Oyama Kaeko, Kawakami Kazuyuki, Maeda Kazuya, Ishiguro Kaname, Watanabe Go
Department of Surgery, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa 920-8641, Japan.
Anticancer Res. 2004 Mar-Apr;24(2B):649-54.
Methylenetetrahydrofolate reductase (MTHFR) plays a critical role in folate metabolism, which is an important pathway of the methyl donor for DNA methylation. The MTHFR gene has genetic variants (C667T and A1298C), which cause reduced enzyme activity. Impaired folate metabolism by these genetic variants of MTHFR could change the methylation pattern of DNA including promoter hypermethylation, which has been frequently observed in cancer. In this study, we compared the MTHFR genotypes and haplotype to the features of colorectal cancer focusing on the promoter methylation of tumor DNA.
Genomic DNA was isolated from 194 colorectal cancer tissues and subjected to MTHFR genotyping by PCR-based restriction fragment length polymorphism analysis. The MTHFR haplotype was determined by combination of C667T and A1298C genotype and classified into 2 groups, high (H-haplotype) or low (L-haplotype) enzymatic activity of MTHFR. The methylation level of tumor suppressor genes (CDKN2A, hMLH1, ARF and TIMP3) was measured by a fluorescence-based, real-time methylation specific PCR method.
There was no significant association of the clinicopathological features with either C667T genotype, A1298C genotype or haplotype of MTHFR. The methylation level of CDKN2A was higher in cancer with the L-haplotype of MTHFR than in that with the H-haplotype when cancers of proximal origin were considered (p=0.029). hMLH1 methylation also tended to be higher in proximal colon cancers of MTHFR L-haplotype (p=0.059). In addition, the proximal colon cancers showing CpG island methylator phenotype (CIMP) were significantly more frequent in L-haplotype than in H-haplotype.
These results suggest that the haplotype with low enzymatic activity of MTHFR is linked with promoter hypermethylation and consequently modifies the risk of CIMP(+) proximal colon cancer development in the Japanese people. The relationship between MTHFR polymorphism and DNA methylation in the Japanese is contrary to the previous results in Caucasians. Further study is needed focusing on ethnic variations in the relationships among MTHFR polymorphism, DNA methylation and the development of CIMP(+) colorectal cancer.
亚甲基四氢叶酸还原酶（MTHFR）在叶酸代谢中起关键作用，叶酸代谢是DNA甲基化甲基供体的重要途径。MTHFR基因存在基因变异（C667T和A1298C），可导致酶活性降低。MTHFR的这些基因变异导致的叶酸代谢受损可能会改变DNA的甲基化模式，包括启动子高甲基化，这在癌症中经常观察到。在本研究中，我们比较了MTHFR基因分型和单倍型与结直肠癌特征的关系，重点关注肿瘤DNA的启动子甲基化。
从194例结直肠癌组织中分离基因组DNA，并通过基于聚合酶链反应的限制性片段长度多态性分析进行MTHFR基因分型。MTHFR单倍型通过C667T和A1298C基因型组合确定，并分为两组，即MTHFR酶活性高（H-单倍型）或低（L-单倍型）。通过基于荧光的实时甲基化特异性聚合酶链反应方法测量肿瘤抑制基因（CDKN2A、hMLH1、ARF和TIMP3）的甲基化水平。
MTHFR的C667T基因型、A1298C基因型或单倍型与临床病理特征均无显著关联。当考虑近端起源的癌症时，MTHFR L-单倍型癌症中CDKN2A甲基化水平高于H-单倍型癌症（p=0.029）。MTHFR L-单倍型近端结肠癌中hMLH1甲基化也有升高趋势（p=0.059）。此外，显示CpG岛甲基化表型（CIMP）的近端结肠癌在L-单倍型中比在H-单倍型中显著更常见。
这些结果表明，MTHFR酶活性低的单倍型与启动子高甲基化有关，从而改变了日本人群中CIMP（+）近端结肠癌发生的风险。日本人中MTHFR多态性与DNA甲基化之间的关系与之前白种人的结果相反。需要进一步研究关注MTHFR多态性、DNA甲基化与CIMP（+）结直肠癌发生之间关系的种族差异。
