Polymorphisms of folate metabolic genes and susceptibility to bladder cancer: a case-control study

Jie Lin, Margaret R. Spitz, Yunfei Wang, Matthew B. Schabath, Ivan P. Gorlov, Ladia M. Hernandez, Patricia C. Pillow, H. Barton Grossman1 and Xifeng Wu1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies have shown an association between low folate intake and an increased cancer risk. Major genes involved in folate metabolism include methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS). We investigated joint effects of polymorphisms of the MTHFR (677 C->T, 1298A->C) and MS genes (2756 A->G), dietary folate intake and cigarette smoking on the risk of bladder cancer in a case-control study. The study population consisted of 457 bladder cancer patients and 457 healthy controls, matched to the cases in terms of age, gender and ethnicity. Genotype data were analyzed in a subset of 410 Caucasian cases and 410 controls. Compared with individuals carrying the MTHFR 677 wild-type (CC) and reporting a high folate intake, those carrying the variant genotype (CT or TT) and reporting a low folate intake were at a significantly 3.51-fold increased risk of bladder cancer (95% CI: 1.59–6.52). In contrast, individuals carrying a variant genotype and reporting a high folate intake were at only a 1.39-fold increased risk (95% CI: 0.71–2.70), and those carrying the wild-type and reporting a low folate intake were at only 1.56-fold increased risk (95% CI: 0.82–2.97). The interaction between genetic polymorphisms and folate intake was significant on the multiplicative scale (P = 0.01). When analyzed in the context of smoking status, compared with never smokers with the MTHFR 677 wild-type, the risk increased to 6.56-fold (95% CI: 3.28–13.12) in current smokers carrying the variant genotype. Analyses of the MTHFR 1298, MS 2756 genes revealed similar results. In addition, age at cancer onset in former smokers increased as the proportion of the heteromorphic haplotype in the individual increased (P = 0.005). Our results strongly suggest that polymorphisms of the MTHFR and MS genes act together with low folate intake and smoking to increase bladder cancer risk. These results have important implications for cancer prevention in susceptible populations.

Abbreviations: DFE, dietary folate equivalent; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosylmethionine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies have demonstrated an association between folate deficiency and an increased risk of a variety of cancers (1,2). The crucial role of folate as the donor of one-carbon groups in both DNA methylation and DNA synthesis may explain some of these observations (3).

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 65–84 years who were followed up for 10 years, Heijmans et al. (27) found that there was a 5-fold (95% CI: 1.67–18.0) 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
In this ongoing case-control study that was initiated in July 1999, patients with incident urinary bladder cancer are being recruited from The University of Texas M.D. Anderson Cancer Center in Houston, Texas, without age, gender, ethnicity or tumor stage restrictions. Cases are defined as those who are newly diagnosed and histologically confirmed with urinary bladder cancer, and who have received no previous chemotherapy or radiotherapy. A brief eligibility questionnaire is administered to assess previous cancer therapy and their willingness to participate in the epidemiological study. Healthy controls without a history of cancer (except non-melanoma skin cancer) are recruited from a large database of potential volunteers from the Kelsey-Seybold clinics, a community-based multi-specialty physician practice in the Houston metropolitan area. Control subjects are frequency matched to the cases on the basis of age (±5 years), gender and ethnicity.

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 1994–1996, 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 PCR–RFLP method was used to determine the MTHFR C677T and A1298C polymorphisms. The procedures were adapted from Skibola et al. (15). The C->T 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 A->C 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 {chi}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 Hardy–Weinberg equilibrium was tested by the goodness-of-fit {chi}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– Smith–Donnelly (SSD) algorithm (37). The SSD approach incorporates concepts from population genetics theory in a Markov chain–Monte Carlo technique, which allows haplotypes to be effectively predicted for individuals with incomplete information (38).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The demographic characteristics of cases and controls are summarized in Table I. Males constituted 76.6% of the patient population and Caucasians constituted 91.5% of all patients. African Americans and Mexican Americans represented only 4.8 and 3.7% of the patient population, respectively. The mean age of the cases was 63.4 years (median 65 years, range 18–86 years), and this was 62.7 years for controls (median 64 years, range 21–89). Cases and controls were well matched in terms of gender, ethnicity and age (±5 years). As expected, there were significant differences in smoking status between the cases and controls (P < 0.001). For example, 23.8% of cases as opposed to 8.1% of controls were current smokers; in contrast, 25.2% of cases and 47.0% of controls were never smokers. In addition, the self-reported median pack-years in smokers were significantly higher in the cases than in the controls (36.4 for cases and 20.0 for controls, P < 0.001). Further, controls had a significantly higher intake than cases in terms of both food folate (median 136.2 versus 124.8 µg/ 1000 kcal/day, P < 0.001) and DFE from food (median 248.5 versus 226.9 µg DFE/1000 kcal/day, P < 0.001). In addition, the total DFE was significantly higher in controls than in cases (median 857.6 versus 416.9 µg DFE/1000 kcal/day, P < 0.001) (Table I).


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Table I. Selected characteristics of cases and controls

 
As shown in Table II, for Caucasians, the frequency of the MTHFR 677 T allele was 0.35 for the cases and 0.32 for the controls. The observed frequencies of the three MTHFR 677 genotypes among controls (CC: 47.8%; CT: 40.0%; TT: 12.2%) did not differ from those expected under the Hardy–Weinberg equilibrium ({chi}2 = 2.88, P = 0.09). There was also no significant difference in the genotype distribution between the cases and controls ({chi}2 = 2.13, P = 0.35). The MTHFR 1298 C allele frequency was 0.30 for the Caucasian cases and 0.31 for the controls. The observed frequencies of the three MTHFR 1298 genotypes among controls (AA: 46.2%; AC: 45.0%; CC: 8.8%) were also in agreement with the Hardy–Weinberg equilibrium ({chi}2 = 0.87, P = 0.35). Again, the difference in the genotype distribution between cases and controls did not reach statistical significance ({chi}2 = 0.61, P = 0.74). Similar results were noted for the frequency of the MS 2756 G allele, which was 0.18 for the cases and 0.19 for the controls.


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Table II. Distribution of MTHFR 677, MTHFR 1298 and MS 2756 genotypes among cases and controls in different ethnic groups

 
Evaluation of other ethnic groups did not reveal significant differences in the genotype distribution between cases and controls or a significant deviation in variant allele frequencies from the Hardy–Weinberg equilibrium (Table II). However, there was an appreciable difference in the variant allele frequencies of MTHFR 677, MTHFR 1298 and MS 2756 in controls among the three ethnic groups: the frequencies of the MTHFR 677 T allele were 0.32, 0.36 and 0.12 for Caucasians, African Americans and Mexican Americans, respectively (P < 0.001). The frequencies of the C allele of MTHFR 1298 were 0.31, 0.19 and 0.21 for Caucasians, African-Americans and Mexican Americans, respectively (P = 0.007). A significant difference in the frequencies of the G allele of MS 2756 among ethnic groups was also noted (P = 0.01).

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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Table III. MTHFR haplotype frequencies in cases and controlsa

 
Because both the MTHFR 677 CT and MTHFR 677 TT genotypes were associated with ORs >1, we combined them in the subsequent analyses to increase the statistical power. In addition, because the proportions of both African Americans and Mexican Americans in the study population were too small for meaningful statistical analysis and there were appreciable differences in the allele frequencies between these two groups and Caucasians (Table II), we restricted the subsequent analyses to Caucasians only. Overall, we found that the combined heterozygote MTHFR 677CT and variant MTHFR 677TT genotypes were associated with a borderline significantly increased risk of bladder cancer (OR: 1.22; 95% CI: 0.92–1.60) (Table IV). The increased risk remained unchanged after adjusting for smoking status, food folate and alcohol consumption (adjusted OR: 1.23; 95% CI: 0.91–1.66). We did not find significant associations between either the MTHFR 1298 or the MS 2756 polymorphisms and bladder cancer risk with and without adjustment for confounders (Table IV).


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Table IV. Risk estimates for MTHFR 677, MTHFR 1298, and MS 2756 polymorphisms in Caucasiansa

 
Because all genes are important in folate metabolism, we examined the joint effects of the various genotypes in conjunction with dietary folate status. We divided food folate into three categories on the basis of the percentile values in controls: high (above the 75th percentile value), medium (from the 25th percentile to the 75th percentile) and low (below the 25th percentile). Using subjects with the wild-type genotype and who reported a high food folate intake as the reference group, we found elevated risks for all those with other MTHFR 677 genotypes and any folate status (Table V). Compared with individuals with the MTHFR 677 wild-type and who reported a high food folate intake, individuals carrying a variant genotype (MTHFR 677CT/TT) and with high food folate intake were at 1.39-fold increased risk (95% CI: 0.71–2.70); the risk was 1.56 (95% CI: 0.82–2.97) for those with the MTHFR 677 wild-type and low food folate intake. The highest risk group consisted of those reporting low food folate intake and a variant genotype (adjusted OR: 3.51; 95% CI: 1.59–6.52). Furthermore, the interaction between this genetic polymorphism and food folate intake was statistically significant on the multiplicative scale (P = 0.01). Similar results were obtained for the MTHFR 1298 and MS 2756 polymorphisms. Moreover, when we categorized individuals by their MTHFR 677, MTHFR 1298 and MS 2756 genotypes, we observed an inverse relationship between the levels of folate intake and bladder cancer risk (all P for trend <0.05) (Table V). Similar results were noted when folate intake was measured in terms of both the DFE from food sources only and total folate (results not shown).


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Table V. Joint effects of MTHFR 677, MTHFR 1298 and MS 2756 polymorphisms and food folate intake on bladder cancer risk in Caucasiansa

 
We also examined the joint effects of genotype and smoking, a well-established risk factor for bladder cancer. The reference group consisted of never smokers with the wild-type genotype. We observed a 2.15-fold (95% CI: 1.30–3.53) elevated risk of bladder cancer for former smokers with the MTHFR 677 wild-type and a 2.30-fold increased risk (95% CI: 1.43–3.68) for former smokers carrying at least one variant allele (Table VI). Interestingly, the risk increased to 4.65 (95% CI: 2.30–9.40) for current smokers with the MTHFR 677 wild-type and reached the highest value of 6.56 (95% CI: 3.28–13.12) in current smokers carrying at least one MTHFR 677 variant allele (Table VI). Similar results were obtained for the MTHFR 1298 and MS 2756 genotypes. For example, former smokers carrying at least one variant C allele of MTHFR 1298 had a 1.72-fold increased risk (95% CI: 1.07–2.76), while current smokers with the MTHFR 1298 wild-type were at a 3.30-fold increased risk (95% CI: 1.71–6.37). The highest risk was found in current smokers carrying at least one variant C allele (adjusted OR: 4.94; 95% CI: 2.37–10.30). Similar results were obtained for the MS 2756 genotypes, with the highest OR occurring in current smokers carrying at least one variant allele (adjusted OR: 4.39; 95% CI: 2.02–9.58) (Table VI). We also noted a significant dose–response relationship between the ORs and smoking status (all P trend <0.001) (Table VI). A similar analysis was performed for pack-years smoked. Compared with the reference group, never smokers with the MTHFR 677 wild-type, light smokers (pack-years <42) with a variant genotype (MTHFR 677CT/TT) had a 2.09-fold increased risk (95% CI: 1.28–3.40) (Table VII), while heavy smokers (pack-years ≥42) with a MTHFR 677 wild-type had a 4.30-fold increased risk (95% CI: 2.28–8.11). The group at the highest risk consisted of heavy smokers with the variant genotype (adjusted OR: 5.54; 95% CI: 3.07–9.98) (Table VII). Further analysis revealed a significant dose–response trend in the OR with pack years (Table VII). Similar results were noted for the MTHFR 1298 and MS 2756 genotypes (Table VII).


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Table VI. Joint effects of MTHFR 677, MTHFR 1298 and MS 2756 polymorphisms and smoking status on bladder cancer risk in Caucasiansa

 

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Table VII. Joint effects of MTHFR 677, MTHFR 1298 and MS 2756 polymorphisms and tobacco use on bladder cancer risk in Caucasiansa

 
An interesting finding of the above analysis was that in stratified analysis by tumor stage (superficial versus invasive), the joint effects of polymorphisms and environmental factors (i.e. folate intake, cigarette smoking) were much stronger in the invasive stratum than in the superficial stratum, suggesting the gene–environment interaction was more evident among the higher histological stage (data not shown). For example, the stratified analyses showed that risk associated with joint effects of MTHFR 677 variant genotype (CC/CT) and low folate intake was higher in the invasive stratum (OR: 4.81; 95% CI: 1.81–12.79) than in the superficial stratum (OR: 2.99; 95% CI: 1.24–7.17). In addition, the joint effects of the MTHFR 677 variant genotype (CC/CT) and current smoking were much stronger in the invasive group (OR: 23.89; CI: 5.89–96.83) than in the superficial group (OR: 3.16; 95% CI: 1.31–7.61). Similar findings were obtained for joint effects of MTHFR 1298, MS 2756 and smoking.

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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Table VIII. Age at bladder cancer onset among former smokers with different diplotypes (haplotype combinations)

 


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Fig. 1. Age at the bladder cancer diagnosis associated with different haplotypes. (a) Cumulative percentage of cases among normal homozygotes (W-W/W-W) (dashed line) and among homozygotes for heteromorphic haplotypes (W-M/W-M or M-W/M-W) (solid line). Those with the W-M/W-M and M-W/M-W diplotypes are characterized by approximately the same age at onset (see Table VIII). (b) Dependence of age at onset on the proportion of heteromorphic haplotypes.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We investigated the association between folate metabolic genes (MTHFR and MS) and bladder cancer risk in a case-control study. The effects of these genotypes were evaluated in conjunction with dietary folate intake and smoking. Overall, we found only a borderline significantly increased risk of bladder cancer in individuals carrying at least one variant MTHFR 677 T allele. However, this risk was significantly increased for people with low folate intake and for current and heavy smokers.

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. 15–19).

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: 0.27–0.87) 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: 2.0–12.2) 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: 1.3–2.9) 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: 1.23–16.02) 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 A->G 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: 0.5–17.4) and 3.3 (95% CI: 0.6–19.3) 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: 0.27–1.27). 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 host–cell 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.


    Acknowledgments
 
This study was supported by CA 85576, CA 91846, CA 74880 and CA 86390.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 29, 2003; revised March 15, 2004; accepted April 20, 2004.