Detection of aberrant CpG island methylation in fecal DNA using an electrochemical DNA array chip as a molecular screening test for alimentary tract cancer

作为样本的准备，从1997年解剖的结直肠癌、胃癌和食道癌的新鲜冰冻切片中提取了DNA。甲基化特异性聚合酶链式反应包括24个基因的CpG岛(p16、TSP1、hMLH1、H-cad、RARbeta、XAF1、Slit2、GATA5、TIMP3、RASSF1A、CACNA1G、DLC1、SFRP2、WIF1、HLTF、MGMT、SLC13A5、TSLC1、KIRREL2、HRK、LOX、CDH4、ID4和RASSF4)。我们已经研究了结直肠癌和正常大肠粘膜中12个基因(p16、TSP1、hMLH1、HCAD、RARbeta、XAF1、SLIT2、GATA5、TIMP3、RASSF1A、CACNA1G、DLC1和SFRP2)的异常甲基化，我们在88.1%的癌症中检测到至少一个基因的异常甲基化。我们一直在检测结直肠癌中其余12个基因以及胃癌和食道癌中24个基因的异常甲基化。我们一直在检查这些被认为对诊断有用的基因的组合。我们已经设计并建立了检测结直肠癌的候选基因之一p16的CpG岛的基因芯片，对照实验正在进行中。Nest检查计划包括从粪便样本中提取DNA，使用电化学芯片检测粪便DNA中的异常甲基化，并验证这种分子诊断的敏感性和特异性。

As a preparation of samples, DNA has been extracted from the fresh frozen sections of the colorectal, the gastric, and the esophageal cancers that have been dissected in 1997. The fresh frozen sections of both of the colorectal cancer and normal mucosa and the stool samples before and after the cancer resection from the patients who had colorectal cancers and accepted oral and written informed consent were also collected and processed.Primers for methylation-specific PCR included the CpG islands of 24 genes (p16,TSP1,hMLH1,H-cad,RARbeta,XAF1,SLIT2,GATA5,TIMP3,RASSF1A,CACNA1G,DLC1,SFRP2,WIF1,HLTF,MGMT,SLC13A5,TSLC1,KIRREL2,HRK,LOX,CDH4,ID4,and RASSF4). We already studied colorectal cancers and normal colorectal mucosa for aberrant DNA methylation of 12 genes (p16,TSP1,hMLH1,HCAD,RARbeta,XAF1,SLIT2,GATA5,TIMP3,RASSF1A,CACNA1G,DLC1,and SFRP2), and we detected aberrant methylation of at least one gene in 88.1% of cancers. We have been detecting an aberrant methylation of the remaining 12 genes in colorectal cancers and of 24 genes in the gastric and esophageal cancers. We have been examining the combination of these genes thought to be useful for the diagnosis now. We already designed and established the gene chip for CpG island of p16,one of the candidate genes for detection of colorectal cancers, and the control experiment is under going. The nest examination schedule includes DNA extraction from the fecal sample, detection of an aberrant methylation in fecal DNA using an electrochemical chip, and verifying the sensitivity and the specificity of this molecular diagnostics.