MLH1、MSH2基因mRNA突变分析与遗传性非息肉性结直肠癌的基因诊断

The analysis for mRNA mutation of MLH1, MSH2 genes and the gene diagnosis for hereditary nonpolyposis colorectal cancer

目的检测胚系MLH1和MSH2基因mRNA突变,确立遗传性非息肉性结直肠癌(hereditarynonpolyposis colorectal cancer,HNPCC)家系。方法收集符合Amsterdam标准Ⅱ的12个家系14名家庭成员外周血,用特异引物和耐热性逆转录酶特异地逆转录MLH1和MSH2的RNA;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。提取外周血的DNA,设计与利用上述方法检测出突变对应外显子的特异性引物,利用TaqDNA聚合酶扩增测序,以检测上述方法的有效性。结果利用基于外周血mRNA的方法,在6个家系中检出6个胚系突变,4个MLH1突变和2个MSH2突变,MLH1突变分别位于第8、12、16和第19外显子;MSH2突变分别位于第1和第2外显子。利用基于外周血DNA的方法,上述突变均在MLH1和MSH2相应的外显子中得到验证。突变类型为4个错义突变、1个同义突变和1个非编码区突变;其中5个突变国际上尚未报道;6个突变中有5个为病理性,分布于5个不同家系,该5个家系被确诊为HNPCC家系。结论基于外周血MLH1和MSH2mRNA异常的检测能确诊HNPCC家系;该方法敏感、省时、节约成本。

Objective To identify hereditary nonpolyposis colorectal cancer （HNPCC） families based on the gennline mutations of MLH1 and MSH2 mRNA. Methods RNA was extracted from the peripheral blood of the 14 members from 12 different families fulfilling Amsterdam Criteria Ⅱ. The germline mutations of MLH1 and MSH2 mRNA were detected by cDNA sequencing analysis following reverse transcription-PCR（RT-PCR） with special primers, heat-resistance reverse transcriptase, and expand long template PCR. DNA was extracted from the peripheral blood of the 14 members, the corresponding exons, in which mutations were found using the above method, were amplified with Taq enzyme, sequencing analysis was followed. Results Six gennline mutations were detected and identified from the 6 different families based on mRNA, 4 of them to be in MLH1 , the other 2 in MSH2 . The MLH1 mutations distribute in the exon 8, 12, 16, and 19. TheMSH2 mutations distribute in exons 1 and 2. The 6 mutations were identified from the corresponding exons respectively in genomic DNA sequencing analysis. The mutation types involve in 4 missense, 1 silent, and 1 non-coding area mutations. Five out of the 6 mutations have not been reported previously. Five out of the 6 mutations were pathological, involving in 5 different families. The five families were identified to HNPCC families. Conclusion HNPCC family can be identified with RNA-based sequencing of MLH1 andMSH2 from peripheral blood, which has the advantages of beth cost, time saving and high sensitivity.