 3'-UTR 6 bp insertion/5'-UTR 3Rg haplotype, which may produce a high level of TS; however, these authors reported confusing results showing that the decreased risk of ESCC and GCA was also associated with the TS 3'-UTR 6-bp deletion/5'-UTR 2R haplotype, which theoretically produces a low level of TS. The reasons for these contrary results from previous studies with different cancer sites and different ethnic populations are not clear. However, all of these studies except that of Zhang et al. (46) did not further type the G/C SNP within the second repeat of the 3R allele, which might have made misclassification of genotypes in terms of function and biased their results because a sizeable fraction of subjects with the 3R/3R genotype demonstrated low TS expression due to the SNP (14,15). Therefore, it would be helpful for these studies to re-evaluate the association between the tandem repeat polymorphism and the cancer risk by taking into account the G/C SNP. In addition, because subjects' folate status may be crucial in evaluating the risk of cancer related to genetic polymorphisms in folate metabolizing enzymes, analysis of gene–folate interaction would immensely enhance the accurate estimation.

Our study may have some limitations. Because it was a hospital-based, case–control study the patients may not be representative of the population at large. It would therefore be important to confirm these results in population-based studies. In addition, although the TS polymorphism investigated is a constitutive marker, the folate concentration in serum is a one-time measurement, which may not be reflective of long time folate status, a problem often experienced by retrospective studies. Moreover, one may argue that the low serum folate concentrations among patients could result from the disease status. However, because our patients were initially diagnosed and their blood samples were taken before surgery or other anti-cancer treatment, it could be unlikely that the low serum folate was caused by an ingestion problem or disease treatment. The low serum folate concentrations among patients may thus be indicative of folate deficiency because previous studies in Chinese and other populations have consistently shown an inverse association between consumption of vegetables and fruits, a major source of folate, and risk of gastroesophageal cancer (25,23,24). Population-based prospective studies will be helpful to clarify this gene–nutrient interaction between the TS polymorphism and folate status.

In summary, our study demonstrated an association between TS polymorphism and risk of ESCC and GCA. We found that the low expression TS alleles alone, or in interaction with low serum folate status, significantly increased the risk of cancer. These data extend our previous findings showing a strong association between the elevated risk of ESCC and GCA and genetic polymorphisms in MTHFR, another key enzyme in folate metabolism and further support the hypothesis that folate deficiency and impaired folate metabolism may play an important role in the etiology of ESCC and GCA.


    Acknowledgments
 
This work was supported by grants from Beijing Municipal Commission of Science and Technology (No. H0209-20030130) and National Natural Science Foundation (No. 30270598).

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 28, 2005; revised March 27, 2005; accepted March 29, 2005.





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