Detection of germline mutations of hMLH1 and hMSH2 based on cDNA sequencing in China.
OBJECTIVE
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.


RESULTS
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.


CONCLUSIONS
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.
Wang
Chao-Fu
CF
Laboratory of Molecular Pathology, Cancer Hospital of Fudan University, Shanghai 20032, China.
Zhou
Xiao-Yan
XY
Zhang
Tai-Ming
TM
Sun
Meng-Hong
MH
Shi
Da-Ren
DR
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000097:blood / Q000235:genetics*; D002681:China; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D004252:DNA Mutational Analysis; D004260:DNA Repair; D018095:Germ-Line Mutation; D006801:Humans; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000097:blood / Q000235:genetics*; D009687:Nuclear Proteins / Q000097:blood / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein