Department of Epidemiology and 1 Department of Urology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA
2 To whom correspondence should be addressed Email: xwu{at}mdanderson.org
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Abstract |
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Abbreviations: DFE, dietary folate equivalent; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosylmethionine
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Introduction |
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Major genes involved in the metabolism of the methyl group include methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS). The gene MTHFR is located at chromosome 1p36.3 (6). MTHFR catalyzes the reduction of 5,10-methylenetetrahydrolate to 5-methyltetrahydrofolate, the major circulatory form of folate in the body and a carbon donor for the conversion of homocysteine to methionine (4,5). As a precursor of S-adenosylmethionine (SAM), methionine is the universal methyl donor for DNA methylation. MTHFR is also involved in dTMP production and plays a role in DNA synthesis. Thus, a defect in the MTHFR gene could influence both DNA methylation and DNA synthesis. A common polymorphism in the MTHFR gene, a C to T change at position 677 in exon 4, has been identified (6), leading to an alanine to valine conversion (7), and individuals carrying the variant MTHFR 677TT genotype have only 30% of the enzyme activity in vitro as compared with the CC wild-type. Heterozygotes (CT) show nearly 65% of normal enzyme activity (7). The homozygous variant TT genotype is associated with DNA hypomethylation (8), a characteristic that may promote carcinogenesis because insufficient methylation of DNA may induce genomic instability, and thereby activate oncogenes (913). Another common polymorphism in the MTHFR gene, an A to C change at position 1298 in exon 7, which cause an alanine to glutamate change (14), is also associated with decreased enzymatic activity (14). It has been shown that these two loci of the MTHFR gene are in strong linkage disequilibrium (1519).
Other genes involved in folate metabolism include methionine synthase (MS) and cystathionine ß-synthase (CBS). MS, which is reported to have a polymorphism in 2756 A to G, catalyzes the remethylation of homocysteine to methionine. The gene is located at chromosome 1q43 (20) and the A to G transition at nucleotide 2756 is in the protein binding region of the gene, resulting in a substitution of aspartic acid by glycine (20,40). The polymorphism results in homocysteine elevation and DNA hypomethylation (20).
Although the association between the polymorphisms of the MTHFR gene and cancer risk has been examined in many studies (1517,2126), only a few studies have examined how the MTHFR polymorphisms influence the risk of bladder cancer, the fourth leading type of cancer among men in the USA. In a cohort study of 860 men aged 6584 years who were followed up for 10 years, Heijmans et al. (27) found that there was a 5-fold (95% CI: 1.6718.0) age-adjusted increased risk of bladder and kidney cancer in those with the homozygous variant genotype of MTHFR 677 as compared with the homozygous wild-type carriers. However, in a case-control study of transitional cell carcinoma (TCC) of the urinary bladder, Kimura et al. (28) found no significant difference in the MTHFR 677 allele frequency between case patients and control subjects and concluded that the MTHFR 677 genotypes do not contribute to susceptibility to TCC of the bladder. To our knowledge, no studies have examined the relationship between the MTHFR 1298, MS 2756 polymorphisms and bladder cancer risk in the context of dietary folate intake data.
In this molecular epidemiological study, we investigated the association between MTHFR and MS polymorphisms and bladder cancer risk in a case-control study. We hypothesized that MTHFR 677, MTHFR 1298 and MS 2756 polymorphisms are associated with increased bladder cancer risk and because these genes are all involved in folate metabolism, we further hypothesized that dietary folate intake might modulate the effect. Moreover, because cigarette smoking is a well-established risk factor for bladder cancer (29,30), we also examined the joint effects of smoking and folate metabolic genes on bladder cancer risk. Finally, because recent studies have shown that the two MTHFR loci are in strong linkage disequilibrium, we also tested the linkage disequilibrium of these two loci and investigated the association between bladder cancer risk and the MTHFR 677/MTHFR 1298 haplotypes.
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Materials and methods |
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Epidemiological data
Once case patients and control subjects agree to participate, informed consent is obtained prior to the collection of epidemiological data. All participants then undergo a 45-min personal interview administered by M.D.Anderson staff interviewers in conformance with institutional guidelines for studies including human subjects. Data are collected on demographical characteristics (age, gender, ethnicity, etc.), alcohol consumption and history of tobacco use.
In addition, a 60-min food-frequency questionnaire was administered to assess diet during the year prior to diagnosis in the cases and the year prior to the interview in the control subjects. The food-frequency questionnaire is derived from a modified version of the Health Habits and History Questionnaire developed by the National Cancer Institute (31,32). The intake of folate was estimated from this food questionnaire. DIETSYS+Plus (version 5.9 Block Dietary Data Systems, Berkeley, CA, 1999) was used to perform dietary analysis. The primary source for folate variables was derived from the Nutrient Database for Standard References, Release 14 (SR14) (United States Department of Agriculture, Agriculture Research Service. USDA Nutrient Database for Standard Reference, Release 14. Nutrient Data Laboratory Home 2001. Available at http://www.nal.usda.gov/fnic/foodcomp). Because the DIETSYS+Plus did not contain separate values for naturally occurring folate and folic acid supplied by food fortification, post-fortification values for natural food folate and folic acid added to foods, and dietary folate equivalents (DFE) from food sources were obtained from SR14 and added to the nutrient data file. For multi-ingredient food items that were not available in SR14, appropriate recipes from the Continuing Survey of Food Intakes 19941996, 1998 were used to obtain folate intake (33). Recipe adjustments for nutrient loss and moisture changes due to cooking were made as needed. Folate intake was categorized by: (i) food folate, folate naturally occurring in food; (ii) DFE from food, which include both the folate occurring naturally in food and folic acid in fortified foods; and (iii) total DFE, which is the sum of the DFE from food and folic acid from supplemental sources.
Immediately after the interview, a 40-ml blood sample was drawn into coded heparinized tubes and transported directly to a research laboratory for molecular analysis. An individual who has never smoked or has smoked less than 100 cigarettes in his or her lifetime is defined as a never smoker. A former smoker was a person who had quit smoking at least 1 year prior to diagnosis (cases) or who had quit smoking at least 1 year prior to the interview (controls). A current smoker was someone who was currently smoking or who had stopped <1 year prior to being diagnosed with bladder cancer (cases) or interview (controls).
MTHFR and MS genotyping
The PCRRFLP method was used to determine the MTHFR C677T and A1298C polymorphisms. The procedures were adapted from Skibola et al. (15). The CT base pair substitution creates a HinfI restriction site. Briefly, primers 5'-TGA AGG AGA AGG TGT CTG CGG GA-3' and 5'-AGG ACG GTG CGG TGA GAG TG-3' were used to amplify the portion of the MTHFR sequence from 100 ng of human genomic DNA. PCR thermal cycling conditions included a 5-min denaturalization period at 94°C and 35 cycles of the following: 94°C for 30 s, 62°C for 30 s and 72°C for 30 s. This was followed by a 5-min extension at 72°C. One 25-µl reaction mixture contained 2.5 µl of 10x PCR buffer, 0.2 mM dNTP, 2.5 mM of MgCl2, 1.5 U Taq DNA polymerase, 100 ng of DNA template, 0.2 µM of each primer. The 15 µl of PCR products were mixed with 10 U HinfI for digestion at 37°C overnight. Digestion products were visualized after electrophoresis on a 4% agarose gel with ethidium bromide. The MTHFR 677 wild-type (CC) homozygotes produce a 198 bp fragment; the heterozygotes (CT) produce 198, 175 and 23 bp fragments; and the variant homozygotes (TT) produce 175 and 23 bp fragments.
The AC substitution at MTHFR 1298 abolishes the MboII restriction site. Primers (5'-CTT TGG GGA GCT GAA GGA CTA CTA C-3' and 5'-CAC TTT GTG ACC ATT CCG GTT TG-3') were used to amplify the target sequence from 100 ng of human genomic DNA. PCR thermal cycling conditions included a 5-min denaturalization period at 94°C and 35 cycles of the following: 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. This was followed by a 5-min extension at 72°C. One 25-µl reaction mixture contained 2.5 µl of 10x PCR buffer, 0.2 mM dNTP, 2.5 mM MgCl2, 1.5 U Taq DNA polymerase, 100 ng DNA template, 0.12 µM each primer. The 15 µl of PCR product was mixed with 1 µl of MboII for digestion at 37°C overnight. Digestion products were then visualized after electrophoresis on a 4% agarose gel with ethidium bromide. The MTHFR 1298 wild-type homozygotes (AA) produce five fragments: 56, 31, 30, 28 and 18 bp; the heterozygotes (AC) produce 84, 56, 31, 30, 28 and 18 bp fragments; the variant homozygotes (CC) produce 84, 31, 30 and 18 bp fragments. The 84 and 56 bp fragments can be successfully separated and visualized with the 4% agarose gel.
The MS genotyping procedure was adapted from Paz et al. (34) and Chen et al. (35). In brief, the primers used for PCR amplification were 5'-GAA CTA GAA GAC AGA AAT TCT CTA-3' and 5'-CAT GGA AGA ATA TCA AGA TAT TAG A-3'. PCR thermal cycling conditions included a 5-min denaturalization period at 94°C and 35 cycles of the following: 94°C for 30 s, 46°C for 30 s and 72°C for 30 s. This was followed by a 5-min extension at 72°C. The 15 µl of PCR product was mixed with 10 U HaeIII for digestion at 37°C overnight. Digestion products were then visualized after electrophoresis on a 4% agarose gel with ethidium bromide. The wild-type homozygotes (AA) produce a single 189 bp fragment; the heterozygotes (AG) produce 189, 159 and 30 bp fragments; the variant homozygotes (GG) produce 159 and 30 bp fragments.
Statistical analysis
Distributions in demographic variables, including gender, ethnicity and smoking status between cases and controls were evaluated by the 2 test. Differences between cases and controls in age, folate intake, and self-reported pack-years were tested using the non-parametric Wilcoxon rank sum test. The HardyWeinberg equilibrium was tested by the goodness-of-fit
2 test. Linkage disequilibrium analysis of the MTHFR 677 and MTHFR 1298 was performed for both the controls and the cases. The parameter D' (from 0 to 1) was calculated as a standardized measure of linkage disequilibrium (36).
Conditional logistic regression was used to assess the bladder cancer risk from MTHFR and MS polymorphisms. The odds ratio (OR) and the 95% confidence intervals were derived from both univariate and multivariate models. Because age and gender were among the matching variables, we controlled for possible confounders other than the matching variables, such as smoking status, food folate intake and alcohol consumption in the multivariate model. Conditional logistic regression was also performed to assess the joint effects of genetic polymorphisms, smoking status and pack-years smoked. Because data on folate intake were not available for all study subjects (missing nutrition data), unconditional logistic regression was used to assess the joint effects of dietary folate intake and genetic polymorphisms. Possible confounding effects were controlled in a multivariate model. A trend test was performed to test for a linear trend in the ORs. A likelihood ratio test was used to test for interactions among variables. This test compares the likelihood of a full model including the interaction term with a reduced model without the interaction term. All statistical tests were two-sided with a type I error rate of 5%. Statistical analyses were done with the STATA software (Version 7, College Station, TX).
Haplotype analysis
Haplotypes were determined directly from genotype data for individuals who were heterozygous at only one site or at no sites. For individuals with an unknown linkage phase, the haplotypes were inferred using the Stephens SmithDonnelly (SSD) algorithm (37). The SSD approach incorporates concepts from population genetics theory in a Markov chainMonte Carlo technique, which allows haplotypes to be effectively predicted for individuals with incomplete information (38).
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Results |
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No subject in our study was a homozygous variant (677TT/1298CC) at both MTHFR 677 and MTHFR 1298 loci; in addition, no subject was a homozygous variant at one site and heterozygous at the other site (677TT/1298AC or 677CT/1298CC) (data not shown). The linkage test showed that these two loci were in strong linkage disequilibrium among both the cases and controls (D' = 0.99 for both groups).
The two SNPs in the MTHFR gene gave rise to four possible haplotypes: W-W, W-M, M-W and M-M, where W stands for the normal wild-type allele. For example, the W-M haplotype consists of a normal nucleotide C at the position 677 and the variant nucleotide C at position 1298. Overall, no significant differences in the haplotype frequencies were found between the cases and controls (Table III). There were also no significant differences in haplotype frequencies when the data were stratified by smoking status and ethnicity (Table III).
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Another characteristic we were interested in with respect to the interaction of genetic factors and smoking was the age at bladder cancer onset associated with different haplotypes. Among former smokers, we found a significant difference in the age of onset depending on the haplotypes (Table VIII). Specifically, former smokers with the W-M or M-W haplotypes showed a significantly later onset of the bladder cancer as compared with normal homozygotes. The protective effect of the heteromorphic haplotypes was still highly significant after the Bonferroni correction for multiple comparisons was applied (P = 0.005). A survival analysis with the age of onset as an event also showed a highly significant difference between those with a normal haplotype and the two groups with heteromorphic haplotypes (Figure 1a). Furthermore, the age of cancer onset gradually increased as the proportion of heteromorphic haplotypes in the individual increased (Figure 1b).
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Discussion |
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In our analysis of the frequencies of the polymorphisms in cases and controls, the MTHFR 677 T allele frequency is 0.35 for cases and 0.32 for controls, similar to the values for Caucasian populations published elsewhere (15,2124,39). The MTHFR 1298 C allele frequency is 0.30 and 0.31 for cases and controls, respectively, which is also in agreement with published ranges for Caucasians (14,17,24). The frequency of the MS 2756 G allele (0.18 and 0.19 for cases and controls, respectively) is similar to the 0.17 and 0.19 reported by Ma et al. (40) and Le Marchand et al. (41). We further found that all cases and controls with the homozygous variant MTHFR 677 genotype (TT) were homozygous wild-type (AA) of the MTHFR1298, and those with the homozygous variant MTHFR 1298 (CC) were always MTHFR 677 wild-type (CC). The linkage tests showed that these two loci are in strong linkage disequilibrium, as has been observed in many other studies (e.g. 1519).
Studies of the association between the MTHFR 677 polymorphism and cancer susceptibility have generated conflicting results. For example, increased risk associated with the MTHFR 677 polymorphism has been found in several studies of various cancers, including gastric cancer (16), cervical dysplasia (42), esophageal squamous cell carcinoma (25) and gastric cardia adenocarcinoma (26). In contrast, studies examining the effects of the MTHFR 677 polymorphisms in colorectal cancer have shown a protective effect. For example, Ma et al. (22) reported an OR of 0.49 (CI: 0.270.87) for colorectal cancer in men with the homozygous variant genotype in a case-control study nested within the Physicians' Health Study. The protective effect was even more evident for those with adequate folate levels. In the Health Professionals Follow-up Study, Chen et al. (21) also observed a reduced risk of colorectal cancer in association with the MTHFR 677 homozygous variant genotype, especially when dietary folate intake was high. The reduced risk was also observed in studies of breast cancer (43) and acute lymphocytic leukemia (15).
These conflicting results may be explained by the metabolic role of the MTHFR enzyme, which is involved in both DNA methylation and DNA synthesis. Because individuals carrying the variant T allele would be less efficient in converting 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the resultant lower level of 5-methyltetrahydrofolate would lead to reduced levels of SAM, which donates the methyl group for DNA methylation in the cell. Reduced levels of SAM, in turn, would result in DNA hypomethylation (8), which may promote carcinogenesis by inducing genomic instability and altering the expression of oncogenes and tumor suppressor genes (913). Thus, from the standpoint of DNA methylation, variant genotypes are associated with an increased risk of cancer. The protective effect of the variant T allele may result from the fact that the less efficient conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate potentially prevents the depletion of 5,10-methylenetetrahydrofolate. Because 5,10-methylenetetrahydrofolate acts a cofactor for the de novo synthesis of nucleotides necessary for DNA synthesis (especially dTMP), an abundance of 5,10-methylenetetrahydrofolate might lessen the likelihood of the dTMP stress. This stress results from a short supply in nucleotide precursors required for DNA synthesis and thus increases genetic instability (21,22). There may be a balance between the donation of methyl for DNA methylation and the supply of bases for DNA synthesis, i.e. a balance between beneficial and deleterious effects of the MTHFR 677 T allele (23,44).
Several authors have suggested that whether the variant T allele is beneficial or deleterious depends on environmental factors, particularly on dietary folate intake. That is, the allele is a protective factor when the dietary folate level is adequate and a risk factor when the folate level is deficient (18,2123,25,27,45). Consistent with the hypothesis that the MTHFR 677 T allele is a risk factor when the folate level is low, we observed a significantly increased risk (>3-fold) of bladder cancer in those with at least one T allele and those whose folate intake was in the lowest quartile. Studies in patients with cancer of other sites have yielded similar results. For example, Goodman et al. (42) found a significant OR of 5.0 (CI: 2.012.2) associated with cervical dysplasia in women with at least one variant MTHFR 677 T allele who reported low folate intake. Likewise, in a population-based cohort study of several common cancers, Heijmans et al. (27) found that among men with low folate intake, the MTHFR 677TT genotype was associated with 2.64-fold increased risk of all types of cancer combined. Further, Keku et al. (24) observed an OR of 1.9 (95% CI: 1.32.9) for colon cancer risk in a Caucasian population with the combination of either the MTHFR 677 CC or CT genotypes and a low folate intake.
One factor that at least partially explains the different ORs reported in the literature is the different methods used to determine folate intake. For example, in the study of Heijmans et al. (27), daily folate intake was estimated using a computerized version of The Netherlands food table and the folate content was determined from recent liquid chromatography data. In the Physicians Health Study (22) and Health Professionals Follow-Up Study (21), plasma folate level was used instead of the food folate level. Dietary supplements were included in some studies (e.g. 24,41,42) but not in others (e.g. 39). In the study of Le Marchand et al. (41), which included dietary folate from supplements, the median levels of folate intake in both cases and controls (369 and 450 mg/day, respectively) are consistently higher than the food folate intake in our study (124.8 and 136.2 mg/day, respectively), which did not include dietary supplements or the synthetic form of folate. In Slattery et al.'s study (39), which did not include supplements, the cut-off point for a high folate level was 205 mg/day, which is close to the cut-off point for the 25th quartile of food DFE (207 mg/day) in our study.
Although the risk of bladder cancer was significantly increased for those with the MTHFR 677CT/TT genotypes and whose folate was in the first quartile (the lowest quartile), we did not find significant effects of the MTHFR 1298 polymorphism on bladder cancer risk in the overall analysis. Such non-significant results were also seen in studies of gastric cancer (16), gastric cardia adenocarcinoma (26), lung cancer (17) and malignant lymphoma (18). Indeed, the effects of the MTHFR 1298 AC and CC genotypes do not profoundly impair MTHFR enzyme activity as do the MTHFR 677 CT or TT genotypes (1416). Nevertheless, Song et al. (25) found a significantly increased risk of esophageal squamous cell carcinoma (OR: 4.43; 95% CI: 1.2316.02) in those with the MTHFR 1298 CC genotype relative to those with the wild-type genotype. However, the C allele frequency in the Chinese population they studied was 14% in the cases, which is lower than a frequency of 16% seen in cases in two other studies conducted in Chinese populations (16,26). The C allele frequency of 17% in controls was between the values reported in Shen et al. (16) and Miao et al. (26) (18 and 16%, respectively). However, since different cancer sites were involved in these studies, the effects of the MTHFR 1298 polymorphisms may be cancer-type specific.
Only a few studies have evaluated the effects of the MS polymorphism on cancer risk. MS is a vitamin B12-dependent enzyme important for maintaining the intracellular level of methionine, the precursor of SAM. The AG change at codon 2756 in the MS gene results in a deficiency of MS and possibly reduced SAM levels, elevated homocysteine levels and DNA hypomethylation (8,20). Le Marchand et al. (41) reported ORs of 2.9 (95% CI: 0.517.4) and 3.3 (95% CI: 0.619.3) for the MS 2756 AG and GG genotypes, respectively, in colorectal cancer. However, Ma et al. (40) found an inverse association between the colorectal cancer risk and the homozygous variant MS 2756 genotype (OR: 0.59; 95% CI: 0.271.27). None of these results reached statistical significance. The non-significant results reported in these studies and in our study suggest that polymorphisms in the MS gene may not play as important a role as the MTHFR in cancer predisposition. However, when we analyzed the genetic data jointly with folate intake, we consistently found that those with variant genotypes and a low folate intake were at a significantly increased risk of bladder cancer, strengthening the view that low dietary folate intake in conjunction with genetic polymorphisms acts as a risk factor in bladder cancer.
In summary, a consistent and important finding is that individuals with variant genotypes and with low folate intake are at the highest risk of bladder cancer. It has been shown that a low dietary folate intake is associated with a suboptimal cellular DNA repair capacity, as assessed by the hostcell reactivation assay (46). As a result of insufficient folate intake, individuals with a reduced DNA repair capacity may be more sensitive to carcinogenetic exposures, such as genetically predisposed defects in metabolic enzymes, and thus at higher risk of DNA instability and consequently cancer.
Our results also showed a significantly increased risk of bladder cancer among current smokers and heavy smokers regardless of genotype (Tables VI and VII), thus further supporting the notion that smoking is an important risk factor for bladder cancer, as has been established in many previous studies (29,30). More importantly, our study demonstrated that current or heavy smokers with adverse metabolic genotypes are at the highest risk of bladder cancer (Tables VI and VII), suggesting a joint effect of cigarette smoking and polymorphisms of the folate metabolic genes. No previous studies have examined the effects of smoking and the effect of folate metabolic genes jointly, probably because most studies in the literature examined polymorphisms of these genes in relation to cancers such as colorectal cancer, for which smoking may not be a primary risk factor. Thus, our study may be the first to show a joint effect of smoking and polymorphisms of these genes on bladder cancer risk.
To the best of our knowledge, no previous studies have examined age at bladder cancer onset in relation to MTHFR haplotypes. Our data showed that the age at bladder cancer onset increased as the proportion of the heteromorphic haplotypes in the individual increased. It is interesting that this protective effect was only evident among former smokers. Perhaps the effect of smoking overwhelms the genetic effect in current smokers.
In conclusion, our study showed that low folate intake and smoking, in concert with polymorphisms in the MTHFR and MS genes, increase the bladder cancer risk. These results have important implications for cancer prevention in susceptible populations.
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Acknowledgments |
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References |
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