摘要
目的初步评价遗传性非息肉性结直肠癌(HNPCC)胚系MLH1基因突变中新突变的病理性。方法收集符合AmsterdamⅡ标准的12个不同家系的12例患者外周血,用特异引物和耐热性逆转录酶特异地逆转录MLH1的mRNA;利用长模板PCR扩增酶扩增逆转录产物(cDNA);测序分析扩增产物;利用PCRGenescan技术和免疫组织化学染色分别检测有新突变患者肿瘤组织的5个微卫星位点(BAT26,BAT25,D5S346,D2S123和Mfd15)和MLH1蛋白的表达。结果在4例患者中检出4个MLH1突变,其中2个突变为第12外显子的第384密码子(1151bp处)GTT→GAT的突变,该突变是已报道的病理性突变;另外2个突变分别是第8外显子的第217密码子(649bp处)CGC→TGC突变和第16外显子的第581密码子(1742bp处)CCG→CTG突变;后两者为尚未报道的新突变。2个新突变的患者肿瘤组织均呈高度微卫星不稳定性,两者的瘤组织MLH1蛋白均失表达。结论MLH1第8外显子的第217密码子突变和第16外显子的第581密码子的两个新突变很可能为病理性突变。
Objective To explore germline mutations of MLH1 in hereditary nonpolypesis colorectal cancer （HNPCC）, and to investigate the pathobiology of novel detectable mutations of MLH1. Method RNA was extracted from the peripheral blood of 12 patients from 12 different families fulfilling the Amsterdam Ⅱ Criteria of HNPCC. Germline mutations of MLH1 were determined by RT-PCR with gene specific primers, heat-resistance reverse transcriptase and long-template PCR polymerase, followed by cDNA sequencing analysis. PCR-Genescan analysis was used to further investigate microsatellite instability with a panel of 5 microsatellite markers （BAT26, BAT25, D5S346, D2S123 and Mfdl5 ）, along with immunohistochemistry staining to detect the expression of MLH1 protein in the tumor tissues. Results Four germline mutations were found in 4 patients, 2 of which were previously reported GTT→GAT mutation at codon 384 of exon 12, and the other two were novel mutations： CGC→TGC at codon 217 of exon 8 and CCG→CTG at codon 581 of exon 16. Two tumors with the novel mutations had high frequency microsatellite instability showing more than 2 instable loci （ RER ＋ phenotype）, and both tumors lost their MLH1 protein expression. Conclusion The two novel germline mutations of MLH1 identified in this study, i.e. CGC→TGC at codon 217 of exon 8 and CC→CTG at codon 581 of exon 16, are very likely to have pathological significance.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2006年第2期68-72,共5页
Chinese Journal of Pathology
基金
上海市医学重点学科项目(05Ⅲ004)
