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Abstract

We have used single-strand conformational polymorphism (SSCP) and heteroduplex analysis to examine DNA from 50 colorectal carcinoma patients coming from families meeting the Amsterdam criteria for hereditary nonpolyposis colorectal cancer (HNPCC) or having developed colorectal carcinoma at age 45 or younger. We identified mutations in 12 of these patients, with seven of these being novel mutations. We examined four of the truncating mutations using in vitro transcription and translation (IVTT) assays and determined that the mutation causing an in-frame deletion of exon 5 could easily be detected by the IVTT assay, but the three mutations resulting in premature translation termination were not detected because the steady-state levels of the mutant allele transcripts are too low. Our findings suggest that some but not all mutant hMSH2 alleles have significantly lower steady-state mRNA levels than the normal allele. Under ideal circumstances, where lymphoblastoid cell lines are available for RNA extraction, IVTT may be useful for detecting truncating mutations. However, our data suggest that caution should be taken in using IVTT in routine screening of clinical samples for truncating HNPCC mutations, as many mutations may go undetected.