亚甲基四氢叶酸还原酶基因多态性检测在肿瘤患者中的临床价值

摘 要：亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase MTHFR)是叶酸代谢、DNA甲基化的关键酶。MTHFR的单核苷酸多态性(single nucleotide polymorphism,SNP),包括677 C→T和1298A→C突变,可使MTHFR酶活性下降,从而影响细胞内叶酸的正常代谢。全文分析MTHFR 基因多态性对叶酸代谢和DNA合成的影响及其在恶性肿瘤发生、诊治和预后的研究进展,阐述恶性肿瘤患者中检测MTHFR基因多态性的价值及存在争议。     

叶酸代谢相关酶基因多态性与消化系统肿瘤的关系

叶酸的主要生物学功能是作为甲基供体参与脱氧核糖核酸合成和细胞内甲基化反应.亚甲基四氢叶酸还原酶(methylene tetrahydrofolate reductase,MTHFR)、甲硫氨酸合成酶(methionine synthase,MTR)、甲硫氨酸合成酶还原酶(methionine synthase reductase,MTRR)、胸苷酸合成酶(thymidylate synthase,TS)和亚甲基四氢叶酸脱氢酶(methylenetetrahydrofolate dehydrogenase,MTHFD)等是叶酸代谢通路中重要的酶.叶酸代谢相关酶基因变异、叶酸缺乏或代谢障碍与肿瘤的发生和发展有关.本文就叶酸代谢相关酶基因多态性与消化系统肿瘤间的关系进行简要综述.     

叶酸与结直肠肿瘤

流行病学和试验研究提示叶酸缺乏及叶酸代谢异常和结直肠肿瘤发病风险升高有关。亚甲基四氢叶酸还原酶（MTHFR）是调节叶酸代谢的关键酶，MTHFRC677T基因多态及饮酒影响叶酸代谢，从而影响结直肠肿瘤的发病风险。     

亚甲基四氢叶酸还原酶基因多态性与乳腺癌相关性的研究进展

乳腺癌是一种复杂的全身性的疾病,其发病机制尚未完全清楚,但受基因及环境相互作用的影响。亚甲基四氢叶酸还原酶（MTHFR）基因位于1号染色体短臂的末端（1p36.6）,是叶酸代谢途径的关键酶,参与DNA合成、修复及甲基化过程。MTHFR基因多态性的分布在不同的种族及区域存在差异,其基因的单核苷酸突变会使该酶的生物活性及热稳定性下降,参与肿瘤的发生及发展过程,多项研究显示 MTHFR基因多态性与乳腺癌发生、发展及预后密切相关。     

5,10-亚甲基四氢叶酸还原酶基因多态性与肺癌的相关性研究

叶酸代谢在肺癌发生过程中占有重要的地位,因为叶酸的代谢同时影响了DNA与核苷酸的合成。5,10-亚甲基四氢叶酸还原酶（methylenetetrahydrofolate reductase,MTHFR）是一种非常重要的叶酸还原酶,它不可逆地将5,10-亚甲基四氢叶酸催化生成5-甲基四氢叶酸,而后者正是同型半胱氨酸甲基化生成蛋氨酸反应的甲基供体。MTHFR基因的多态性（C677T,A1298C）会造成酶的活性降低,从而影响疾病的发病率。已有研究表明MTHFR的基因多态性与肺癌的发病率显著相关,文章对MTHFR基因多态性与肺癌的关系作一综述。     

亚甲基四氢叶酸还原酶基因单核苷酸多态与乳腺癌风险

目的 内研究亚甲基四氢叶酸还原酶(MTHFR)基因单核苷酸多态与女性乳腺癌风险的关系。方法 以聚合酶链反应(PCR)和限制性片段长度多态性(RFLF)分析方法,检测了217例乳腺癌患者和218例配对的正常对照者MTHFR因C677T和A1298C基因型,并比较不同基因型与乳腺癌风险的关系。结果 677TT基因型频率在乳腺癌患者和正常对照中的分布差异有显著性(32．7％比24．8％,P=0．02)。携带MTHFR 677TT基因犁者与携带MTHFR 677CC基因型者比较,前者罹患乳腺癌的风险增加1,84倍(95％ C：1．09～3．14)。MTHFR 677CT基因型以及MTHFR A1298C多态与乳腺癌风险不相关。结论 MTHFR基因677C→T突变是女性乳腺癌的遗传易感因素。     

云南人亚甲基四氢叶酸还原酶基因型多态性与乳腺癌易感性的关联

 目的亚甲基四氢叶酸还原酶(MTHFR)是叶酸代谢的关键性酶,其催化作用决定了DNA甲基化与DNA合成之间的平衡。MTHFR基因多态性可能会影响叶酸代谢结局,从而构成肿瘤风险因子。研究分析了MTHFR各基因型在云南籍乳腺癌人群和正常人群中的分布差异,初步探索了MTH-FR多态性与乳腺癌易感性之间的关系。方法以多重PCR-RFLP技术,对125例云南籍乳腺癌患者和103例正常人群MTHFR677位点1298位点多态性进行筛查。结果未发现MTHFRC677T和A1298C基因型频率在乳腺癌和对照样本之间存在显著差异。结论在目前样本条件下,上述两个MTHFR位点基因型多态性与云南籍人群的乳腺癌易感性之间无明显相关性。     

食管鳞状细胞癌中MGMT基因异常甲基化与MTHFR C677T基因多态的关系

背景与目的:作为基因失活的主要原因之一,肿瘤相关基因启动子区异常甲基化正日益受到关注,但是在食管癌方面研究尚十分有限.本研究的目的在于探讨DNA损伤修复基因MGMT异常甲基化与食管鳞状细胞痛临床特征和叶酸代谢酶基因MTHFR C677T多态之间的联系.方法:选择2005年1月至2006年3月间新发的、经病理学检查确诊并在江苏省扬中市人民医院进行手术治疗的食管鳞癌患者开展流行病学问卷调查,并采集其外周静脉血标本、癌组织和癌旁正常组织标本.应用甲基化特异性PcR法检测MGMT基因启动子区CpG岛甲基化状态.应用限制性片断长度多态(restriction fragment length polymorphism,RFLP)技术检测叶酸代谢酶基因MTHFR C677T多态.并分析和探讨食管粘膜组织中MGMT基因甲基化分布规律及其与MTHFR C677T基因多态间的联系.结果:125例食管鳞状细胞癌组织中MGMT基因甲基化频率达27.2%,癌旁组织甲基化频率11.2%,10例正常成人健康对照的食管粘膜均呈去甲基化状态.患者淋巴结转移与否与DNA甲基化频率有关,存在淋巴结转移的患者癌组织中MGMT基因甲基化频率(37.3%)高于无淋巴结转移的患者(18.2%).患者性别、年龄、吸烟、饮酒、饮茶等因素与DNA异常甲基化之间的相关性无统计学意义(P>0.05).在调整了相关潜在混杂因素后,携带MTHFR变异基因型CT和TT者食管癌组织中MGMT基因甲基化频率增高,与野生基因型CC比较,比值比OR分别为3.34(95%Cl:1.07～10.39)和3.83(95%CI:1.13～12.94).结论:食管鳞状细胞癌中MGMT基因启动子区异常甲基化与MTHFR基因多态有关,携带MTHFRC677基因变异基因型CT与TT的癌组织中MGMT基因异常甲基化频率更高.     

MTHFR C677T基因多态性与晚期非小细胞肺癌化疗不良反应的关系

背景与目的：亚甲基四氢叶酸还原酶(methylene tetrahydrofolate reductase,MTHFR)是叶酸代谢的关键酶,在DNA甲基化中起重要作用。本研究旨在探讨MTHFR C677T多态性与晚期非小细胞肺癌(nonsmall cell lung cancer,NSCLC)化疗不良反应的关系。方法：收集2007年6月-2009年5月在浙江省肿瘤医院经病理学确诊的晚期NSCLC患者100例。所有患者均接受铂类药物联合吉西他滨的方案化疗。用等位基因特异-PCR技术检测患者MTHFR基因型。结果：100例晚期NSCLC患者中,MTHFR C677T T/T、T/C和C/C基因型频率分别为20%、44%和36%。在血液学不良反应中,C/C基因型血小板减少发生率较T/T、T/C基因型低,差异有统计学意义(P=0.039)。本研究未发现MTHFR各基因型与化疗后恶心、呕吐不良反应相关。结论：MTHFRC677T基因多态性对预测晚期NSCLC含铂类药物方案化疗后不良反应有临床意义。     

MTHFR基因遗传多态与食管癌贲门癌易感性的关系

目的:探讨食管癌高发区亚甲基四氢叶酸还原酶(MTHFR)基因单核苷酸多态与食管癌、贲门癌发病风险的关系。方法:以聚合酶链反应和限制性片段长度多态方法,分析584例食管癌患者、467例贲门癌患者和540例正常对照的MTHFR基因C677T基因型及其与食管癌、贲门癌发病风险的相关性。结果:在正常对照中,MTH-FR677CC、CT、TT基因型频率分别为22.1%、43.3%和34.6%,在食管癌患者中分别为12.5%、45.0%和42.5%(P=0.000);在贲门癌患者中分别为15.8%、43.5%和40.7%(P=0.024)。多因素分析发现,携带677TT基因型和677CT基因型的个体发生食管癌的风险分别是677CC基因型的2.36倍和1.76倍,发生贲门癌的风险分别是1.34倍和1.23倍。结论:MTHFR单核苷酸多态是食管癌高发区食管癌和贲门癌的遗传易感性因素。     

The methylenetetrahydrofolate reductase C677T mutation induces cell‐specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site‐specific cancer risk modification

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one‐carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one‐carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA‐MB‐435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA‐MB‐435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA‐MB‐435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site‐specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation. © 2008 Wiley‐Liss, Inc.     

Folate restriction and methylenetetrahydrofolate reductase 677T polymorphism decreases adoMet synthesis via folate-dependent remethylation in human-transformed lymphoblasts.

The homozygous mutation (677TT) in the methylenetetrahydrofolate reductase (MTHFR) gene reduces enzyme activity and alters cellular folate composition. Previous epidemiological studies reported a potential protective effect of MTHFR677C --> T against acute lymphocytic leukemia and malignant lymphoma, but the mechanism remains to be determined. We investigated the biochemical impacts of MTHFR677C --> T on cellular S-adenosyl methionine (adoMet) synthesis, global DNA methylation, and de novo purine synthesis, all of which are potential regulatory pathways involved in tumorigenesis. Metabolic fluxes of homocysteine remethylation and de novo purine synthesis were compared between Epstein-Barr virus-transformed lymphoblasts expressing MTHFR 677C and MTHFR 677T using stable isotopic tracers and GCMS. MTHFR TT genotype significantly reduced folate-dependent remethylation under folate restriction, reflecting limited methylated folates under folate restriction. Data also suggested increased formylated folate pool and increased purine synthesis when folate is adequate. The impacts of MTHFR 677T polymorphism appeared closely related to folate status, and such alterations may modulate metabolic pathways involved in cancer onset/progression. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in hematological malignancies. These transformed cells are potential models for studying the consequences of human genetic variation and cancer pathogenesis.     

Polymorphisms in genes involved in folate metabolism and plasma DNA methylation in colorectal cancer patients

Aberrant methylation of promoter regions associated with gene silencing is one of the major mechanisms underlying the inactivation of tumor suppressor genes in carcinogenesis. Previous studies have proposed that methylated DNA from tumor cells is released into the circulation and might be widely used as a marker for non-invasive tumor detection. Catalytic activities of folate metabolism-related enzymes and adequate synthesis of methyl donors are important elements for the cellular methylation reaction. In the present study, we sought to determine the following: i) genotype frequencies of MTHFR and MTR involved in folate metabolism in cases and cancer-free controls; and ii) the methylation status of three candidate genes (p16INK4A, p73 and hMLH1) in plasma related to the folate and homocysteine levels. From genotype frequency analysis, individuals homozygous for the MTHFR 677TT genotype had a significantly reduced risk of developing colorectal cancer compared with those harboring the MTHFR 677CC genotype (OR, 0.206; 95% CI, 0.070-0.604; P=0.005), and had a lower plasma folate concentration than those with the MTHFR 677CC+CT genotype (P<0.05). Using methylation-specific PCR, p73 was shown to be more frequently methylated in the high folate group [50% (8 of 16)] than in the medium [16.7% (3 of 18)] or low folate subgroups [11.1% (2 of 18)]. In conclusion, subjects with the variant MTHFR 677TT genotype appeared to have a significantly lower risk for colorectal cancer than those with the MTHFR 677CC genotype, and the methylation status of circulating p73 genomic DNA was substantially altered by the plasma folate level.     

Polymorphisms of methylenetetrahydrofolate reductase and the risk of prostate cancer: a nested case-control study.

It has been proposed that folate and polymorphisms of the enzyme methylenetetrahydrofolate reductase (MTHFR), which regulates influx of folate from DNA synthesis and repair to methylation reactions, are involved in the aetiology of cancer. To relate the MTHFR 677C-->T and 1298A-->C polymorphisms to the risk of prostate cancer, taking into consideration prospective plasma levels of folate, vitamin B12 and homocysteine. The design was a case-control study of 223 prostate cancer cases and 435 matched controls nested within the population-based Northern Sweden Health and Disease Cohort. Neither the MTHFR 677C-->T nor the MTHFR 1298A-->C polymorphism was statistically significantly associated with the risk of prostate cancer in univariate analysis by conditional logistic regression. After adjustment for MTHFR 1298A-->C, plasma folate, vitamin B12, homocysteine, body mass index and smoking, the odds ratios were, for the 677 CT genotype, 1.52 [95% confidence interval (CI) 1.02-2.26], and for TT, 0.91 (95% CI 0.41-2.04). Our previously reported observation of a possible increase in the risk of prostate cancer at high plasma folate levels was attributable in this study to subjects having the MTHFR 677C-->T polymorphism. We found that the MTHFR 677C-->T polymorphism is not likely to have a major role in the development of prostate cancer, although it may possibly increase the risk in combination with high plasma folate levels. Further investigation in larger studies is warranted.     

Genetic polymorphisms of methylenetetrahydrofolate reductase and colorectal cancer and adenoma

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating folate metabolism, which affects DNA methylation and synthesis. Two functional, common polymorphisms (C677T and A1298C) are known in the MTHFR gene. MTHFR activity is lowered in individuals with the 677TT genotype and is somewhat reduced in those with the 1298CC genotype. We reviewed the consistency of reported associations of these polymorphisms with colorectal cancer and adenoma with consideration of the effects of nutritional status. A total of 16 studies have addressed the association between MTHFR C677T polymorphism and colorectal cancer in 10 countries. Decreased risk of colorectal cancer associated with the 677TT genotype has fairly consistently been observed, with few exceptions. This decrease was observable in people with either high or low folate status. Alteration in the thymidylate pool associated with MTHFR activity is postulated as an underlying mechanism. Studies on the A1298C polymorphism are limited, and their results are variable. Almost all of seven studies of colorectal adenoma have found no association between C677T polymorphism and adenoma, but the 677TT genotype seems to be related to increased risk when folate status is poor. Reduced availability of methyl groups for DNA methylation might be more relevant to adenoma formation. Although the underlying mechanisms still remain to be clarified, epidemiological findings regarding MTHFR C677T polymorphism provide strong evidence that adequate folate status confers protection from colorectal cancer.     

The MTHFR 1298A>C polymorphism and genomic DNA methylation in human lymphocytes.

Methylenetetrahydrofolate reductase (MTHFR) balances the pool of folate coenzymes in one-carbon metabolism for DNA synthesis and methylation, both implicated in carcinogenesis. Epidemiologic studies have shown that two functional polymorphisms in MTHFR gene, 677C>T and 1298A>C, are related to increased cancer risk. We aimed to analyze lymphocyte DNA from 198 subjects to evaluate the MTHFR 1298A>C polymorphism and folate status affecting genomic DNA methylation as a possible mechanism underlying the relationship between MTHFR polymorphisms and cancer susceptibility. Carriers of the 1298AA wild-type genotype showed lower genomic DNA methylation compared with 1298AC or 1298CC genotypes [3.72 versus 8.59 or 6.79 ng 5-methyl-2'-deoxycytidine (5-mCyt)/microg DNA, P < 0.0001 and P = 0.007, respectively]. When DNA methylation was evaluated according to plasma folate status, only 1298AA with low folate levels revealed diminished DNA methylation (P < 0.0001). Moreover, when the two MTHFR polymorphisms were concomitantly evaluated at the low folate status, DNA methylation was reduced only in 1298AA/677TT compared with 1298AA/677CC (3.11 versus 7.29 ng 5-mCyt/microg DNA, P = 0.001) and 1298CC/677CC genotypes (3.11 versus 7.14 ng 5-mCyt/microg DNA, P = 0.004). However, the high prevalence of 677TT mutants within the 1298AA group (79%) and the similar biochemical features of 1298AA/677CC and 1298CC/677CC combined genotypes suggest that the gene-nutrient interaction affecting DNA methylation in 1298AA is mainly due to the coexistence of the 677TT genotype and that the 1298A>C polymorphism may convey its protective effect not through this interaction but through another pathway in one-carbon metabolism. Further mechanistic studies are warranted to investigate how single polymorphisms as well as MTHFR combined genotypes exert their effect on cancer susceptibility.     

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.

DNA methylation is an epigenetic feature of DNA that influences cellular development and function, and aberrations of DNA methylation are a candidate mechanism for the development of cancer. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate, the methyl donor for methionine synthesis and the precursor of S-adenosylmethionine. S-adenosylmethionine is the universal methyl donor for methylation reactions, including that of DNA methylation. In the present study, we investigated whether a common C677T mutation in the MTHFR gene, which results in reduced enzyme activity in vitro, affects genomic DNA methylation. We selected 9 subjects homozygous for the wild-type MTHFR and 10 subjects homozygous for the mutation (T/T). Genomic DNA methylation was determined by an established enzymatic assay that measures the capacity of DNA to accept methyl groups in vitro, which is inversely related to endogenous methylation. DNA from subjects with the T/T MTHFR genotype had a significantly higher methyl group acceptance capacity (12,615 +/- 1836 dpm/2 microg of DNA) compared with wild-type MTHFR (7843 +/- 1043 dpm/2 microg of DNA; P < 0.05), indicating DNA hypomethylation in the T/T genotype. Furthermore, DNA methylation was directly and significantly related to RBC folate concentrations in persons with the T/T genotype, but not in those with wild-type MTHFR. These data are consistent with prior observations, which suggest that the T/T genotype is associated with impaired MTHFR activity in vivo and that the cellular impact of this impairment is determined, in part, by folate status. The relationship of genomic DNA hypomethylation in persons with the T/T MTHFR genotype to the development of cancer remains to be defined.     

Localized depletion: the key to colorectal cancer risk mediated by MTHFR genotype and folate?

Dietary folate has been consistently associated with reduced risk of colorectal cancer (CRC). One of the known biochemical roles of folate is donation of methyl moieties. DNA hypomethylation is an early and almost ubiquitous occurrence in tumor tissue. Therefore, it was originally suggested that adequate folate intake contributed to reduced risk of CRC by facilitating methyl-mediated silencing of oncogenes. Methylene tetrahydrofolate reductase (MTHFR) metabolizes 5,10-MTHF (important in DNA synthesis) to 5-MTHF (contributes to downstream methylation reactions by regeneration of methionine from homocysteine). A common polymorphism in the MTHFR gene (C677T) results in a thermolabile phenotype associated with increased homocysteine levels and DNA hypomethylation. Consistent with the folate/methylation hypothesis, it was originally proposed that C677T may increase risk of CRC due to hypomethylation of oncogenes. However, most subsequent studies have reported a reduced risk associated with this polymorphism. This is inconsistent with methylation as the mechanism by which folate and MTHFR genotype mediate CRC risk. The hypothesis presented here proposes that localized folate depletion combined with the effect of the C677T polymorphism on enzyme stability, impacts on the DNA synthesis pathway and accounts for the observed variation in risk associated with genotype and folate status.