Polymorphisms Of Folate Metabolic Genes(MTHFR, MTR) And Aberrant Promoter Hypermethylation Of RASSF1A Gene In Bladder Cancer Risk In A Chinese Population

IntroductionBladder cancer is the ninth most common cancer worldwide. In China, bladder cancer is the tenth most common cancer. In recent years, incidence rate and mortality rate of bladder cancer have an increased tendency.Epidemiological studies have shown that cigarette smoking and occupational or environmental exposure to chemical carcinogens are the strongest known risk factors in bladder cancer development. Although many people have been exposed to these risk factors, only a fraction of exposed individuals develop bladder cancer in their lifetime, suggesting individual susceptibility to bladder carcinogenesis. In the recent years, interest in the genetic susceptibility to cancers has led to a growing attention to the study of polymorphisms of genes involved in tumourigenesis.Epidemiological studies have shown an association between low folate intake and an increased cancer risk. Folate deficiency is thought to increase the risk of cancer through impaired DNA repair synthesis and disruption of DNA methylation.Methylenetetrahydrofolate reductase (MTHFR) and Methionine synthase (MTR) play an important role in the folate metabolic pathway. 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. 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. Theoretically, the varied activities of folate metabolic enzyme caused by MTHFR and MTR polymorphisms would influence the individual susceptibility to cancers and methylation status including global hypomethylation and hypermethylation in specific genes (tumor suppressor gene).In this study, we conducted a hospital-based case-control study of the MTHFR and MTR polymorphisms and bladder cancer risk in a Chinese population. Additionally, we investigated the association between the polymorphisms of MTHFR and the promoter hypermethylation in RASSF1A gene in bladder cancer.Materials and MethodsIn this hospital-based case-control study including 312 bladder cancer cases and 325 cancer-free controls. All subjects were genetically unrelated ethnic Han Chinese and were from Shenyang city and surrounding regions in northeast China. Cases who were newly diagnosed with incident bladder cancer according to histopathology, were consecutively recruited from the Shengjing Hospital of China Medical University, without the restrictions of age, sex and tumor stage. Those who were previous cancer and previous radiotherapy or chemotherapy were excluded. In order to perform a study of the methylation status, surgically excised, fresh frozen bladder cancer tissues (n= 45) were randomly selected from the 312 bladder cancer cases mentioned above. The cancer-free controls were recruited from those who were seeking health care for conditions other than cancer. Control subjects are frequency matched to the cases on the basis of age (±5 years), sex and ethnicity.The MTHFR (C677T, A1298C) and MTR A2756G polymorphisms were determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The methylation at the promoter region of RASSF1A was determined by methylation-specific PCRResults1. The MTHFR (C677T, A1298C) polymorphisms and bladder cancer risk.(1) The frequencies of CC,CT and TT genotype of MTHFR C677T is 26.28%,54.17%and 19.55%for the cases,34.77%,52.31%and 12.92%for the controls; The frequencies of T allele of MTHFR C677T is 46.64%for the cases and 39.08%for the controls. The difference in the frequencies of genotypes and alleles between the cases and controls was statistically significant (P=0.017 for genotypes and P=0.006 for alleles). The frequencies of AA,AC and CC genotype of MTHFR A1298C is 68.91%,29.17%and 1.92%for the cases,69.54%,28.31%and 2.15%for the controls; The frequencies of C allele of MTHFR A1298C is 16.51%for the cases and16.31%for the controls. The difference in the frequencies of genotypes and alleles between the cases and controls was not statistically significant (P=0.955 for genotypes and P=0.924 for alleles).(2) The MTHFR 677TT genotype was statistically significantly associated with an increased risk of bladder cancer (OR= 2.00,95%CI:1.23-3.25) compared with the MTHFR 677CC genotype. The MTHFR A1298C polymorphism was not associated with risk of bladder cancer.(3)Combined MTHFR C677T and MTHFR A1298C genotypes, The frequencies of MTHFR TT/AA (677TT+1298AA) genotype is 19.55%for the cases and12.92%for the controls, the difference between the cases and controls was statistically significant (P=0.003).(4) Combined MTHFR C677T and MTHFR A1298C genotypes, it shows that genotype 677TT/1298AA (MTHFR 677CT+1298AA) was statistically significantly associated with an increased risk of bladder cancer (OR= 2.27,95%CI:1.33-3.90) compared with the genotype 677CC/1298AA (MTHFR 677CC+1298AA).(5) The reference group consisted of never smokers with the MTHFR 677 CC genotype. We observed a 2.89-fold (95%CI:1.41-5.95) elevated risk of bladder cancer for former smokers with the MTHFR 677 CC genotype and a 3.81-fold increased risk (95%CI:2.09-6.93) for former smokers carrying at least one variant allele. Interestingly, the risk increased to 5.02 (95%CI:2.39-10.54) for current smokers with the MTHFR 677CC and reached the highest value of 7.36 (95%CI:3.88-14.15) in current smokers carrying at least one variant allele.(6) The reference group consisted of never smokers with the MTHFR 1298AA genotype. We observed a 1.60-fold (95%CI:1.02-2.51) elevated risk of bladder cancer for former smokers with the MTHFR 1298AA genotype and a 2.89-fold increased risk (95%CI:1.60-5.21) for former smokers carrying at least one variant allele. Interestingly, the risk increased to 3.15 (95%CI:1.96-5.09) for current smokers with the MTHFR 1298AA and reached the highest value of 5.27 (95%CI:2.34-11.85) in current smokers carrying at least one variant allele.(7) Compared with the reference group, never smokers with the MTHFR 677CC genotype, We observed a 2.44-fold (95%CI:1.19-5.02) elevated risk of bladder cancer for light smokers (pack-years< 35) with the MTHFR 677CC genotype and a 4.47-fold increased risk (95%CI:2.50-8.00) for light smokers (pack-years< 35) carrying at least one variant allele. Interestingly, the risk increased to 6.20 (95%CI:2.91-13.23) for heavy smokers (pack-years≥35) with the MTHFR 677 CC and reached the highest value of 6.79 (95%CI:3.30-13.97) in heavy smokers (pack-years≥35) carrying at least one variant allele.(8) Compared with the reference group, never smokers with the MTHFR 1298AA genotype, We observed a 1.88-fold (95%CI:1.24-2.86) elevated risk of bladder cancer for light smokers (pack-years< 35) with the MTHFR 1298AA genotype and a 3.13-fold increased risk (95%CI:1.68-5.85) for light smokers (pack-years< 35) carrying at least one variant allele. Interestingly, the risk increased to 3.25 (95%CI: 1.85-5.72)for heavy smokers (pack-years≥35) with the MTHFR 1298AA and reached the highest value of 4.17 (95%CI:2.04-8.51) in heavy smokers (pack-years≥35) carrying at least one variant allele.2. The MTR A2756G polymorphism and bladder cancer risk(1) The frequencies of AA,AG and GG genotype of MTR A2756G is 83.97%,13.14% and 2.88% for the cases,86.46%,12.31% and 1.23% for the controls; The frequencies of G allele of MTR A2756G is 9.46% for the cases and 7.38% for the controls. The difference in the frequencies of genotypes and alleles between the cases and controls was not statistically significant (P=0.311 for genotypes and P=0.183 for alleles).(2) The MTR A2756G polymorphism was not associated with risk of bladder cancer.(3) The reference group consisted of never smokers with the MTR 2756AA genotype. We observed a 2.09-fold (95%CI:1.40-3.13) elevated risk of bladder cancer for former smokers with the MTR 2756AA genotype and a 2.75-fold increased risk (95%CI:1.32-5.73) for former smokers carrying at least one variant allele. Interestingly, the risk increased to 3.98 (95%CI:2.55-6.22) for current smokers with the MTR 2756AA and reached the highest value of 4.09 (95%CI:1.69-9.89) in current smokers carrying at least one variant allele.(4) Compared with the reference group, never smokers with the MTR 2756AA genotype, We observed a 2.36-fold (95%CI:1.61-3.47) elevated risk of bladder cancer for light smokers (pack-years< 35) with the MTR 2756AA genotype and a 2.60-fold increased risk (95%CI:1.32-5.15) for light smokers (pack-years< 35) carrying at least one variant allele. Interestingly, the risk increased to 3.85 (95%CI:2.35-6.30) for heavy smokers (pack-years≥35) with the MTR 2756AA and reached the highest value of 5.39 (95%CI:1.87-15.50) in heavy smokers (pack-years≥35) carrying at least one variant allele.3. Methylation status of RASSF1A promoter and the MTHFR (C677T, A1298C) polymorphism(1) Among 45 cases,28 (62.22%) were methylated and 17 (37.78%) unmethylated for RASSF1A gene, the difference between the cases and controls was statistically significant (P=0.001).(2) For the MTHFR C677T and A1298C polymorphisms, there were no statistically significant difference in the genotypes distribution between the unmethylated and hypermethylated RASSF1A promoter cases.Conclution1. The MTHFR 677TT genotype had an effect on increasing the risk of bladder cancer compared with the MTHFR 677CC genotype. MTHFR A1298C and MTR A2756G polymorphisms were not associated with risk of bladder cancer.2. In combined MTHFR genotypes, we found that 677TT/1298AA may contribute to the risk of bladder cancer compared with the 677CC/1298AA genotype.3. Our results also showed a significantly increased risk of bladder cancer among current smokers and heavy smokers (pack-years≥35) regardless of genotype, thus further supporting the notion that smoking is an important risk factor for bladder cancer, as has been established in many previous studies. More importantly, our study showed that current and heavy smokers (pack-years≥35) with adverse metabolic genotypes are at the highest risk of bladder cancer, suggesting a joint effect of cigarette smoking and polymorphisms of the folate metabolic genes.4. Compared to normal bladder tissues, RASSF1 A gene promoter showed a higher proportion hypermethylation in bladder cancer tissues.5. The polymorphisms of MTHFR (C677T, A1298C) were not associated with RASSF1 A gene promoter hypermethylation.