遗传性弥漫型胃癌家系上皮型钙黏素基因的分析

 摘要：目的 研究在遗传性弥漫型胃癌家系中是否存在上皮型钙黏素基因(CDH1)以及基因表达的异常。 方法 收集符合遗传性弥漫型胃癌(HDGC)诊断标准的1个家系中15例成员的外周血和组织标本。通过免疫组化和Western blot 的方法检测组织标本CDH1蛋白表达; 提取基因组DNA, 通过PCR扩增DNA直接测序检测CDH1基因16个外显子突变。用克隆测序法，鉴定CDH1基因启动子区CpG位点甲基化状况。 结果 先证者和另一胃癌患者(家系2号成员)的癌旁胃粘膜上皮细胞CDH1蛋白表达较正常胃粘膜减弱, 两者肿瘤组织的蛋白表达几乎为阴性。包括先证者在内的11例家系成员第14外显子mRNA水平2377位点存在一个C?T的单核苷酸多态性(single nucleotide polymorphism, SNP), 但未检测到16个外显子的胚系突变。相对于正常胃粘膜, 先证者和家系2号成员的胃癌组织均有CDH1基因启动子的高甲基化, 其瘤旁粘膜也有高甲基化。结论 此HDGC家系中,CDH1基因表达丢失可能和胃癌发生有关, CDH1基因外显子突变不是其肿瘤组织上皮型钙黏素蛋白表达丢失的直接原因, 基因启动子区的甲基化可能是导致其失活的原因之一。

Analysis of CDH1 gene expression in hereditary diffuse gastric cancer

Objective To detect the expression of CDH1,screen the germ-line mutation of CDH1 exons and to evaluate CDH1 promoter methylation status in a family with hereditary diffuse gastric cancer(HDGC) in China.MethodsFifteen members of a family with HDGC were visited,peripheral blood samples and tumor specimens were collected.The expression of CDH1 gene was detected by immunohistochemistry and Western blot.By PCR and direct sequencing germ-line mutation of 16 CDH1 exons were screened.PCR and clone sequencing were used to investigate the status of CDH1 promoter methylation.Results In proband and another gastric cancer patient(number 2 member),the protein expression of CDH1 was reduced in mucosae near the tumors,and lost in the tumors.In 11 members(including proband),a single nucleotide substitution of C→T(SNP) in exon 14(mRNA 2377 locus) was found.No germ-line mutation of 16 exons was detected in all members.Compare with normal mucosa,hypermethylation was found in the tumors and mucosae near the tumor of proband and number 2 member.Conclusions In this family with HDGC,germ-line mutation of 16 exons was excluded as the cause of gastric cancer,CDH1 promoter hypermethylation may be one of the reasons inducing inactivation of the gene.