Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

The most sensitive technique for the detection of germline mutations is er; on by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity l imitations that can, at least in part, be overcome by RNA-based analysis. G ermline mutations of MLH1 and MSH2 are the most frequent cause of the inher ited susceptibility to colorectal and other epithelial cancers known as her editary non-polyposis colorectal cancer (HNPCC). We compared the analysis o f the MLH1 and MSH2 genes using mRNA and genomic DNA. as starting material from 21 HNPCC patients. All samples mere 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, r espectively Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrela ted patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel misse nse mutation (K751R) in MLH1 was detected using this method. One nonsense m utation (E205X) in MSH2 was only detectable when RT was performed using MSH 2 gene-specific printers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT pr imers were employed to generate template, except in one case where exon ski pping was observed for exons 9 and 10. In this report we demonstrate that p rimers specific for RT of MLH1 and MSH2 are crucial for increasing the sens itivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA se quencing. Hum Mutat 17:52-60, 2001. (C) 2001 Wiley-Liss, Inc.