遗传性弥漫型胃癌家系上皮型钙黏素基因的分析

Analysis of CDH1 gene expression in hereditary diffuse gastric cancer

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。用免疫组化和Westernblot法检测组织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)的癌旁胃黏膜上皮细胞CDH1蛋白表达较正常胃黏膜减弱,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2 377位点存在一个C→T的单核苷酸多态性(SNP),但未检测到16个外显子的胚系突变。相对于正常胃黏膜,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,其瘤旁黏膜也有高甲基化。结论此HDGC家系中,CDH1基因外显子胚系突变不是其致病原因,基因启动子区的甲基化可能是导致基因失活的原因之一。

Objective To detect the expression of CDH1, screen the germ-line mutation of CDH1 exons and to e- valuate CDH1 promoter methylation status in a family with hereditary diffuse gastric cancer （HDGC） in China. Methods Fifteen 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 di- rect sequencing germ-line mutation of 16 CDH1 exons were screened. PCR and clone sequencing were used to in- vestigate 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, hypermethy- lation 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 1 6 exons was excluded as the cause of gastric cancer, CDH 1 promoterhypermethylation may be one of the reasons inducing inactivation of the gene.