INHIBITION OF NONSENSE-MEDIATED MESSENGER-RNA DECAY IN CLINICAL-SAMPLES FACILITATES DETECTION OF HUMAN MSH2 MUTATIONS WITH AN IN-VIVO FUSION PROTEIN ASSAY AND CONVENTIONAL TECHNIQUES

Germ-line mutations in the human MSH2 (hMSH2) gene account for about 4 0% of known defects in kindreds with hereditary nonpolyposis colon can cer, We describe a simple fusion protein assay for detection of hMSH2 nonsense mutations in yeast, Detection of nonsense mutations with this assay is severely compromised in many cases by nonsense-mediated mRNA decay, a physiological process that destabilizes the mutant RNA. Trig gering of nonsense-mediated decay requires mRNA scanning by the riboso me to detect the stop codon. We show that treatment of cells with the translation inhibitor puromycin suppresses nonsense-mediated decay and facilitates the detection of nonsense mutations in clinical samples b y cDNA sequencing, in vitro protein truncation tests, and the yeast fu sion protein assay, Given the prevalence of chain-terminating mutation s in human disease genes, puromycin treatment of blood samples should improve the signal-to-noise ratio and hence the sensitivity of many RN A-based diagnostic tests, Paradoxically, the yeast hMSH2::ADE2 fusion protein assay also detects some in-frame mutations, presumably through an effect on the folding of the fusion protein.