Case–control analysis of thymidylate synthase polymorphisms and risk of lung cancer

Qiuling Shi, Zhengdong Zhang, Ana S. Neumann, Guojun Li, Margaret R. Spitz and Qingyi Wei1

Department of Epidemiology, The University of Texas M.D.Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA

1 To whom correspondence should be addressed. Tel: +1 713 792 3020; Fax: +1 713 792 0807; Email: qwei{at}mdanderson.org


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although tobacco smoking is the primary risk factor for lung cancer, low dietary folate intake and suboptimal DNA repair capacity also contribute to lung cancer risk. Thymidylate synthase (TYMS) is involved in the metabolism of folate and the provision of nucleotides needed for DNA synthesis and repair. Thus, a variation in TYMS functions likely plays a role in the etiology of lung cancer. The TYMS gene has a tandem repeat polymorphism (two or three 28 bp) in the TYMS enhancer region (TSER) and a 6 bp deletion/insertion polymorphism in the TS 3'-untranslated region (TS3'UTR or 1494del6). We investigated the frequencies of these two polymorphisms in a hospital-based case–control study of 1055 lung cancer patients and 1140 cancer-free controls in a non-Hispanic white population and genotyped for these two polymorphisms. We found that the TS3'UTR, but not the TSER, variant was associated with the risk of lung cancer. Compared with homozygotes for the TS3'UTR 6 bp deletion (0bp/0bp), the 6bp/0bp+6bp/6bp genotypes were associated with a significantly increased risk of lung cancer [odds ratio (OR) = 1.52, 95% confidence interval (CI) = 1.12–2.06]. In stratification analysis the risk associated with the 0bp/6bp+6bp/6bp genotype was more pronounced in subjects who were >55 years old (OR = 1.57, 95% CI = 1.10–2.23), males (OR = 1.88, 95% CI = 1.22–2.89), current (OR = 2.04, 95% CI = 1.26–3.29) and heavy smokers (OR = 1.73, 95% CI = 1.10–2.70) and current drinkers (OR = 3.17, 95% CI = 1.78–5.64).Furthermore, significant gene–dietary interactions were found between TS3'UTR and alcohol consumption and between TSER and vitamin B12 intake. In conclusion, the polymorphisms of TYMS are likely to contribute to the risk of lung cancer in non-Hispanic whites and interact with dietary factors in lung cancer development.

Abbreviations: ALL, acute lymphocyte leukemia; CI, confidence interval; OR, odds ratio; TSER, thymidylate synthase enhancer region; TS3'UTR, thymidylate synthase 3'-untranslated region; TYMS, thymidylate synthase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lung cancer is the most common cause of cancer-related deaths in the USA, accounting for an estimated 173 770 new cases and 160 440 deaths in 2004 (1). Although overwhelming epidemiological evidence exists that smoking is the primary risk factor for lung cancer (2), <20% of lifetime smokers develop lung cancer, suggesting that interindividual variation in genetic susceptibility to lung cancer exists in the general population (3). Suboptimal DNA repair capacity for removing tobacco-induced DNA adducts has been shown to be a risk factor for lung cancer and could account for part of this interindividual variation in susceptibility (3,4). Low folate intake leads to an increase in DNA damage (5) and DNA repair capacity may be modulated by dietary folate intake (6), providing a biologically plausible link between phenotypically inefficient DNA repair and dietary factors in lung cancer risk (38). By identifying biomarkers for underlying genetic susceptibility to tobacco carcinogenesis, risk assessment of lung cancer in smokers can be facilitated.

Thymidylate synthase (TYMS), a key enzyme of folate metabolism, catalyzes the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate and 5,10-methylenetetrahydrofolate to dihydrofolate (9). Deoxyuridine monophosphate is essential for the provision of thymidine, a nucleotide needed for DNA synthesis and repair (10). Not surprisingly, therefore, impairment of the TYMS enzyme is associated with chromosome damage and fragile site induction (11,12), suggesting that DNA repair mechanisms can be affected by nucleotide availability. In addition, TYMS is a target for chemotherapeutic drugs such as 5-fluorouracil and TYMS mRNA and protein expression levels are considered prognostic indicators for several cancers (1316). Therefore, genetic variation and in vivo regulation of TYMS are likely to be important in both cancer etiology and outcome.

In humans, a polymorphic tandem repeat element in the TYMS promoter enhancer region (TSER) has been identified near the initiation start site. This variant is most frequently present as either two (2R) or three (3R) repeats (17), although more rare alleles such as 4R, 5R and 9R also exist (18). TSER*3R confers a higher translational efficiency than does TSER*2R and, indeed, an in vitro study in HeLa S3 cells has demonstrated that the TSER*2R allele is associated with less than half the TYMS protein expression as the TSER*3R allele (17). In vivo TYMS mRNA levels in tumor tissue were 3-fold lower among subjects with 2R/2R homozygotes than among subjects with 3R/3R homozygotes (19). Furthermore, the 3R/3R homozygous genotype has been associated with increased levels of TYMS protein expression and higher absolute enzyme activity (20). In turn, lower expression levels of TYMS may reduce the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate, thereby increasing the chance of uracil misincorporation into DNA, which could increase the number of DNA double-strand breaks in rapidly proliferating tissues. Recently, a second TYMS polymorphism was identified (21): a 6 bp deletion/insertion at bp 1494 in the 3'-untranslated region of the TYMS gene (TS3'UTR or 1494del6, dbSNP accession no. rs16430). To date, several reported studies have investigated a possible association between the TS3'UTR 6 bp deletion/insertion and risk of colon cancer, but no consistent results were demonstrated (2123).

Although the protective effect of the TSER tandem repeat polymorphism in lung cancer chemotherapy has been well studied (24,25), the association between TYMS polymorphisms and risk for lung cancer has not been studied. Therefore, in this study, as part of an ongoing hospital-based case–control study of lung cancer, we tested our hypothesis that the TSER and TS3'UTR polymorphisms are associated with lung cancer risk and that these polymorphisms may interact with nutrient intake.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
Patients were recruited between September 1995 and November 2003 without restrictions as to age, sex, stage or histology from an ongoing molecular epidemiological study of lung cancer conducted by the Department of Epidemiology of the The University of Texas M.D.Anderson Cancer Center (Houston, TX). The study has been described in detail previously (3,4,6). All patients included in this analysis had newly diagnosed, histopathologically confirmed and previously untreated (by radiotherapy or chemotherapy) lung cancer without a reported history of any other cancer and all patients were non-Hispanic whites. The controls were selected from a pool of cancer-free subjects recruited at the largest multispecialty physician practice, the Kelsey Seybold Clinic, which has multiple clinics throughout the Houston metropolitan area. The controls were frequency matched to the cases with regard to age (±5 years), sex, ethnicity and smoking status (i.e. current, former and never). Exclusion criteria for all control participants were previous treatment with radiotherapy or chemotherapy and previous cancer. The participation response rate was 77.4% among the cases and 73.3% among the controls. Subjects were interviewed after providing signed informed consent. We used a structured questionnaire to collect information on demographic data and risk factors, such as smoking status and pack-years smoked. Participants who had smoked <100 cigarettes in their lifetimes were categorized as never smokers and the rest were categorized as ever smokers; those ever smokers who had quit smoking more than 1 year previously were considered former smokers and the rest were considered current smokers. Participants who had drunk alcoholic beverages at least once a week for 1 year or more were categorized as ever drinkers and the rest were defined as never drinkers; those ever drinkers who had quit were defined as former drinkers and the rest were categorized as current drinkers. Dietary nutrient assessments have been described in detail in previous studies (7). Trained interviewers collected dietary data using a modified version of the National Cancer Institute Health Habits and History Questionnaire, which assessed the dietary intake in the year prior to diagnosis for the cases and in the previous year for the controls. Total folate intake was adjusted according to daily calorie intake and expressed as total folate as µg/1000 kcal/day. The study was approved by the institutional review boards of the M.D.Anderson Cancer Center and the Kelsey Seybold Clinic.

Genotyping
Using 1 ml of whole blood from each blood sample, a leukocyte pellet was obtained from the 200 µl buffy coat fraction by centrifugation. Genomic DNA was extracted using a Qiagen DNA Blood Mini Kit (Qiagen, Valencia, CA). DNA purity and concentrations were determined by spectrophotometric measurement of absorbance at 260 and 280 nm.

To analyze the TSER 28 bp repeat polymorphism, we amplified the fragment using the following primers: TSER forward primer 5'-GTGGCTCCTGCGTTTCCCCC-3'; reverse primer 5'-GGCTCCGAGCCGGCCACAGGCATGGCGCGG-3' (17). PCR was performed in a total volume of 20 µl of 1x PCR buffer (50 mM KCl, 10 mM Tris–HCl and 0.1% Triton X-100), 1.5 mM MgCl2, 0.15 mM deoxyribonucleotide triphosphates, 100 nM each primer, 10% dimethylsulfoxide, 1.5 U Taq polymerase (Sigma-Aldrich, St Louis, MO) and ~50 ng genomic DNA. The cycling conditions consisted of: one cycle of 95°C for 5 min; 35 cycles of 95°C for 30 s, 63°C for 45 s and 72°C for 45 s; a final extension at 72°C for 10 min. The amplified fragments were separated in a 3% Nusieve agarose gel (Biowhittaker Molecular Applications, Rockland, ME). The fragments containing 3R and 2R repeats were 243 bp and 215 bp, respectively.

The TS3'UTR polymorphism was analyzed on the basis of PCR–restriction fragment length polymorphism. A fragment containing the 6 bp deletion/insertion was amplified using the primers 5'-CAAATCTGAGGGAGCTGAGT-3' and 5'-CAGATAAGTGGCAGTACAGA-3' (21) in 20 µl of reaction mixture containing 1x PCR buffer (50 mM KCl, 10 mM Tris–HCl and 0.1% Triton X-100), 1.5 mM MgCl2, 0.15 mM deoxyribonucleotide triphosphates, 100 nM each primer, 1.5 U Taq ploymerase (Sigma) and ~50 ng genomic DNA. The cycling conditions consisted of: a 5 min denaturation period at 95°C; 35 cycles of 95°C for 30 s, 58°C for 45 s and 72°C for 60 s; a 10 min extension at 72°C. The amplified fragments were digested with DraI (New England BioLabs, Beverly, MA) and the products separated in a 3% Nusieve agarose gel. The expected fragment sizes were 70 and 88 bp for the 6 bp inserted allele and 152 bp for the 6 bp deleted allele. More than 10% of the samples was randomly selected for repeat assays and the results were in 100% concordance.

Statistical analyses
The mean values of body mass index, total energy intake, total folate intake, dietary intake of vitamins B6 and B12 and pack-years smoked in cases and controls were compared using Student's t-test. Differences in categorized demographic variables, smoking status, alcohol consumption and allele and genotype frequencies between the cases and controls were tested by using the {chi}2 test. The associations between genotypes and risk of lung cancer were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) from both univariate and multivariate unconditional logistic regression analyses. The reference groups were subjects carrying TSER 2R2R or TS3'UTR 0bp/0bp. Stratification analysis was used to estimate risk for subgroups by age, sex, smoking status, pack-years smoked, alcohol consumption and dietary intake of folate, vitamin B6 and vitamin B12. The tertile values of continuous variables were used as cut-off points. P values for interaction were determined by the likelihood ratio test for the models with and without a multiplicative interaction term. All tests were two-sided and all statistical analyses were performed with Statistical Analysis System software version 8e (SAS Institute, Cary, NC).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 1055 cases and 1140 controls were available for this analysis. Because DNA samples from four cases failed to be amplified, the final analysis included 1051 cases and 1140 controls (Table I). No significant differences in age and sex were found between the cases and controls, suggesting that the cases and controls had been adequately frequency matched. However, there were more current smokers among the cases than among controls (P = 0.021) and the mean number of pack-years smoked in the cases (43.93 ± 35.11 years) was significantly higher than that of the controls (35.36 ± 30.74 years, P < 0.0001). Among all cases there were 509 (48.4%) adenocarcinoma, 226 (21.5%) squamous carcinoma, 204 (19.4%) non-small cell carcinoma, 73 (7.0%) small cell carcinoma and 39 (3.7%) unknown histological cell types.


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Table I. Frequency distributions of selected variables in lung cancer cases and cancer-free controls

 
Because some participants did not provide information on nutrient intake and alcohol consumption, only 957 cases and 1070 controls were included in the nutrient intake analysis, and 962 cases and 1072 controls were included in the alcohol analysis. The cases had a significantly lower mean body mass index (26.13 ± 5.00 kg/m2) than the controls (27.36 ± 5.03 kg/m2, P < 0.0001). Likewise, the cases in general reported statistically significantly lower dietary total folate intake (208.01 ± 69.36 µg/day/kcal) and vitamin B6 intake (1.70 ± 0.62 mg/day) than did the controls (total folate 220.50 ± 86.56 µg/day/kcal, vitamin B6 1.79 ± 0.76 mg/day; P = 0.0003 for folate and 0.003 for vitamin B6) (Table I). There were fewer (31.9%) current drinkers among the cases than among the controls (44.8%; P < 0.0001).

The TSER and TS3'UTR allele and genotype distributions among the cases and controls are shown in Table II. The TSER 2R allele frequencies were 0.466 and 0.458 in the cases and controls, respectively, and the TS3'UTR 6bp insertion allele frequencies were 0.704 and 0.678 in the cases and controls, respectively. These differences between the cases and controls were not statistically significant (P = 0.444 for TSER and P = 0.058 for TS3'UTR 6bp), although the 2R and 6bp insertion allele frequencies were slightly higher in the cases than in the controls. The distributions of these two genotypes in the controls were in Hardy–Weinburg equilibrium (P = 0.062 for TSER; P = 0.071 for TS3'UTR). The distributions of the three major TSER genotypes (3R3R, 3R2R and 2R2R) did not differ between the cases and controls, whereas the distributions of the three TS3'UTR genotypes differed significantly between the cases and controls (P = 0.043): the frequencies of the 0bp/0bp, 6bp/0bp and 6bp/6bp genotypes were 8.4, 42.4 and 49.2%, respectively, for the cases and 11.6, 41.3 and 47.1%, respectively, for the controls, suggesting that the 6bp insertion allele may be a risk allele.


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Table II. TSER and TS3'UTR genotypes and allele frequencies of the cases and cancer-free controls and their association with risk of lung cancer

 
Because of the uncertain function of the TSER 4R allele and its rare frequency, subjects with this allele were excluded from the following analysis related to TSER. In the univariate logistic regression both the 6bp/0bp and 6bp/6bp genotypes were associated with a significantly increased risk of lung cancer (OR = 1.42, 95% CI = 1.05–1.92 and OR = 1.44, 95% CI = 1.08–1.94, respectively) when compared with the TS3'UTR 0bp/0bp genotype. In the multivariate logistic analysis with adjustment for age, sex, smoking status, square root of pack-years, body mass index, alcohol intake, daily intake of energy, total folate and vitamin B6 and vitamin B12 intakes both 6bp/0bp and 6bp/6bp genotypes were associated with a significantly increased risk of lung cancer (OR = 1.52, 95% CI = 1.10–2.09 and OR = 1.52, 95% CI = 1.11–2.09, respectively) compared with the TS3'UTR 0bp/0bp genotype, suggesting that the 6bp insertion allele has a dominant effect (Table II). The adjusted OR for the combined TS3'UTR (6bp/0bp+6bp/6bp) genotype was 1.52 (95% CI = 1.12–2.06). However, the combined TSER (2R3R+2R2R) genotype was not associated with an elevated risk of lung cancer (adjusted OR = 1.03, 95% CI = 0.85–1.25) (Table II), although these two TYMS polymorphisms were in linkage disequilibrium (P < 0.0001; data not shown). Furthermore, the lung cancer risk factors included in this study were not associated with any of the genotypes (data not shown).

Results of analyses stratified by age, sex, smoking and alcohol use, daily dietary intake of folate, vitamin B6 and vitamin B12 are also summarized in Table III. Although no significant main effect of the TSER variant genotypes was found in each stratum, subjects >55 years old with one or two TS3'UTR 6bp insertion alleles had a significantly higher risk of lung cancer than did subjects with the 0bp/0bp genotype (OR = 1.57, 95% CI = 1.10–2.23). Similar results were also found for men (OR = 1.88, 95% CI = 1.22–2.89), current smokers (OR = 2.04, 95% CI = 1.26–3.29) and heavy smokers (OR = 1.73, 95% CI = 1.10–2.70) and current alcohol users (OR = 3.17, 95% CI = 1.78–5.64) (Table III). For nutrient intake, the subjects were categorized into low, medium and high intake groups on the basis of tertiles of the continuous variable in controls used as the cut-off points. As shown in Table III, for individuals with low vitamin B12 intake the 2R allele was associated with a significantly increased lung cancer risk (OR = 1.43, 95% CI = 1.02–2.01), whereas the 2R allele was associated with decreased risk of lung cancer in individuals with medium and high vitamin B12 intake (OR = 0.83, 95% CI = 0.58–1.17 for high vitamin B12 intake; OR = 0.88, 95% CI = 0.63–1.24 for medium vitamin B12 intake). These data were consistent with a significant interaction between this polymorphism and vitamin B12 intake (Figure 1) that was not evident for other nutrient intakes. Furthermore, we also found a significant interaction between the TS3'UTR 6bp variant genotypes and alcohol intake (Figure 2). For never and former drinkers the 6bp variant genotypes were not associated with an increased risk for lung cancer (OR = 1.16, 95% CI = 0.68–1.98 for never drinkers; OR = 1.01, 95% CI = 0.59–1.75 for former drinkers), whereas a >3-fold higher risk was found in current drinkers with the 6bp variant genotypes (OR = 3.17, 95% CI = 1.78–5.64) (Table III). This interaction was not evident for intake of nutrients. For different histological cell types the TS3'UTR 6bp insertion allele was only associated with a significantly higher risk of squamous cell lung cancer (OR = 2.05, 95% CI = 1.09–3.87) (Table III).


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Table III. Stratification analysis of the TYMS polymorphisms among lung cancer cases and cancer-free controls

 


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Fig. 1. Risk of lung cancer associated with the TSER polymorphism, stratified by vitamin B12 intake. Subjects with the TSER 3R3R genotype who reported high vitamin B12 intake were considered as the reference group. The P value for a gene–diet interaction was determined by likelihood ratio testing of models with and without a multiplicative interaction item.

 


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Fig. 2. Risk of lung cancer associated with the TS3'UTR polymorphism, stratified by alcohol consumption. Never drinkers with the 0bp/0bp genotype were considered as the reference. The P value for a gene–diet interaction was determined by likelihood ratio testing of models with and without a multiplicative interaction item.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study we found that, overall, subjects with one or two TS3'UTR 6bp insertion alleles had a nearly 1.5-fold greater risk of lung cancer than those without the 6bp insertion allele. In addition, we found this association to be stronger among subjects >55 years old, men and current and heavy smokers. Moreover, we found evidence for significant interactions between the TS3'UTR polymorphism and alcohol consumption and between the TSER polymorphism and dietary vitamin B12 intake. These findings suggest that the TS3'UTR 6 bp insertion allele and TSER polymorphisms are likely to contribute to susceptibility to lung cancer and their effects may be modulated by alcohol consumption and vitamin B12 intake.

Because of the critical role TYMS plays in folate metabolism and DNA repair, TYMS polymorphisms have recently drawn special interest from laboratory, epidemiological and clinical investigators. Several lines of evidence support a role of variation in TYMS and the risk of cancer. For example, TYMS enzymatic activity and the level of TYMS mRNA expression were found to be significantly higher in tumor tissue than in normal tissue, particularly for stomach and colorectal cancers (26). A functional analysis involving the use of a transient expression assay with cell lines showed that the TSER tandem repeat may contribute to the efficiency of expression of the TYMS gene (17). Individuals with the TYMS 2R2R genotype had lower plasma folate levels than did those with the TYMS 3R3R genotype (27). However, most of the published studies on the association of TYMS have been with regard to colorectal cancer, with the two largest studies being in Caucasian populations.

In a relatively large, hospital-based, case–control study of 510 patients with colorectal cancer and 604 polyp-free controls, Ulrich et al. (23) noted that the TYMS polymorphisms and dietary folate intake had an interactive effect on risk. Specifically, among individuals with the 3R3R genotype high levels of folate intake were associated with a reduced colorectal cancer risk, while among individuals with the 2R2R genotype high levels of folate intake were associated with an increased risk. No association was observed between the risk of colorectal cancer and the TYMS 6bp deletion/insertion polymorphism (23). These findings were not supported by a later prospective nested case–control study of 270 cases of colorectal cancer and 454 controls, in which the 2R/2R genotype was found to be associated with reduced risk (27).

To date, no case–control study of an association between the TYMS polymorphisms and lung cancer risk has been reported, although other related descriptive studies are available. For example, it was found that high TYMS activity, as measured by immunohistochemistry, was associated with higher proliferative activity in non-small cell lung tumors (28) and that lung cancer tissue also tended to have higher TYMS mRNA levels than did normal lung tissue (29). In addition, the TSER 3R3R genotype was associated with a significantly higher level of TYMS mRNA than were other genotypes in non-small cell lung cancer tissue (30).

The possible effect of the TYMS polymorphisms may be inferred from the published data on other cancers. However, in contrast to the findings for colorectal cancer, a recent study of 71 cases of acute lymphocyte leukemia (ALL) in Caucasians and 114 matched Caucasian controls found that compared with the TSER 3R3R genotype, the TSER 2R2R genotype was associated with an increased risk for adult ALL (31). Similarly, a case–control study of 180 lymphoma cases and 494 controls showed that individuals with at least one TSER 2R allele had an increased risk for malignant lymphoma, compared with those without the TSER 2R allele (32).

To the best of our knowledge, the present large case–control study is the first one to investigate an association between the TSER polymorphism and lung cancer risk. If TYMS enzyme activity mediates folate metabolism and high folate levels reduce DNA damage or facilitate DNA repair, then it is expected that the 2R2R genotype would be associated with an increased risk of lung cancer. However, studies of the association between the TSER polymorphism and serum folate levels in both healthy individuals and cancer patients have yielded mixed results, suggesting that the effect of the TSER polymorphism and folate pathway on cancer may be specific to certain cancers or ethnic groups (27,33,34). The absence of an association between the TSER tandem repeat polymorphism and risk of lung cancer suggests that this polymorphism does not modify the risk of lung cancer associated with low dietary folate intake as observed in the present study. Since vitamin B12 is also involved in DNA methylation, the unexpected finding of an increased risk of lung cancer associated with the TSER variant genotype in the presence of lower vitamin B12 intake suggests that vitamin B12 intake modifies the risk of lung cancer. However, it is unclear to what extent this effect may be biased by intake of other nutrients or supplemental vitamins.

Although the significance of mutations in the 3'-untranslated region is not as obvious as mutations resulting in amino acid changes, the mRNA turnover rate can be affected by this kind of sequence variation. Differences in mRNA turnover alter the stability of a given mRNA, which in turn determines protein expression levels (35). Therefore, it is possible that the TS3'UTR polymorphism affects mRNA stability or secondary mRNA structures, leading to altered protein levels or responses to up-regulation of this enzyme. In our study, because the TS3'UTR (0bp/6bp) polymorphism was found to be in linkage disequilibrium with the TSER (2R3R) polymorphism, the 6bp insertion allele might similarly decrease enzyme activity as does the 2R allele. This hypothesis can partly explain our finding of a higher risk of lung cancer in individuals with the 6bp insertion allele. In addition, this association was more pronounced among older individuals (>55 years), men and heavy smokers. Older males in this study were likely to be heavy smokers who may have incurred more smoking-induced DNA damage. Because a suboptimal repair capacity for removing smoking-related carcinogen-induced adducts has been shown to be associated with an increased risk of lung cancer (4), our data suggest that the TS3'UTR polymorphism may play a role in modifying the risk associated with smoking by decreasing thymidine supply for DNA synthesis and repair. Recently, one functional study on the biological significance of the TS3'UTR polymorphism showed that the 6bp deletion allele had an ~50% lower mRNA expression than did the 6bp insertion allele (36), which is inconsistent with our results. This suggests that other mechanisms are involved in this oncogenic pathway that need to be further elucidated.

With regard to the interaction between the TS3'UTR polymorphism and diet, the 6bp insertion allele was associated with a higher risk of lung cancer only in current alcohol drinkers. Although the mechanisms by which alcohol consumption interacts with the effect of TYMS in lung carcinogenesis are not known, there are several causal pathways that can explain this. First, chronic alcohol exposure impairs folate absorption (37). However, we do not have data on folate status measured in plasma, serum or tissue to evaluate this hypothesis. Second, alcohol itself may be associated with a risk of lung cancer. In particular, elevated DNA adducts of acetaldehyde, the primary oxidative metabolite of ethanol, were found in peripheral blood cells among alcoholics (38) and impaired DNA repair was also observed among alcohol-fed rats with DNA damage induced by carcinogens (39). These findings suggest that the role of TYMS in DNA repair is implicated in alcohol-induced DNA damage. Our data also suggested that alcohol intake may interact with cigarette smoking, because these two exposures tended to be highly correlated and there were significantly more ever drinkers among ever smokers (72.3%) than among never smokers (40.9%; P < 0.0001) in our study population. Furthermore, alcohol may act as a solvent of tobacco carcinogens (40) and may change the oxidative capacity of liver microsomes, leading to a reduced ability to metabolize tobacco carcinogens (41), or it may affect cellular metabolism, resulting in an increased metabolic activation of procarcinogens (41,42). However, we did not find interactive effects between these two polymorphisms with folate intake. This may be due to possible misclassification of dietary folate intake assessed by the questionnaire. Therefore, further studies with additional assessment of serum or plasma folate levels are warranted to better evaluate these gene–nutrition interactions.

Possible limitations in our hospital-based study design could have introduced a selection bias. However, the genotype distributions of our study were similar to those seen in other studies. For example, the frequencies of the TSER 3R3R, 2R3R and 2R2R genotypes among our 1140 non-Hispanic white controls from Texas were 30.8, 46.3 and 22.6%, respectively, compared with 26, 52 and 21%, respectively, for 623 population-based Caucasian controls from Minnesota (21) and 29, 48 and 23% for 454 cohort controls (93% Caucasians) in the Physicians' Health Study (26). Also, the frequencies of TS3'UTR 0bp/0bp, 6bp/0bp and 6bp/6bp among our 1140 controls were 11.6, 41.3 and 47.1%, respectively, compared with 10, 40 and 50%, respectively, for 623 Caucasian controls from Minnesota (21) and 14, 42 and 44% for the 454 controls in the Physicians' Health Study (26). Because our TYMS genotype frequency estimates in the hospital-based controls are similar to those in population-based and cohort-based controls, any selection bias is unlikely to be substantial. However, in a recent study of a Chinese population the frequencies of TS3'UTR 0bp/0bp, 6bp/0bp and 6bp/6bp among 348 Chinese controls were 45.7, 44.5 and 9.8%, respectively (43), suggesting a substantial ethnicity-related difference in the distribution of the TS3'UTR polymorphism. However, the 6bp/6bp genotype was associated with an increased risk for esophageal cancer in the Chinese population (43), a finding similar to that in the present study of lung cancer.

There were also some limitations in the dietary data analysis. For instance, the data did not include information on the intake of specific supplemental vitamins and folate and, therefore, we could not adjust for other possible confounding effects, which could also explain the unexpected interactions between vitamin B12 or alcohol consumption and the TYMS variant genotypes. Like all case–control studies of diet and cancer risk, the diet information obtained was retrospective and was for the year preceding the lung cancer diagnosis. However, several studies have examined the reproducibility of food frequency questionnaires under a wide variety of conditions and have shown correlations generally ranging from 0.5 to 0.7 for nutrient intakes measured at periods of 1–10 years apart (4446). The average reduction in the intake of nutrients was 0.07 over a 5 year period, suggesting that dietary changes over time were not substantial and repeated administration of the questionnaire was not necessary (45). It is also possible that the low intake of dietary vitamins and folate among the cases may be due to the symptomatic effects of the cancer. However, in this study >80% of the cases were interviewed within 15 days after diagnosis, thus reducing potential measurement errors attributable to recall bias as well as recent dietary changes after diagnosis.

In conclusion, in a large non-Hispanic white population we have demonstrated that the TS3'UTR polymorphism may be associated with an increased risk of lung cancer and that this association may be modified by alcohol consumption, an interaction that was also observed for the TSER polymorphism and vitamin B12 intake. Once our findings are replicated, these polymorphisms may be used to identify individuals at risk of developing lung cancer. However, the underlying biological relevance of these TYMS polymorphisms and the mechanisms of gene–diet interactions warrant further study.


    Acknowledgments
 
We thank Susan Honn for assistance in recruiting the subjects, Wayne Gosbee for data management, Li-E Wang and Zhensheng Liu for technical support, Jianzhong He, John I.Calderon and Kejin Xu for laboratory assistance, Joanne Sider for manuscript preparation and Richel Williams (Department of Scientific Publications) for scientific editing. This study was supported in part by National Institutes of Health grants CA55769 and CA86390 (to M.R.S.), ES 11740 (to Q.W.) and CA16672 (to M.D.Anderson Cancer Center).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 28, 2004; revised November 11, 2004; accepted November 21, 2004.