RESUMO
Germ-line mutations in the human MSH2 (hMSH2) gene account for about 40% of known defects in kindreds with hereditary nonpolyposis colon cancer. 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. Triggering of nonsense-mediated decay requires mRNA scanning by the ribosome 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 by cDNA sequencing, in vitro protein truncation tests, and the yeast fusion protein assay. Given the prevalence of chain-terminating mutations in human disease genes, puromycin treatment of blood samples should improve the signal-to-noise ratio and hence the sensitivity of many RNA-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.
Assuntos
Códon sem Sentido/efeitos dos fármacos , Códon de Terminação/análise , Proteínas de Ligação a DNA , Testes Genéticos/métodos , Proteínas Proto-Oncogênicas/genética , Puromicina/farmacologia , Western Blotting , Células Cultivadas , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Humanos , Linfócitos/química , Proteína 2 Homóloga a MutS , Mutação , LevedurasAssuntos
Aerococcus/isolamento & purificação , Infecções por Bactérias Gram-Positivas/diagnóstico , Infecções por Bactérias Gram-Positivas/epidemiologia , Técnicas de Diagnóstico Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aerococcus/química , Aerococcus/genética , Idoso , Técnicas Bacteriológicas/métodos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Feminino , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Masculino , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genéticaRESUMO
About 80% of the mutations identified to date in the Adenomatous Polyposis Coli (APC) gene have been found in the 5' half of the coding sequence, the vast majority of which (>95%) are nonsense or frameshift mutations that result in the loss of the carboxyl terminus of APC protein. Using a stop codon assay in yeast recently developed by others (Ishioka et al., 1997), we have screened the 5' half of the APC gene for mutations in 7 unrelated families affected with Familial Adenomatous Polyposis. The assay relies on the expression of a yeast reporter gene fused in frame to one of 3 contiguous segments of the APC open reading frame. Here we report on the detection by this assay of 5 germline mutations, 4 of which lie upstream of exon 15, where lesions appear to be sometimes difficult to detect by standard methods.