[The analysis for mRNA mutation of MLH1, MSH2 genes and the gene diagnosis for hereditary nonpolyposis colorectal cancer].
OBJECTIVE
To identify hereditary nonpolyposis colorectal cancer (HNPCC) families based on the germline 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 germline 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. The MSH2 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.


CONCLUSIONS
HNPCC family can be identified with RNA-based sequencing of MLH1 and MSH2 from peripheral blood, which has the advantages of both cost, time saving and high sensitivity.
Wang
Chao-fu
CF
Laboratory of Molecular Pathology, Cancer Hospital of Fudan University, Shanghai 200032, P.R.China.
Zhou
Xiao-yan
XY
Zhang
Tai-ming
TM
Sun
Meng-hong
MH
Xu
Ye
Y
Shi
Da-ren
DR
D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000175:diagnosis / Q000235:genetics*; D005260:Female; D018095:Germ-Line Mutation; D006801:Humans; D008297:Male; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis
D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein