叶酸代谢酶基因MTHFR、MTR多态性与膀胱癌遗传易感性及与RASSF1A基因启动子区甲基化关系的研究
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摘要
前言
世界范围内,膀胱癌发病率居恶性肿瘤的第九位,在我国,膀胱癌发病率居恶性肿瘤的第十位,近年来膀胱癌发病率及死亡率有增高的趋势。大量流行病学研究发现吸烟和接触化学致癌物是膀胱癌的主要致病因素。其它可能的致病因素还包括人造甜味剂、咖啡、染发剂、长期饮用砷含量高的饮用水等。然而接触同样的环境危险因素,却只有小部分人发生膀胱癌,这提示不同的个体对膀胱癌有遗传易感性。体内与肿瘤相关基因DNA序列的改变赋予个体不同的遗传背景,引起个体或群体对恶性肿瘤易感性的差异,近年来,随着对肿瘤遗传易感性研究的增加,肿瘤相关基因多态性的研究越来越被学者重视。
流行病学研究发现低叶酸摄入水平会增加肿瘤的发病风险,这主要是因为低叶酸摄入会导致DNA合成障碍和DNA甲基化状态紊乱。叶酸在体内的代谢过程即是一碳单位的转运过程,一方面,叶酸以5-甲基四氢叶酸(5-THF)的形式合成S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)以提供甲基基团用于DNA甲基化,SAM是体内通用甲基供体；另一方面,叶酸以5,10-亚甲基四氢叶酸(5,10-methylenetetrahydrolate)的形式参与DNA的合成过程。叶酸这两个代谢过程对于机体极其重要,若此代谢过程出现异常并长期存在,则很有可能导致癌变。
叶酸代谢酶基因存在众多的单核苷酸多态,这些基因单核苷酸多态可通过改变酶活性进而影响叶酸在体内的合成和代谢。因此我们假设叶酸代谢酶基因MTHFR和MTR多态位点的存在可能会影响叶酸的合成和代谢,进而可导致DNA的合成障碍和DNA的甲基化状态紊乱。DNA甲基化状态紊乱包括全基因组低甲基化或特异性基因高甲基化,而全基因组低甲基化又常常伴有肿瘤抑癌基因的高甲基化,这都有可能导致膀胱癌的发生。
本课题探讨叶酸代谢酶基因MTHFR和MTR多态性与膀胱癌遗传易感性的关系,同时初步探讨抑癌基因RASSF1A基因启动子区甲基化状态与叶酸代谢酶基因MTHFR多态性之间的关系。
材料和方法
本课题采用以医院为基础的病例对照研究设计,研究对象为东北地区的汉族居民,病例组膀胱癌患者312例,对照组325例。病例组是在中国医科大学附属盛京医院泌尿外科收治的原发性膀胱癌患者,均经病理确诊,既往无肿瘤史,术前未经放疗及化疗。对照组为同期收治的其他非肿瘤患者及健康体检者。病例组与对照组在年龄(土5岁)及性别相匹配。
采用聚合酶链反应—限制性片段长度多态性(Polymerase Chain Reaction-Restriction Fragment Length Polymorphism, PCR-RFLP)技术,检测MTHFR (C677T, A1298C)及MTR (A2756G)基因位点的多态性。采用甲基化特异性PCR((Methylation specific PCR, MSP)方法,在病例组中随机选取45例膀胱癌组织,检测RASSF1A基因启动子区甲基化情况。
结果
一、MTHFR (C677T, A1298C)与膀胱癌遗传易感性
1、病例组中MTHFR C677T的三种基因型CC、CT及TT的频率分别为26.28%、54.17%和19.55%,对照组分别为34.77%、52.31%和12.92%,两组间比较有统计学差异(P=0.017)；病例组与对照组MTHFR C677T的T等位基因频率分别为46.64%和39.08%,有统计学差异(P=0.006)。病例组中MTHFR A1298C的三种基因型AA、AC及CC的频率分别为68.91%、29.17%和1.92%,对照组分别为69.54%、28.31%和2.15%,两组间比较无统计学差异(P=0.955)；病例组与对照组MTHFR A1298C的C等位基因频率分别为16.51%和16.31%,无统计学差异(P=0.924)。
2、携带MTHFR 677TT基因型的个体发生膀胱癌的风险是携带677CC基因型者2.00倍(95%CI：1.23-3.25),MTHFR A1298C基因多态与膀胱癌遗传易感性无显著关联。
3、MTHFR C677T与MTHFR A1298C联合分析,病例组与对照组中同时携带MTHFR TT/AA (677TT+1298AA)基因型的频率分别为19.55%和12.92%,两组间比较有统计学差异(P=0.003)。
4、携带MTHFR TT/AA (677TT+1298AA)基因型者发生膀胱癌的风险是携带CC/AA(677CC+1298AA)基因型者2.27倍(95% CI：1.33-3.90)。
5、以携带MTHFR 677CC基因型不吸烟的个体为参照,在既往吸烟者中,携带MTHFR 677CC基因型的个体和携带MTHFR 677CT或TT基因型的个体发生膀胱癌的风险分别增加到2.89倍(95% CI：1.41-5.95)和3.81倍(95% CI：2.09-6.93),而在现时吸烟者中,风险分别增加到5.02倍(95% CI：2.39-10.54)和7.36倍(95% CI：3.88-14.15)。
6、当以携带MTHFR 1298AA基因型不吸烟的个体为参照,在既往吸烟者中,携带MTHFR 1298AA基因型的个体和携带MTHFR1298AC或CC基因型的个体发生膀胱癌的风险分别增加到1.60倍(95% CI：1.02-2.51)和2.89倍(95% CI：1.60-5.21),而在现时吸烟者中,风险分别增加到3.15倍(95% CI：1.96-5.09)和5.27倍(95% CI：2.34-11.85)。
7、以携带MTHFR 677CC基因型吸烟量为0包年的个体为参照,吸烟量<35包年的个体,携带MTHFR 677CC基因型者和携带MTHFR 677CT或TT基因型者发生膀胱癌的风险分别增加到2.44倍(95% CI：1.19-5.02)和4.47倍(95% CI：2.50-8.00),而在吸烟量≥35包年的个体中,风险分别增加到6.20倍(95% CI：2.91-13.23)和6.79倍(95% CI：3.30-13.97)。
8、当以携带MTHFR 1298AA基因型吸烟量为0包年的个体为参照,吸烟量<35包年的个体,携带MTHFR 1298AA基因型的个体和携带MTHFR1298AC或CC基因型的个体发生膀胱癌的风险分别增加到1.88倍(95% CI：1.24-2.86)和3.13倍(95% CI：1.68-5.85),而在吸烟量≥35包年的个体中,风险分别增加到3.25倍(95% CI：1.85-5.72)和4.17倍(95% CI：2.04-8.51)。
二、MTR A2756G基因多态与膀胱癌遗传易感性
1、病例组中MTR A2756G的三种基因型AA、AG及GG的频率分别为83.97%、13.14%和2.88%,对照组分别为86.46%、12.31%和1.23%,两组间比较有无统计学差异(P=0.311)；病例组与对照组MTR A2756G的G等位基因频率分别为9.46%和7.38%,两组间比较无统计学差异(P=0.183)。
2、MTR A2756G基因多态与膀胱癌遗传易感性无显著关联。
3、当以携带MTR 2756AA基因型不吸烟的个体为参照,在既往吸烟者中,携带MTR 2756AA基因型的个体和携带MTR2756AG或GG基因型的个体发生膀胱癌的风险分别增加到2.09倍(95% CI：1.40-3.13)和2.75倍(95% CI：1.32-5.73),而在现时吸烟者中,风险分别增加到3.98倍(95% CI：2.55-6.22)和4.09倍(95% CI：1.69-9.89)。
4、当以携带MTR 2756AA基因型吸烟量为0包年的个体为参照,吸烟量<35包年的个体,携带MTR 2756AA基因型的个体和携带MTR2756AG或GG基因型的个体发生膀胱癌的风险分别增加到2.36倍(95% CI：1.61-3.47)和2.60倍(95% CI：1.32-5.15),而在吸烟量≥35包年的个体中,风险分别增加到3.85倍(95% CI：2.35-6.30)和5.39倍(95% CI：1.87-15.50)。
三、RASSF1A基因启动子区甲基化状态与MTHFR (C677T, A1298C)基因多态性
1、45例膀胱移形细胞癌组织中28例检测到RASSF1A甲基化,8例正常膀胱组织中均未检测到RASSF1A甲基化,两组间甲基化率存在统计学学差异(P=0.001)。
2、MTHFR (C677T,A1298C)基因多态与RASSF1A启动子区高甲基化无明显关联。
结论
1、与MTHFR 677CC基因型相比,携带MTHFR 677TT基因型的个体膀胱癌发病风险增加。MTHFR A1298C和MTRA2756G基因多态与膀胱癌发病风险无明显关联。
2、MTHFR C677T与MTHFR A1298C联合分析：与MTHFR CC/AA (677CC+1298AA)基因型相比,携带MTHFR TT/AA (677TT+1298AA)基因型者发生膀胱癌的风险增加。
3、MTHFR (C677T, A1298C)和MTR A2756G基因多态与吸烟联合作用研究发现,现时吸烟者和大量吸烟者(≥35包年)无论携带任何基因型,都会增加膀胱癌发病风险；同时,我们发现,现时吸烟者和大量吸烟者(≥35包年)如携带不利的基因型(如MTHFR 677TT),发生膀胱癌的风险将明显增加,提示吸烟与叶酸代谢酶基因多态性存在联合作用。
4、膀胱癌组织RASSF1A基因启动子区甲基化率明显高于正常膀胱组织。
5、MTHFR (C677T, A1298C)基因多态与膀胱癌组织RASSF1A基因启动子区高甲基化无明显关联。
Introduction
Bladder 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 Methods
In 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 PCR
Results
1. 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.
Conclution
1. 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.
世界范围内,膀胱癌发病率居恶性肿瘤的第九位,在我国,膀胱癌发病率居恶性肿瘤的第十位,近年来膀胱癌发病率及死亡率有增高的趋势。大量流行病学研究发现吸烟和接触化学致癌物是膀胱癌的主要致病因素。其它可能的致病因素还包括人造甜味剂、咖啡、染发剂、长期饮用砷含量高的饮用水等。然而接触同样的环境危险因素,却只有小部分人发生膀胱癌,这提示不同的个体对膀胱癌有遗传易感性。体内与肿瘤相关基因DNA序列的改变赋予个体不同的遗传背景,引起个体或群体对恶性肿瘤易感性的差异,近年来,随着对肿瘤遗传易感性研究的增加,肿瘤相关基因多态性的研究越来越被学者重视。
流行病学研究发现低叶酸摄入水平会增加肿瘤的发病风险,这主要是因为低叶酸摄入会导致DNA合成障碍和DNA甲基化状态紊乱。叶酸在体内的代谢过程即是一碳单位的转运过程,一方面,叶酸以5-甲基四氢叶酸(5-THF)的形式合成S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)以提供甲基基团用于DNA甲基化,SAM是体内通用甲基供体；另一方面,叶酸以5,10-亚甲基四氢叶酸(5,10-methylenetetrahydrolate)的形式参与DNA的合成过程。叶酸这两个代谢过程对于机体极其重要,若此代谢过程出现异常并长期存在,则很有可能导致癌变。
叶酸代谢酶基因存在众多的单核苷酸多态,这些基因单核苷酸多态可通过改变酶活性进而影响叶酸在体内的合成和代谢。因此我们假设叶酸代谢酶基因MTHFR和MTR多态位点的存在可能会影响叶酸的合成和代谢,进而可导致DNA的合成障碍和DNA的甲基化状态紊乱。DNA甲基化状态紊乱包括全基因组低甲基化或特异性基因高甲基化,而全基因组低甲基化又常常伴有肿瘤抑癌基因的高甲基化,这都有可能导致膀胱癌的发生。
本课题探讨叶酸代谢酶基因MTHFR和MTR多态性与膀胱癌遗传易感性的关系,同时初步探讨抑癌基因RASSF1A基因启动子区甲基化状态与叶酸代谢酶基因MTHFR多态性之间的关系。
材料和方法
本课题采用以医院为基础的病例对照研究设计,研究对象为东北地区的汉族居民,病例组膀胱癌患者312例,对照组325例。病例组是在中国医科大学附属盛京医院泌尿外科收治的原发性膀胱癌患者,均经病理确诊,既往无肿瘤史,术前未经放疗及化疗。对照组为同期收治的其他非肿瘤患者及健康体检者。病例组与对照组在年龄(土5岁)及性别相匹配。
采用聚合酶链反应—限制性片段长度多态性(Polymerase Chain Reaction-Restriction Fragment Length Polymorphism, PCR-RFLP)技术,检测MTHFR (C677T, A1298C)及MTR (A2756G)基因位点的多态性。采用甲基化特异性PCR((Methylation specific PCR, MSP)方法,在病例组中随机选取45例膀胱癌组织,检测RASSF1A基因启动子区甲基化情况。
结果
一、MTHFR (C677T, A1298C)与膀胱癌遗传易感性
1、病例组中MTHFR C677T的三种基因型CC、CT及TT的频率分别为26.28%、54.17%和19.55%,对照组分别为34.77%、52.31%和12.92%,两组间比较有统计学差异(P=0.017)；病例组与对照组MTHFR C677T的T等位基因频率分别为46.64%和39.08%,有统计学差异(P=0.006)。病例组中MTHFR A1298C的三种基因型AA、AC及CC的频率分别为68.91%、29.17%和1.92%,对照组分别为69.54%、28.31%和2.15%,两组间比较无统计学差异(P=0.955)；病例组与对照组MTHFR A1298C的C等位基因频率分别为16.51%和16.31%,无统计学差异(P=0.924)。
2、携带MTHFR 677TT基因型的个体发生膀胱癌的风险是携带677CC基因型者2.00倍(95%CI：1.23-3.25),MTHFR A1298C基因多态与膀胱癌遗传易感性无显著关联。
3、MTHFR C677T与MTHFR A1298C联合分析,病例组与对照组中同时携带MTHFR TT/AA (677TT+1298AA)基因型的频率分别为19.55%和12.92%,两组间比较有统计学差异(P=0.003)。
4、携带MTHFR TT/AA (677TT+1298AA)基因型者发生膀胱癌的风险是携带CC/AA(677CC+1298AA)基因型者2.27倍(95% CI：1.33-3.90)。
5、以携带MTHFR 677CC基因型不吸烟的个体为参照,在既往吸烟者中,携带MTHFR 677CC基因型的个体和携带MTHFR 677CT或TT基因型的个体发生膀胱癌的风险分别增加到2.89倍(95% CI：1.41-5.95)和3.81倍(95% CI：2.09-6.93),而在现时吸烟者中,风险分别增加到5.02倍(95% CI：2.39-10.54)和7.36倍(95% CI：3.88-14.15)。
6、当以携带MTHFR 1298AA基因型不吸烟的个体为参照,在既往吸烟者中,携带MTHFR 1298AA基因型的个体和携带MTHFR1298AC或CC基因型的个体发生膀胱癌的风险分别增加到1.60倍(95% CI：1.02-2.51)和2.89倍(95% CI：1.60-5.21),而在现时吸烟者中,风险分别增加到3.15倍(95% CI：1.96-5.09)和5.27倍(95% CI：2.34-11.85)。
7、以携带MTHFR 677CC基因型吸烟量为0包年的个体为参照,吸烟量<35包年的个体,携带MTHFR 677CC基因型者和携带MTHFR 677CT或TT基因型者发生膀胱癌的风险分别增加到2.44倍(95% CI：1.19-5.02)和4.47倍(95% CI：2.50-8.00),而在吸烟量≥35包年的个体中,风险分别增加到6.20倍(95% CI：2.91-13.23)和6.79倍(95% CI：3.30-13.97)。
8、当以携带MTHFR 1298AA基因型吸烟量为0包年的个体为参照,吸烟量<35包年的个体,携带MTHFR 1298AA基因型的个体和携带MTHFR1298AC或CC基因型的个体发生膀胱癌的风险分别增加到1.88倍(95% CI：1.24-2.86)和3.13倍(95% CI：1.68-5.85),而在吸烟量≥35包年的个体中,风险分别增加到3.25倍(95% CI：1.85-5.72)和4.17倍(95% CI：2.04-8.51)。
二、MTR A2756G基因多态与膀胱癌遗传易感性
1、病例组中MTR A2756G的三种基因型AA、AG及GG的频率分别为83.97%、13.14%和2.88%,对照组分别为86.46%、12.31%和1.23%,两组间比较有无统计学差异(P=0.311)；病例组与对照组MTR A2756G的G等位基因频率分别为9.46%和7.38%,两组间比较无统计学差异(P=0.183)。
2、MTR A2756G基因多态与膀胱癌遗传易感性无显著关联。
3、当以携带MTR 2756AA基因型不吸烟的个体为参照,在既往吸烟者中,携带MTR 2756AA基因型的个体和携带MTR2756AG或GG基因型的个体发生膀胱癌的风险分别增加到2.09倍(95% CI：1.40-3.13)和2.75倍(95% CI：1.32-5.73),而在现时吸烟者中,风险分别增加到3.98倍(95% CI：2.55-6.22)和4.09倍(95% CI：1.69-9.89)。
4、当以携带MTR 2756AA基因型吸烟量为0包年的个体为参照,吸烟量<35包年的个体,携带MTR 2756AA基因型的个体和携带MTR2756AG或GG基因型的个体发生膀胱癌的风险分别增加到2.36倍(95% CI：1.61-3.47)和2.60倍(95% CI：1.32-5.15),而在吸烟量≥35包年的个体中,风险分别增加到3.85倍(95% CI：2.35-6.30)和5.39倍(95% CI：1.87-15.50)。
三、RASSF1A基因启动子区甲基化状态与MTHFR (C677T, A1298C)基因多态性
1、45例膀胱移形细胞癌组织中28例检测到RASSF1A甲基化,8例正常膀胱组织中均未检测到RASSF1A甲基化,两组间甲基化率存在统计学学差异(P=0.001)。
2、MTHFR (C677T,A1298C)基因多态与RASSF1A启动子区高甲基化无明显关联。
结论
1、与MTHFR 677CC基因型相比,携带MTHFR 677TT基因型的个体膀胱癌发病风险增加。MTHFR A1298C和MTRA2756G基因多态与膀胱癌发病风险无明显关联。
2、MTHFR C677T与MTHFR A1298C联合分析：与MTHFR CC/AA (677CC+1298AA)基因型相比,携带MTHFR TT/AA (677TT+1298AA)基因型者发生膀胱癌的风险增加。
3、MTHFR (C677T, A1298C)和MTR A2756G基因多态与吸烟联合作用研究发现,现时吸烟者和大量吸烟者(≥35包年)无论携带任何基因型,都会增加膀胱癌发病风险；同时,我们发现,现时吸烟者和大量吸烟者(≥35包年)如携带不利的基因型(如MTHFR 677TT),发生膀胱癌的风险将明显增加,提示吸烟与叶酸代谢酶基因多态性存在联合作用。
4、膀胱癌组织RASSF1A基因启动子区甲基化率明显高于正常膀胱组织。
5、MTHFR (C677T, A1298C)基因多态与膀胱癌组织RASSF1A基因启动子区高甲基化无明显关联。
Introduction
Bladder 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 Methods
In 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 PCR
Results
1. 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.
Conclution
1. 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.
引文
1 Parkin D, Bray F, Ferlay J, et al. Global cancer statistics,2002. CA Cancer J Clin. 2002;55:74-108.
2 Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008. CA Cancer J Clin.2008;58:71-96.
3 Fortuny J, Johnson C, Bohlke K, et al. Use of anti-inflammatory drugs and lower esophageal sphincter-relaxing drugs and risk of esophageal and gastric cancers. Clin Gastroenterol Hepatol.2007;5:1154-1159.e3.
4 Brockmoller J, Cascorbi I, Kerb R, et al. Combined analysis of inherited polymorphisms in arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1, microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk. Cancer Res. 1996;56:3915-25.
5 Moore LE, Wiencke JK, Bates MN, et al. Investigation of genetic polymorphisms and smoking in a bladder cancer case-control study in Argentina. Cancer Letters.2004;211:199-207.
6 Wang M, Zhu H, Fu G, et al. Polymorphisms of methylenetetrahydrofolate reductase and methionine synthase genes and bladder cancer risk:a case-control study with meta-analysis. Clin Exp Med.2009;9:9-19.
7 Duthie S. Folic acid deficiency and cancer:mechanisms of DNA instability. Br Med Bull. 1999;55:578-92.
8 Choi S, Mason J. Folate and carcinogenesis:an integrated scheme. J Nutr.2000;130:129-32.
9 Wei Q, Shen H, Wang L, et al. Association between low dietary folate intake and suboptimal cellular DNA repair capacity. Cancer Epidemiol Biomarkers Prev.2003;12:963-9.
10 Larsson S, Giovannucci E, Wolk A. Folate intake, MTHFR polymorphisms, and risk of esophageal, gastric, and pancreatic cancer:a meta-analysis. Gastroenterology. 2006;131:1271-83.
11 Cravo M, Pinto A, Chaves P, et al. Effect of folate supplementation on DNA methylation of rectal mucosa in patients with colonic adenomas:correlation with nutrient intake. Clin Nutr. 1998;17:45-9.
12 Blount B, Mack M, Wehr C, et al. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage:implications for cancer and neuronal damage. Proc Natl Acad Sci U S A.1997;94:3290-5.
13 Herman J, Umar A, Polyak K, et al. Incidence and functional consequences of hMLHl promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A 1998;95:6870-5.
14 Kim Y. Folate and carcinogenesis:evidence, mechanisms, and implications. J Nutr Biochem. 1999; 10:66-88.
15 Suzuki T, Matsuo K, Hiraki A, et al. Impact of one-carbon metabolism-related gene polymorphisms on risk of lung cancer in Japan:a case control study. Carcinogenesis. 2007;28:1718-25.
16 Frosst P, Blom H, Milos R, et al. A candidate genetic risk factor for vascular disease:a common mutation in methylenetetrahydrofolate reductase. Nat Genet.1995;10:111-3.
17 Molloy A, Daly S, Mills J, et al. Thermolabile variant of 5,10-methylenetetrahydrofolate reductase associated with low red-cell folates:implications for folate intake recommendations. Lancet.1997;349:1591-3.
18 Ulvik A, Ueland P, Fredriksen A, et al. Functional inference of the methylenetetrahydrofolate reductase 677C> T and 1298A> C polymorphisms from a large-scale epidemiological study. Hum Genet.2007;121:57-64.
19 Stern L, Mason J, Selhub J, et al. Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiol Biomarkers Prev.2000;9:849-53.
20 Bagley P, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc Natl Acad Sci U S A.1998;95:13217-20.
21 Sohn K, Croxford R, Yates Z, et al. Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate. J Natl Cancer Inst.2004;96:134-44.
22 van der Put N, Gabreels F, Stevens E, et al. A second common mutation in the . methylenetetrahydrofolate reductase gene:an additional risk factor for neural-tube defects? Am J Hum Genet.1998;62:1044-51.
23 Stegmann K, Ziegler A, Ngo E, et al. Linkage disequilibrium of MTHFR genotypes 677C/T-1298A/C in the German population and association studies in probands with neural tube defects(NTD). Am J Med Genet.1999;87:23-9.
24 Chen J, Ma J, Stampfer M, et al. Linkage disequilibrium between the 677C>T and 1298A>C polymorphisms in human methylenetetrahydrofolate reductase gene and their contributions to risk of colorectal cancer. Pharmacogenetics.2002;12:339-42.
25 Ouerhani S, Oliveira E, Marrakchi R, et al. Methylenetetrahydrofolate reductase and methionine synthase polymorphisms and risk of bladder cancer in a Tunisian population. Cancer Genetics and Cytogenetics.2007;176:48-53.
26 Harmon D, Shields D, Woodside J, et al. Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations. Genet Epidemiol.1999;17:298-309.
27 Chen J, Giovannucci E, Hankinson S, et al. A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma. Carcinogenesis.1998;19:2129-32.
28 Leclerc D, Campeau E, Goyette P, et al. Human methionine synthase:cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum Mol Genet.1996;5:1867-74.
29 Chen J, Stampfer M, Ma J, et al. Influence of a methionine synthase (D919G) polymorphism on plasma homocysteine and folate levels and relation to risk of myocardial infarction. Atherosclerosis.2001;154:667-72.
30 Tsai M, Bignell M, Yang F, et al. Polygenic influence on plasma homocysteine:association of two prevalent mutations, the 844ins68 of cystathionine beta-synthase and A(2756)G of methionine synthase, with lowered plasma homocysteine levels. Atherosclerosis. 2000;149:131-7.
31 Lin J, Spitz MR, Wang Y, et al. Polymorphisms of folate metabolic genes and susceptibility to bladder cancer:A case-control study. Proceedings of the American Association for Cancer Research Annual Meeting.2004;45:918-919.
32 Shi Y, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res.2005;15:97-8.
33 Platek M, Shields P, Marian C, et al. Alcohol consumption and genetic variation in methylenetetrahydrofolate reductase and 5-methyltetrahydrofolate-homocysteine methyltransferase in relation to breast cancer risk. Cancer Epidemiol Biomarkers Prev. 2009; 18:2453-9.
34 Ma E, Iwasaki M, Kobayashi M, et al. Dietary intake of folate, vitamin B2, vitamin B6, vitamin B12, genetic polymorphism of related enzymes, and risk of breast cancer:a case-control study in Japan. Nutr Cancer.2009;61:447-56.
35 Zhang F, Terry M, Hou L, et al. Genetic polymorphisms in folate metabolism and the risk of stomach cancer. Cancer Epidemiol Biomarkers Prev.2007;16:115-21.
36 Yang C, Matsuo K, Ito H, et al. Gene-environment interactions between alcohol drinking and the MTHFR C677T polymorphism impact on esophageal cancer risk:results of a case-control study in Japan. Carcinogenesis.2005;26:1285-90.
37 Suzuki T, Matsuo K, Sawaki A, et al. Alcohol drinking and one-carbon metabolism-related gene polymorphisms on pancreatic cancer risk. Cancer Epidemiol Biomarkers Prev. 2008; 17:2742-7.
38 Steck S, Keku T, Butler L, et al. Polymorphisms in methionine synthase, methionine synthase reductase and serine hydroxymethyltransferase, folate and alcohol intake, and colon cancer risk. J Nutrigenet Nutrigenomics.2008; 1:196-204.
39 Chen K, Song L, Jin M, et al. Association between genetic polymorphisms in folate metabolic enzyme genes and colorectal cancer:a nested case-control study. Zhonghua Zhong Liu Za Zhi. 2006;28:429-32.
40 Matsuo K, Ito H, Wakai K, et al. One-carbon metabolism related gene polymorphisms interact with alcohol drinking to influence the risk of colorectal cancer in Japan. Carcinogenesis. 2005;26:2164-71.
41 Tong S, Lee J, Song E, et al. The effects of polymorphisms in methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR) on the risk of cervical intraepithelial neoplasia and cervical cancer in Korean women. Cancer Causes Control.2010;21:23-30.
42 Marchal C, Redondo M, Reyes-Engel A, et al. Association between polymorphisms of folate-metabolizing enzymes and risk of prostate cancer. Eur J Surg Oncol.2008;34:805-10.
43 Lee K, Lan Q, Kricker A, et al. One-carbon metabolism gene polymorphisms and risk of non-Hodgkin lymphoma in Australia. Hum Genet.2007;122:525-33.
44 Matsuo K, Hamajima N, Suzuki R, et al. Methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms and reduced risk of malignant lymphoma. Am J Hematol.2004;77:351-7.
45 Heijmans B, Boer J, Suchiman H, et al. A common variant of the methylenetetrahydrofolate reductase gene (lp36) is associated with an increased risk of cancer. Cancer Res. 2003;63:1249-53.
46 Kimura F, Florl A, Steinhoff C, et al. Polymorphic methyl group metabolism genes in patients with transitional cell carcinoma of the urinary bladder. Mutat Res.2001;458:49-54.
47 Karagas MR, Park S, Nelson HH, et al. Methylenetetrahydrofolate reductase (MTHFR) variants and bladder cancer:A population-based case-control study. International Journal of Hygiene and Environmental Health.2005;208:321-327.
48 Moore LE, Malats N, Rothmans N, et al. Polymorphisms in one-carbon metabolism and trans-sulfuration pathway genes and susceptibility to bladder cancer. International Journal of Cancer.2007;120:2452-2458.
49 Rouissi K, Ouerhani S, Oliveira E, et al. Polymorphisms in one-carbon metabolism pathway genes and risk for bladder cancer in a Tunisian population. Cancer Genet Cytogenet. 2009; 195:43-53.
50 Ouerhani S, Rouissi K, Marrakchi R, et al. Combined effect of NAT2, MTR and MTHFR genotypes and tobacco on bladder cancer susceptibility in Tunisian population. Cancer Detect Prev.2009;32:395-402.
51 Matsuo K, Suzuki R, Hamajima N, et al. Association between polymorphisms of folate-and methionine-metabolizing enzymes and susceptibility to malignant lymphoma. Blood. 2001;97:3205-9.
52 Chen J, Giovannucci E, Kelsey K, et al. A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer. Cancer Res.1996;56:4862-4.
53 Ma J, Stampfer M, Giovannucci E, et al. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res.1997;57:1098-102.
54 Ulrich C, Kampman E, Bigler J, et al. Colorectal adenomas and the C677T MTHFR polymorphism:evidence for gene-environment interaction? Cancer Epidemiol Biomarkers Prev.1999;8:659-68.
55 Song C, Xing D, Tan W, et al. Methylenetetrahydrofolate reductase polymorphisms increase risk of esophageal squamous cell carcinoma in a Chinese population. Cancer Res. 2001;61:3272-5.
56 Levine A, Siegmund K, Ervin C, et al. The methylenetetrahydrofolate reductase 677C->T polymorphism and distal colorectal adenoma risk. Cancer Epidemiol Biomarkers Prev. 2000;9:657-63.
57 Giovannucci E, Chen J, Smith-Warner S, et al. Methylenetetrahydrofolate reductase, alcohol dehydrogenase, diet, and risk of colorectal adenomas. Cancer Epidemiol Biomarkers Prev. 2003;12:970-9.
58 Piyathilake C, Macaluso M, Hine R, et al. Local and systemic effects of cigarette smoking on folate and vitamin B-12. Am J Clin Nutr.1994;60:559-66.
59 Laird P, Jaenisch R. DNA methylation and cancer. Hum Mol Genet.1994;3 Spec No:1487-95.
60 Dammann R, Li C, Yoon J, et al. Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet.2000;25:315-9.
61 Catto J, Azzouzi A, Rehman I, et al. Promoter hypermethylation is associated with tumor location, stage, and subsequent progression in transitional cell carcinoma. J Clin Oncol. 2005;23:2903-10.
62 Lin H, Ke H, Huang S, et al. Increase sensitivity in detecting superficial, low grade bladder cancer by combination analysis of hypermethylation of E-cadherin, p16, p14, RASSF1A genes in urine. Urol Oncol.2009.
63 Pfeifer G, Dammann R. Methylation of the tumor suppressor gene RASSF1A in human tumors. Biochemistry (Mosc).2005;70:576-83.
64 Das P, Singal R. DNA methylation and cancer. J Clin Oncol.2004;22:4632-42.
65 Auerkari E. Methylation of tumor suppressor genes pl6(INK4a), p27(Kipl) and E-cadherin in carcinogenesis. Oral Oncol.2006;42:5-13.
66 Filion G, Defossez P. Epigenetics and cancer. Bull Cancer 2006;93:343-7.
67 Laird P. Cancer epigenetics. Hum Mol Genet.2005;14 Spec No 1:R65-76.
68 Kang S, Kim J, Kang G, et al. Polymorphism in folate-and methionine-metabolizing enzyme and aberrant CpG island hypermethylation in uterine cervical cancer. Gynecol Oncol. 2005;96:173-80.
69 Siraj A, Ibrahim M, Al-Rasheed M, et al. Genetic polymorphisms of methylenetetrahydrofolate reductase and promoter methylation of MGMT and FHIT genes in diffuse large B cell lymphoma risk in Middle East. Ann Hematol.2007;86:887-95.
70 Wang J, Sasco A, Fu C, et al. Aberrant DNA methylation of P16, MGMT, and hMLHl genes in combination with MTHFR C677T genetic polymorphism in esophageal squamous cell carcinoma. Cancer Epidemiol Biomarkers Prev.2008; 17:118-25.
71 Holliday R. The inheritance of epigenetic defects. Science.1987;238:163-70.
72 Glickman J, Flynn J, Reich N. Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase. Biochem Biophys Res Commun. 1997;230:280-4.
73 Agathanggelou A, Honorio S, Macartney D, et al. Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene. 2001;20:1509-18.
74 Loginov V, Maliukova A, Seregin I, et al. [Methylation of the promoter region of the RASSF1A gene, a candidate tumor suppressor, in primary epithelial tumors]. Mol Biol (Mosk). 2004;38:654-67.
75 张家浩,姜昊文,顾静文等.RASSF1A基因在前列腺癌中甲基化检测及表达.Shanghai Med J.2005,5:366-369.
76 Oliveira C, Velho S, Domingo E, et al. Concomitant RASSF1A hypermethylation and KRAS/BRAF mutations occur preferentially in MSI sporadic colorectal cancer. Oncogene. 2005;24:7630-4.
77 Guo W, Dong Z, Chen Z, et al. Aberrant CpG island hypermethylation of RASSF1A in gastric cardia adenocarcinoma. Cancer Invest.2009;27:459-65.
78 Yanagawa N, Tamura G, Oizumi H, et al. Promoter hypermethylation of RASSF1A and RUNX3 genes as an independent prognostic prediction marker in surgically resected non-small cell lung cancers. Lung Cancer.2007;58:131-8.
79 Bagadi S, Prasad C, Kaur J, et al. Clinical significance of promoter hypermethylation of RASSF1A, RARbeta2, BRCA1 and HOXA5 in breast cancers of Indian patients. Life Sci. 2008;82:1288-92.
80 Yeo W, Wong N, Wong W, et al. High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma. Liver Int.2005;25:266-72.
81 Kee S, Lee J, Kim M, et al. Hypermethylation of the Ras association domain family 1A (RASSF1A) gene in gallbladder cancer. Mol Cells.2007;24:364-71.
82 Tokinaga K, Okuda H, Nomura A, et al. Hypermethylation of the RASSF1A tumor suppressor gene in Japanese clear cell renal cell carcinoma. Oncol Rep.2004;12:805-10.
83 Pallares J, Velasco A, Eritja N, et al. Promoter hypermethylation and reduced expression of RASSF1A are frequent molecular alterations of endometrial carcinoma. Mod Pathol. 2008;21:691-9.
84 Choi Y, Kang S, Shin Y, et al. Aberrant hypermethylation of RASSF1A promoter in ovarian borderline tumors and carcinomas. Virchows Arch.2006;448:331-6.
85 Graziano F, Kawakami K, Ruzzo A, et al. Methylenetetrahydrofolate reductase 677C/T gene polymorphism, gastric cancer susceptibility and genomic DNA hypomethylation in an at-risk Italian population. Int J Cancer.2006;118:628-32.
1 Parkin D, Bray F, Ferlay J, Pisani P. Global cancer statistics,2002. CA Cancer J Clin;55:74-108.
2 Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008. CA Cancer J Clin;58:71-96.
3 Fortuny J, Johnson C, Bohlke K, et al. Use of anti-inflammatory drugs and lower esophageal sphincter-relaxing drugs and risk of esophageal and gastric cancers. Clin Gastroenterol Hepatol 2007;5:1154-1159.e3.
4 Brockmoller J, Cascorbi I, Kerb R, Roots I. Combined analysis of inherited polymorphisms in arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1, microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk. Cancer Res 1996;56:3915-25.
5 Moore LE, Wiencke JK, Bates MN, Zheng SC, Rey OA, Smith AH. Investigation of genetic polymorphisms and smoking in a bladder cancer case-control study in Argentina. Cancer Letters 2004;211:199-207.
6 Wang M, Zhu H, Fu G, Zhang Z, Lu Q, Wang S. Polymorphisms of methylenetetrahydrofolate reductase and methionine synthase genes and bladder cancer risk:a case-control study with meta-analysis. Clin Exp Med 2009;9:9-19.
7 郝玲,孔志新.我国部分地区成年人血浆叶酸的地区和季节差异比较.中华预防医学杂志.2002,36：308-310.
8 Choi S, Mason J. Folate and carcinogenesis:an integrated scheme. J Nutr 2000; 130:129-32.
9 Mayne S, Risch H, Dubrow R, et al. Nutrient intake and risk of subtypes of esophageal and gastric cancer. Cancer Epidemiol Biomarkers Prev 2001;10:1055-62.
10 Cravo M, Mason J, Dayal Y, et al. Folate deficiency enhances the development of colonic neoplasia in dimethylhydrazine-treated rats. Cancer Res 1992;52:5002-6.
11 Baggott J, Vaughn W, Juliana M, Eto I, Krumdieck C, Grubbs C. Effects of folate deficiency and supplementation on methylnitrosourea-induced rat mammary tumors. J Natl Cancer Inst 1992;84:1740-4.
12 Shrubsole M, Jin F, Dai Q, et al. Dietary folate intake and breast cancer risk:results from the Shanghai Breast Cancer Study. Cancer Res 2001;61:7136-41.
13 Ames B. Micronutrient deficiencies. A major cause of DNA damage. Ann N Y Acad Sci 1999;889:87-106.
14 Pelucchi C, Talamini R, Negri E, et al. Folate intake and risk of oral and pharyngeal cancer. Ann Oncol 2003; 14:1677-81.
15 Tworoger S, Hecht J, Giovannucci E, Hankinson S. Intake of folate and related nutrients in relation to risk of epithelial ovarian cancer. Am J Epidemiol 2006; 163:1101-11.
16 Butterworth CJ, Hatch K, Gore H, Mueller H, Krumdieck C. Improvement in cervical dysplasia associated with folic acid therapy in users of oral contraceptives. Am J Clin Nutr 1982;35:73-82.
17 Keyes M, Jang H, Mason J, et al. Older age and dietary folate are determinants of genomic and p16-specific DNA methylation in mouse colon. J Nutr 2007;137:1713-7.
18 Kraunz K, Hsiung D, McClean M, et al. Dietary folate is associated with p16(INK4A) methylation in head and neck squamous cell carcinoma. Int J Cancer 2006; 119:1553-7.
19 Garcia-Closas R, Garcia-Closas M, Kogevinas M, et al. Food, nutrient and heterocyclic amine intake and the risk of bladder cancer. Eur J Cancer 2007;43:1731-40.
20 Suzuki T, Matsuo K, Hiraki A, et al. Impact of one-carbon metabolism-related gene polymorphisms on risk of lung cancer in Japan:a case control study. Carcinogenesis 2007;28:1718-25.
21 Frosst P, Blom H, Milos R, et al. A candidate genetic risk factor for vascular disease:a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10:111-3.
22 Molloy A, Daly S, Mills J, et al. Thermolabile variant of 5,10-methylenetetrahydrofolate reductase associated with low red-cell folates:implications for folate intake recommendations. Lancet 1997;349:1591-3.
23 Ulvik A, Ueland P, Fredriksen A, et al. Functional inference of the methylenetetrahydrofolate reductase 677C> T and 1298A> C polymorphisms from a large-scale epidemiological study. Hum Genet 2007; 121:57-64.
24 Stern L, Mason J, Selhub J, Choi S. Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiol Biomarkers Prev 2000;9:849-53.
25 Bagley P, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc Natl Acad Sci U S A 1998;95:13217-20.
26 Sohn K, Croxford R, Yates Z, Lucock M, Kim Y. Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate. J Natl Cancer Inst 2004;96:134-44.
27 van der Put N, Gabreels F, Stevens E, et al. A second common mutation in the methylenetetrahydrofolate reductase gene:an additional risk factor for neural-tube defects? Am J Hum Genet 1998;62:1044-51.
28 Stegmann K, Ziegler A, Ngo E, et al. Linkage disequilibrium of MTHFR genotypes 677C/T-1298A/C in the German population and association studies in probands with neural tube defects(NTD). Am J Med Genet 1999;87:23-9.
29 Chen J, Ma J, Stampfer M, Palomeque C, Selhub J, Hunter D. Linkage disequilibrium between the 677C>T and 1298A>C polymorphisms in human methylenetetrahydrofolate reductase gene and their contributions to risk of colorectal cancer. Pharmacogenetics 2002;12:339-42.
30 Ouerhani S, Oliveira E, Marrakchi R, et al. Methylenetetrahydrofolate reductase and methionine synthase polymorphisms and risk of bladder cancer in a Tunisian population. Cancer Genetics and Cytogenetics 2007;176:48-53.
31 Harmon D, Shields D, Woodside J, et al. Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations. Genet Epidemiol 1999;17:298-309.
32 Chen J, Giovannucci E, Hankinson S, et al. A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma. Carcinogenesis 1998; 19:2129-32.
33 Leclerc D, Campeau E, Goyette P, et al. Human methionine synthase:cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum Mol Genet 1996;5:1867-74.
34 Chen J, Stampfer M, Ma J, et al. Influence of a methionine synthase (D919G) polymorphism on plasma homocysteine and folate levels and relation to risk of myocardial infarction. Atherosclerosis 2001; 154:667-72.
35 Tsai M, Bignell M, Yang F, Welge B, Graham K, Hanson N. Polygenic influence on plasma homocysteine:association of two prevalent mutations, the 844ins68 of cystathionine beta-synthase and A(2756)G of methionine synthase, with lowered plasma homocysteine levels. Atherosclerosis 2000;149:131-7.
36 Wilson A, Platt R, Wu Q, et al. A common variant in methionine synthase reductase combined with low cobalamin (vitamin B12) increases risk for spina bifida. Mol Genet Metab 1999;67:317-23.
37 Gaughan D, Kluijtmans L, Barbaux S, et al. The methionine synthase reductase (MTRR) A66G polymorphism is a novel genetic determinant of plasma homocysteine concentrations. Atherosclerosis 2001;157:451-6.
38 Leclerc D, Wilson A, Dumas R, et al. Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc Natl Acad Sci U S A 1998;95:3059-64.
39 Volcik K, Shaw G, Zhu H, Lammer E, Laurent C, Finnell R. Associations between polymorphisms within the thymidylate synthase gene and spina bifida. Birth Defects Res A Clin Mol Teratol 2003;67:924-8.
40 Liu J, Schmitz J, Lin X, et al. Thymidylate synthase as a translational regulator of cellular gene expression. Biochim Biophys Acta 2002;1587:174-82.
41 Hori T, Ayusawa D, Glover T, Seno T. Expression of fragile site on the human X chromosome in somatic cell hybrids between human fragile X cells and thymidylate synthase-negative mouse mutant cells. Jpn J Cancer Res 1985;76:977-83.
42 Horie N, Aiba H, Oguro K, Hojo H, Takeishi K. Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase. Cell Struct Funct 1995;20:191-7.
43 Pullarkat S, Stoehlmacher J, Ghaderi V, et al. Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J 2001; 1:65-70.
44 Glasgow S, Yu J, Carvalho L, Shannon W, Fleshman J, McLeod H. Unfavourable expression of pharmacologic markers in mucinous colorectal cancer. Br J Cancer 2005;92:259-64.
45 Iacopetta B, Grieu F, Joseph D, Elsaleh H. A polymorphism in the enhancer region of the thymidylate synthase promoter influences the survival of colorectal cancer patients treated with 5-fluorouracil. Br J Cancer 2001;85:827-30.
46 Ulrich C, Bigler J, Velicer C, Greene E, Farin F, Potter J. Searching expressed sequence tag databases:discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomarkers Prev 2000;9:1381-5.
47 Mandola M, Stoehlmacher J, Zhang W, et al. A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics 2004;14:319-27.
48 Heijmans B, Boer J, Suchiman H, et al. A common variant of the methylenetetrahydrofolate reductase gene (lp36) is associated with an increased risk of cancer. Cancer Res 2003;63:1249-53.
49 Kimura F, Florl A, Steinhoff C, et al. Polymorphic methyl group metabolism genes in patients with transitional cell carcinoma of the urinary bladder. Mutat Res 2001;458:49-54.
50 Karagas MR, Park S, Nelson HH, et al. Methylenetetrahydrofolate reductase (MTHFR) variants and bladder cancer:A population-based case-control study. International Journal of Hygiene and Environmental Health 2005;208:321-327.
51 Moore LE, Malats N, Rothmans N, et al. Polymorphisms in one-carbon metabolism and trans-sulfuration pathway genes and susceptibility to bladder cancer. International Journal of Cancer 2007; 120:2452-2458.
52 Lin J, Spitz MR, Wang Y, et al. Polymorphisms of folate metabolic genes and susceptibility to bladder cancer:A case-control study. Proceedings of the American Association for Cancer Research Annual Meeting 2004;45:918-919.
53 Rouissi K, Ouerhani S, Oliveira E, et al. Polymorphisms in one-carbon metabolism pathway genes and risk for bladder cancer in a Tunisian population. Cancer Genet Cytogenet 2009; 195:43-53.
54 Ouerhani S, Rouissi K, Marrakchi R, et al. Combined effect of NAT2, MTR and MTHFR genotypes and tobacco on bladder cancer susceptibility in Tunisian population. Cancer Detect Prev 2009;32:395-402.
2 Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008. CA Cancer J Clin.2008;58:71-96.
3 Fortuny J, Johnson C, Bohlke K, et al. Use of anti-inflammatory drugs and lower esophageal sphincter-relaxing drugs and risk of esophageal and gastric cancers. Clin Gastroenterol Hepatol.2007;5:1154-1159.e3.
4 Brockmoller J, Cascorbi I, Kerb R, et al. Combined analysis of inherited polymorphisms in arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1, microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk. Cancer Res. 1996;56:3915-25.
5 Moore LE, Wiencke JK, Bates MN, et al. Investigation of genetic polymorphisms and smoking in a bladder cancer case-control study in Argentina. Cancer Letters.2004;211:199-207.
6 Wang M, Zhu H, Fu G, et al. Polymorphisms of methylenetetrahydrofolate reductase and methionine synthase genes and bladder cancer risk:a case-control study with meta-analysis. Clin Exp Med.2009;9:9-19.
7 Duthie S. Folic acid deficiency and cancer:mechanisms of DNA instability. Br Med Bull. 1999;55:578-92.
8 Choi S, Mason J. Folate and carcinogenesis:an integrated scheme. J Nutr.2000;130:129-32.
9 Wei Q, Shen H, Wang L, et al. Association between low dietary folate intake and suboptimal cellular DNA repair capacity. Cancer Epidemiol Biomarkers Prev.2003;12:963-9.
10 Larsson S, Giovannucci E, Wolk A. Folate intake, MTHFR polymorphisms, and risk of esophageal, gastric, and pancreatic cancer:a meta-analysis. Gastroenterology. 2006;131:1271-83.
11 Cravo M, Pinto A, Chaves P, et al. Effect of folate supplementation on DNA methylation of rectal mucosa in patients with colonic adenomas:correlation with nutrient intake. Clin Nutr. 1998;17:45-9.
12 Blount B, Mack M, Wehr C, et al. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage:implications for cancer and neuronal damage. Proc Natl Acad Sci U S A.1997;94:3290-5.
13 Herman J, Umar A, Polyak K, et al. Incidence and functional consequences of hMLHl promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A 1998;95:6870-5.
14 Kim Y. Folate and carcinogenesis:evidence, mechanisms, and implications. J Nutr Biochem. 1999; 10:66-88.
15 Suzuki T, Matsuo K, Hiraki A, et al. Impact of one-carbon metabolism-related gene polymorphisms on risk of lung cancer in Japan:a case control study. Carcinogenesis. 2007;28:1718-25.
16 Frosst P, Blom H, Milos R, et al. A candidate genetic risk factor for vascular disease:a common mutation in methylenetetrahydrofolate reductase. Nat Genet.1995;10:111-3.
17 Molloy A, Daly S, Mills J, et al. Thermolabile variant of 5,10-methylenetetrahydrofolate reductase associated with low red-cell folates:implications for folate intake recommendations. Lancet.1997;349:1591-3.
18 Ulvik A, Ueland P, Fredriksen A, et al. Functional inference of the methylenetetrahydrofolate reductase 677C> T and 1298A> C polymorphisms from a large-scale epidemiological study. Hum Genet.2007;121:57-64.
19 Stern L, Mason J, Selhub J, et al. Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiol Biomarkers Prev.2000;9:849-53.
20 Bagley P, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc Natl Acad Sci U S A.1998;95:13217-20.
21 Sohn K, Croxford R, Yates Z, et al. Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate. J Natl Cancer Inst.2004;96:134-44.
22 van der Put N, Gabreels F, Stevens E, et al. A second common mutation in the . methylenetetrahydrofolate reductase gene:an additional risk factor for neural-tube defects? Am J Hum Genet.1998;62:1044-51.
23 Stegmann K, Ziegler A, Ngo E, et al. Linkage disequilibrium of MTHFR genotypes 677C/T-1298A/C in the German population and association studies in probands with neural tube defects(NTD). Am J Med Genet.1999;87:23-9.
24 Chen J, Ma J, Stampfer M, et al. Linkage disequilibrium between the 677C>T and 1298A>C polymorphisms in human methylenetetrahydrofolate reductase gene and their contributions to risk of colorectal cancer. Pharmacogenetics.2002;12:339-42.
25 Ouerhani S, Oliveira E, Marrakchi R, et al. Methylenetetrahydrofolate reductase and methionine synthase polymorphisms and risk of bladder cancer in a Tunisian population. Cancer Genetics and Cytogenetics.2007;176:48-53.
26 Harmon D, Shields D, Woodside J, et al. Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations. Genet Epidemiol.1999;17:298-309.
27 Chen J, Giovannucci E, Hankinson S, et al. A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma. Carcinogenesis.1998;19:2129-32.
28 Leclerc D, Campeau E, Goyette P, et al. Human methionine synthase:cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum Mol Genet.1996;5:1867-74.
29 Chen J, Stampfer M, Ma J, et al. Influence of a methionine synthase (D919G) polymorphism on plasma homocysteine and folate levels and relation to risk of myocardial infarction. Atherosclerosis.2001;154:667-72.
30 Tsai M, Bignell M, Yang F, et al. Polygenic influence on plasma homocysteine:association of two prevalent mutations, the 844ins68 of cystathionine beta-synthase and A(2756)G of methionine synthase, with lowered plasma homocysteine levels. Atherosclerosis. 2000;149:131-7.
31 Lin J, Spitz MR, Wang Y, et al. Polymorphisms of folate metabolic genes and susceptibility to bladder cancer:A case-control study. Proceedings of the American Association for Cancer Research Annual Meeting.2004;45:918-919.
32 Shi Y, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res.2005;15:97-8.
33 Platek M, Shields P, Marian C, et al. Alcohol consumption and genetic variation in methylenetetrahydrofolate reductase and 5-methyltetrahydrofolate-homocysteine methyltransferase in relation to breast cancer risk. Cancer Epidemiol Biomarkers Prev. 2009; 18:2453-9.
34 Ma E, Iwasaki M, Kobayashi M, et al. Dietary intake of folate, vitamin B2, vitamin B6, vitamin B12, genetic polymorphism of related enzymes, and risk of breast cancer:a case-control study in Japan. Nutr Cancer.2009;61:447-56.
35 Zhang F, Terry M, Hou L, et al. Genetic polymorphisms in folate metabolism and the risk of stomach cancer. Cancer Epidemiol Biomarkers Prev.2007;16:115-21.
36 Yang C, Matsuo K, Ito H, et al. Gene-environment interactions between alcohol drinking and the MTHFR C677T polymorphism impact on esophageal cancer risk:results of a case-control study in Japan. Carcinogenesis.2005;26:1285-90.
37 Suzuki T, Matsuo K, Sawaki A, et al. Alcohol drinking and one-carbon metabolism-related gene polymorphisms on pancreatic cancer risk. Cancer Epidemiol Biomarkers Prev. 2008; 17:2742-7.
38 Steck S, Keku T, Butler L, et al. Polymorphisms in methionine synthase, methionine synthase reductase and serine hydroxymethyltransferase, folate and alcohol intake, and colon cancer risk. J Nutrigenet Nutrigenomics.2008; 1:196-204.
39 Chen K, Song L, Jin M, et al. Association between genetic polymorphisms in folate metabolic enzyme genes and colorectal cancer:a nested case-control study. Zhonghua Zhong Liu Za Zhi. 2006;28:429-32.
40 Matsuo K, Ito H, Wakai K, et al. One-carbon metabolism related gene polymorphisms interact with alcohol drinking to influence the risk of colorectal cancer in Japan. Carcinogenesis. 2005;26:2164-71.
41 Tong S, Lee J, Song E, et al. The effects of polymorphisms in methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR) on the risk of cervical intraepithelial neoplasia and cervical cancer in Korean women. Cancer Causes Control.2010;21:23-30.
42 Marchal C, Redondo M, Reyes-Engel A, et al. Association between polymorphisms of folate-metabolizing enzymes and risk of prostate cancer. Eur J Surg Oncol.2008;34:805-10.
43 Lee K, Lan Q, Kricker A, et al. One-carbon metabolism gene polymorphisms and risk of non-Hodgkin lymphoma in Australia. Hum Genet.2007;122:525-33.
44 Matsuo K, Hamajima N, Suzuki R, et al. Methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms and reduced risk of malignant lymphoma. Am J Hematol.2004;77:351-7.
45 Heijmans B, Boer J, Suchiman H, et al. A common variant of the methylenetetrahydrofolate reductase gene (lp36) is associated with an increased risk of cancer. Cancer Res. 2003;63:1249-53.
46 Kimura F, Florl A, Steinhoff C, et al. Polymorphic methyl group metabolism genes in patients with transitional cell carcinoma of the urinary bladder. Mutat Res.2001;458:49-54.
47 Karagas MR, Park S, Nelson HH, et al. Methylenetetrahydrofolate reductase (MTHFR) variants and bladder cancer:A population-based case-control study. International Journal of Hygiene and Environmental Health.2005;208:321-327.
48 Moore LE, Malats N, Rothmans N, et al. Polymorphisms in one-carbon metabolism and trans-sulfuration pathway genes and susceptibility to bladder cancer. International Journal of Cancer.2007;120:2452-2458.
49 Rouissi K, Ouerhani S, Oliveira E, et al. Polymorphisms in one-carbon metabolism pathway genes and risk for bladder cancer in a Tunisian population. Cancer Genet Cytogenet. 2009; 195:43-53.
50 Ouerhani S, Rouissi K, Marrakchi R, et al. Combined effect of NAT2, MTR and MTHFR genotypes and tobacco on bladder cancer susceptibility in Tunisian population. Cancer Detect Prev.2009;32:395-402.
51 Matsuo K, Suzuki R, Hamajima N, et al. Association between polymorphisms of folate-and methionine-metabolizing enzymes and susceptibility to malignant lymphoma. Blood. 2001;97:3205-9.
52 Chen J, Giovannucci E, Kelsey K, et al. A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer. Cancer Res.1996;56:4862-4.
53 Ma J, Stampfer M, Giovannucci E, et al. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res.1997;57:1098-102.
54 Ulrich C, Kampman E, Bigler J, et al. Colorectal adenomas and the C677T MTHFR polymorphism:evidence for gene-environment interaction? Cancer Epidemiol Biomarkers Prev.1999;8:659-68.
55 Song C, Xing D, Tan W, et al. Methylenetetrahydrofolate reductase polymorphisms increase risk of esophageal squamous cell carcinoma in a Chinese population. Cancer Res. 2001;61:3272-5.
56 Levine A, Siegmund K, Ervin C, et al. The methylenetetrahydrofolate reductase 677C->T polymorphism and distal colorectal adenoma risk. Cancer Epidemiol Biomarkers Prev. 2000;9:657-63.
57 Giovannucci E, Chen J, Smith-Warner S, et al. Methylenetetrahydrofolate reductase, alcohol dehydrogenase, diet, and risk of colorectal adenomas. Cancer Epidemiol Biomarkers Prev. 2003;12:970-9.
58 Piyathilake C, Macaluso M, Hine R, et al. Local and systemic effects of cigarette smoking on folate and vitamin B-12. Am J Clin Nutr.1994;60:559-66.
59 Laird P, Jaenisch R. DNA methylation and cancer. Hum Mol Genet.1994;3 Spec No:1487-95.
60 Dammann R, Li C, Yoon J, et al. Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet.2000;25:315-9.
61 Catto J, Azzouzi A, Rehman I, et al. Promoter hypermethylation is associated with tumor location, stage, and subsequent progression in transitional cell carcinoma. J Clin Oncol. 2005;23:2903-10.
62 Lin H, Ke H, Huang S, et al. Increase sensitivity in detecting superficial, low grade bladder cancer by combination analysis of hypermethylation of E-cadherin, p16, p14, RASSF1A genes in urine. Urol Oncol.2009.
63 Pfeifer G, Dammann R. Methylation of the tumor suppressor gene RASSF1A in human tumors. Biochemistry (Mosc).2005;70:576-83.
64 Das P, Singal R. DNA methylation and cancer. J Clin Oncol.2004;22:4632-42.
65 Auerkari E. Methylation of tumor suppressor genes pl6(INK4a), p27(Kipl) and E-cadherin in carcinogenesis. Oral Oncol.2006;42:5-13.
66 Filion G, Defossez P. Epigenetics and cancer. Bull Cancer 2006;93:343-7.
67 Laird P. Cancer epigenetics. Hum Mol Genet.2005;14 Spec No 1:R65-76.
68 Kang S, Kim J, Kang G, et al. Polymorphism in folate-and methionine-metabolizing enzyme and aberrant CpG island hypermethylation in uterine cervical cancer. Gynecol Oncol. 2005;96:173-80.
69 Siraj A, Ibrahim M, Al-Rasheed M, et al. Genetic polymorphisms of methylenetetrahydrofolate reductase and promoter methylation of MGMT and FHIT genes in diffuse large B cell lymphoma risk in Middle East. Ann Hematol.2007;86:887-95.
70 Wang J, Sasco A, Fu C, et al. Aberrant DNA methylation of P16, MGMT, and hMLHl genes in combination with MTHFR C677T genetic polymorphism in esophageal squamous cell carcinoma. Cancer Epidemiol Biomarkers Prev.2008; 17:118-25.
71 Holliday R. The inheritance of epigenetic defects. Science.1987;238:163-70.
72 Glickman J, Flynn J, Reich N. Purification and characterization of recombinant baculovirus-expressed mouse DNA methyltransferase. Biochem Biophys Res Commun. 1997;230:280-4.
73 Agathanggelou A, Honorio S, Macartney D, et al. Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene. 2001;20:1509-18.
74 Loginov V, Maliukova A, Seregin I, et al. [Methylation of the promoter region of the RASSF1A gene, a candidate tumor suppressor, in primary epithelial tumors]. Mol Biol (Mosk). 2004;38:654-67.
75 张家浩,姜昊文,顾静文等.RASSF1A基因在前列腺癌中甲基化检测及表达.Shanghai Med J.2005,5:366-369.
76 Oliveira C, Velho S, Domingo E, et al. Concomitant RASSF1A hypermethylation and KRAS/BRAF mutations occur preferentially in MSI sporadic colorectal cancer. Oncogene. 2005;24:7630-4.
77 Guo W, Dong Z, Chen Z, et al. Aberrant CpG island hypermethylation of RASSF1A in gastric cardia adenocarcinoma. Cancer Invest.2009;27:459-65.
78 Yanagawa N, Tamura G, Oizumi H, et al. Promoter hypermethylation of RASSF1A and RUNX3 genes as an independent prognostic prediction marker in surgically resected non-small cell lung cancers. Lung Cancer.2007;58:131-8.
79 Bagadi S, Prasad C, Kaur J, et al. Clinical significance of promoter hypermethylation of RASSF1A, RARbeta2, BRCA1 and HOXA5 in breast cancers of Indian patients. Life Sci. 2008;82:1288-92.
80 Yeo W, Wong N, Wong W, et al. High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma. Liver Int.2005;25:266-72.
81 Kee S, Lee J, Kim M, et al. Hypermethylation of the Ras association domain family 1A (RASSF1A) gene in gallbladder cancer. Mol Cells.2007;24:364-71.
82 Tokinaga K, Okuda H, Nomura A, et al. Hypermethylation of the RASSF1A tumor suppressor gene in Japanese clear cell renal cell carcinoma. Oncol Rep.2004;12:805-10.
83 Pallares J, Velasco A, Eritja N, et al. Promoter hypermethylation and reduced expression of RASSF1A are frequent molecular alterations of endometrial carcinoma. Mod Pathol. 2008;21:691-9.
84 Choi Y, Kang S, Shin Y, et al. Aberrant hypermethylation of RASSF1A promoter in ovarian borderline tumors and carcinomas. Virchows Arch.2006;448:331-6.
85 Graziano F, Kawakami K, Ruzzo A, et al. Methylenetetrahydrofolate reductase 677C/T gene polymorphism, gastric cancer susceptibility and genomic DNA hypomethylation in an at-risk Italian population. Int J Cancer.2006;118:628-32.
1 Parkin D, Bray F, Ferlay J, Pisani P. Global cancer statistics,2002. CA Cancer J Clin;55:74-108.
2 Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008. CA Cancer J Clin;58:71-96.
3 Fortuny J, Johnson C, Bohlke K, et al. Use of anti-inflammatory drugs and lower esophageal sphincter-relaxing drugs and risk of esophageal and gastric cancers. Clin Gastroenterol Hepatol 2007;5:1154-1159.e3.
4 Brockmoller J, Cascorbi I, Kerb R, Roots I. Combined analysis of inherited polymorphisms in arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1, microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk. Cancer Res 1996;56:3915-25.
5 Moore LE, Wiencke JK, Bates MN, Zheng SC, Rey OA, Smith AH. Investigation of genetic polymorphisms and smoking in a bladder cancer case-control study in Argentina. Cancer Letters 2004;211:199-207.
6 Wang M, Zhu H, Fu G, Zhang Z, Lu Q, Wang S. Polymorphisms of methylenetetrahydrofolate reductase and methionine synthase genes and bladder cancer risk:a case-control study with meta-analysis. Clin Exp Med 2009;9:9-19.
7 郝玲,孔志新.我国部分地区成年人血浆叶酸的地区和季节差异比较.中华预防医学杂志.2002,36：308-310.
8 Choi S, Mason J. Folate and carcinogenesis:an integrated scheme. J Nutr 2000; 130:129-32.
9 Mayne S, Risch H, Dubrow R, et al. Nutrient intake and risk of subtypes of esophageal and gastric cancer. Cancer Epidemiol Biomarkers Prev 2001;10:1055-62.
10 Cravo M, Mason J, Dayal Y, et al. Folate deficiency enhances the development of colonic neoplasia in dimethylhydrazine-treated rats. Cancer Res 1992;52:5002-6.
11 Baggott J, Vaughn W, Juliana M, Eto I, Krumdieck C, Grubbs C. Effects of folate deficiency and supplementation on methylnitrosourea-induced rat mammary tumors. J Natl Cancer Inst 1992;84:1740-4.
12 Shrubsole M, Jin F, Dai Q, et al. Dietary folate intake and breast cancer risk:results from the Shanghai Breast Cancer Study. Cancer Res 2001;61:7136-41.
13 Ames B. Micronutrient deficiencies. A major cause of DNA damage. Ann N Y Acad Sci 1999;889:87-106.
14 Pelucchi C, Talamini R, Negri E, et al. Folate intake and risk of oral and pharyngeal cancer. Ann Oncol 2003; 14:1677-81.
15 Tworoger S, Hecht J, Giovannucci E, Hankinson S. Intake of folate and related nutrients in relation to risk of epithelial ovarian cancer. Am J Epidemiol 2006; 163:1101-11.
16 Butterworth CJ, Hatch K, Gore H, Mueller H, Krumdieck C. Improvement in cervical dysplasia associated with folic acid therapy in users of oral contraceptives. Am J Clin Nutr 1982;35:73-82.
17 Keyes M, Jang H, Mason J, et al. Older age and dietary folate are determinants of genomic and p16-specific DNA methylation in mouse colon. J Nutr 2007;137:1713-7.
18 Kraunz K, Hsiung D, McClean M, et al. Dietary folate is associated with p16(INK4A) methylation in head and neck squamous cell carcinoma. Int J Cancer 2006; 119:1553-7.
19 Garcia-Closas R, Garcia-Closas M, Kogevinas M, et al. Food, nutrient and heterocyclic amine intake and the risk of bladder cancer. Eur J Cancer 2007;43:1731-40.
20 Suzuki T, Matsuo K, Hiraki A, et al. Impact of one-carbon metabolism-related gene polymorphisms on risk of lung cancer in Japan:a case control study. Carcinogenesis 2007;28:1718-25.
21 Frosst P, Blom H, Milos R, et al. A candidate genetic risk factor for vascular disease:a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10:111-3.
22 Molloy A, Daly S, Mills J, et al. Thermolabile variant of 5,10-methylenetetrahydrofolate reductase associated with low red-cell folates:implications for folate intake recommendations. Lancet 1997;349:1591-3.
23 Ulvik A, Ueland P, Fredriksen A, et al. Functional inference of the methylenetetrahydrofolate reductase 677C> T and 1298A> C polymorphisms from a large-scale epidemiological study. Hum Genet 2007; 121:57-64.
24 Stern L, Mason J, Selhub J, Choi S. Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiol Biomarkers Prev 2000;9:849-53.
25 Bagley P, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc Natl Acad Sci U S A 1998;95:13217-20.
26 Sohn K, Croxford R, Yates Z, Lucock M, Kim Y. Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate. J Natl Cancer Inst 2004;96:134-44.
27 van der Put N, Gabreels F, Stevens E, et al. A second common mutation in the methylenetetrahydrofolate reductase gene:an additional risk factor for neural-tube defects? Am J Hum Genet 1998;62:1044-51.
28 Stegmann K, Ziegler A, Ngo E, et al. Linkage disequilibrium of MTHFR genotypes 677C/T-1298A/C in the German population and association studies in probands with neural tube defects(NTD). Am J Med Genet 1999;87:23-9.
29 Chen J, Ma J, Stampfer M, Palomeque C, Selhub J, Hunter D. Linkage disequilibrium between the 677C>T and 1298A>C polymorphisms in human methylenetetrahydrofolate reductase gene and their contributions to risk of colorectal cancer. Pharmacogenetics 2002;12:339-42.
30 Ouerhani S, Oliveira E, Marrakchi R, et al. Methylenetetrahydrofolate reductase and methionine synthase polymorphisms and risk of bladder cancer in a Tunisian population. Cancer Genetics and Cytogenetics 2007;176:48-53.
31 Harmon D, Shields D, Woodside J, et al. Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations. Genet Epidemiol 1999;17:298-309.
32 Chen J, Giovannucci E, Hankinson S, et al. A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma. Carcinogenesis 1998; 19:2129-32.
33 Leclerc D, Campeau E, Goyette P, et al. Human methionine synthase:cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum Mol Genet 1996;5:1867-74.
34 Chen J, Stampfer M, Ma J, et al. Influence of a methionine synthase (D919G) polymorphism on plasma homocysteine and folate levels and relation to risk of myocardial infarction. Atherosclerosis 2001; 154:667-72.
35 Tsai M, Bignell M, Yang F, Welge B, Graham K, Hanson N. Polygenic influence on plasma homocysteine:association of two prevalent mutations, the 844ins68 of cystathionine beta-synthase and A(2756)G of methionine synthase, with lowered plasma homocysteine levels. Atherosclerosis 2000;149:131-7.
36 Wilson A, Platt R, Wu Q, et al. A common variant in methionine synthase reductase combined with low cobalamin (vitamin B12) increases risk for spina bifida. Mol Genet Metab 1999;67:317-23.
37 Gaughan D, Kluijtmans L, Barbaux S, et al. The methionine synthase reductase (MTRR) A66G polymorphism is a novel genetic determinant of plasma homocysteine concentrations. Atherosclerosis 2001;157:451-6.
38 Leclerc D, Wilson A, Dumas R, et al. Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc Natl Acad Sci U S A 1998;95:3059-64.
39 Volcik K, Shaw G, Zhu H, Lammer E, Laurent C, Finnell R. Associations between polymorphisms within the thymidylate synthase gene and spina bifida. Birth Defects Res A Clin Mol Teratol 2003;67:924-8.
40 Liu J, Schmitz J, Lin X, et al. Thymidylate synthase as a translational regulator of cellular gene expression. Biochim Biophys Acta 2002;1587:174-82.
41 Hori T, Ayusawa D, Glover T, Seno T. Expression of fragile site on the human X chromosome in somatic cell hybrids between human fragile X cells and thymidylate synthase-negative mouse mutant cells. Jpn J Cancer Res 1985;76:977-83.
42 Horie N, Aiba H, Oguro K, Hojo H, Takeishi K. Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase. Cell Struct Funct 1995;20:191-7.
43 Pullarkat S, Stoehlmacher J, Ghaderi V, et al. Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J 2001; 1:65-70.
44 Glasgow S, Yu J, Carvalho L, Shannon W, Fleshman J, McLeod H. Unfavourable expression of pharmacologic markers in mucinous colorectal cancer. Br J Cancer 2005;92:259-64.
45 Iacopetta B, Grieu F, Joseph D, Elsaleh H. A polymorphism in the enhancer region of the thymidylate synthase promoter influences the survival of colorectal cancer patients treated with 5-fluorouracil. Br J Cancer 2001;85:827-30.
46 Ulrich C, Bigler J, Velicer C, Greene E, Farin F, Potter J. Searching expressed sequence tag databases:discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomarkers Prev 2000;9:1381-5.
47 Mandola M, Stoehlmacher J, Zhang W, et al. A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics 2004;14:319-27.
48 Heijmans B, Boer J, Suchiman H, et al. A common variant of the methylenetetrahydrofolate reductase gene (lp36) is associated with an increased risk of cancer. Cancer Res 2003;63:1249-53.
49 Kimura F, Florl A, Steinhoff C, et al. Polymorphic methyl group metabolism genes in patients with transitional cell carcinoma of the urinary bladder. Mutat Res 2001;458:49-54.
50 Karagas MR, Park S, Nelson HH, et al. Methylenetetrahydrofolate reductase (MTHFR) variants and bladder cancer:A population-based case-control study. International Journal of Hygiene and Environmental Health 2005;208:321-327.
51 Moore LE, Malats N, Rothmans N, et al. Polymorphisms in one-carbon metabolism and trans-sulfuration pathway genes and susceptibility to bladder cancer. International Journal of Cancer 2007; 120:2452-2458.
52 Lin J, Spitz MR, Wang Y, et al. Polymorphisms of folate metabolic genes and susceptibility to bladder cancer:A case-control study. Proceedings of the American Association for Cancer Research Annual Meeting 2004;45:918-919.
53 Rouissi K, Ouerhani S, Oliveira E, et al. Polymorphisms in one-carbon metabolism pathway genes and risk for bladder cancer in a Tunisian population. Cancer Genet Cytogenet 2009; 195:43-53.
54 Ouerhani S, Rouissi K, Marrakchi R, et al. Combined effect of NAT2, MTR and MTHFR genotypes and tobacco on bladder cancer susceptibility in Tunisian population. Cancer Detect Prev 2009;32:395-402.
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