Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes.
The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA-based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non-polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT-PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene-specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing.
Jakubowska
A
A
Department of Genetics and Pathology, Pomeranian Academy of Medicine, Szczecin, Polabska, Poland. aniaj@r1.pam.szczecin.pl
Górski
B
B
Kurzawski
G
G
Debniak
T
T
Hadaczek
P
P
Cybulski
C
C
Kladny
J
J
Oszurek
O
O
Scott
R J
RJ
Lubinski
J
J
D048868:Adaptor Proteins, Signal Transducing; D017398:Alternative Splicing / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D002352:Carrier Proteins; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000175:diagnosis / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins; D018095:Germ-Line Mutation; D006801:Humans; D008239:Lysine / Q000235:genetics; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D020125:Mutation, Missense / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA / Q000379:methods*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D001120:Arginine; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D008239:Lysine