基于cDNA测序在中国检测hMLH1和hMSH2的种系突变。
Detection of germline mutations of hMLH1 and hMSH2 based on cDNA sequencing in China.
作者信息
Wang Chao-Fu, Zhou Xiao-Yan, Zhang Tai-Ming, Sun Meng-Hong, Shi Da-Ren
机构信息
Laboratory of Molecular Pathology, Cancer Hospital of Fudan University, Shanghai 20032, China.
出版信息
World J Gastroenterol. 2005 Nov 14;11(42):6620-3. doi: 10.3748/wjg.v11.i42.6620.
AIM
To detect the germline mutations of hMLH1 and hMSH2 based on mRNA sequencing to identify hereditary non-polyposis colorectal cancer (HNPCC) families.
METHODS
Total RNA was extracted from peripheral blood of 14 members from 12 different families fulfilling Amsterdam criteria II. mRNA of hMLH1 and hMSH2 was reversed with special primers and heat-resistant reverse transcriptase. cDNA was amplified with expand long template PCR and cDNA sequencing analysis was followed.
RESULT
Seven germline mutations were found in 6 families (6/12, 50%), in 4 hMLH1 and 3 hMSH2 mutations (4/12, 33.3%); (3/12, 25%). The mutation types involved 4 missense, 1 silent and 1 frame shift mutations as well as 1 mutation in the non-coding area. Four out of the seven mutations have not been reported previously. The 4 hMLH1 mutations were distributed in exons 8, 12, 16, and 19. The 3 hMSH2 mutations were distributed in exons 1 and 2. Six out of the 7 mutations were pathological, which were distributed in 5 HNPCC families.
CONCLUSION
Germline mutations of hMLH1 and hMSH2 can be found based on cDNA sequencing so as to identify HNPCC family, which is highly sensitive and has the advantages of cost and time saving.
目的
基于mRNA测序检测hMLH1和hMSH2的种系突变，以鉴定遗传性非息肉病性结直肠癌（HNPCC）家系。
方法
从符合阿姆斯特丹标准II的12个不同家系的14名成员的外周血中提取总RNA。使用特殊引物和耐热逆转录酶逆转录hMLH1和hMSH2的mRNA。用扩展长模板PCR扩增cDNA，随后进行cDNA测序分析。
结果
在6个家系（6/12，50%）中发现7种种系突变，其中hMLH1突变4种，hMSH2突变3种（4/12，33.3%；3/12，25%）。突变类型包括4种错义突变、1种沉默突变和1种移码突变以及1种非编码区突变。7种突变中有4种以前未报道过。4种hMLH1突变分布在第8、12、16和19外显子。3种hMSH2突变分布在第1和2外显子。7种突变中有6种是病理性的，分布在5个HNPCC家系中。
结论
基于cDNA测序可发现hMLH1和hMSH2的种系突变，从而鉴定HNPCC家系，该方法灵敏度高，具有节省成本和时间的优点。
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