APC mutation analysis by chemical cleavage of mismatch and a protein truncation assay in familial adenomatous polyposis
| Autor: | J. A. Fantes, A. Condie, J Prosser, Andrew H. Wyllie, Malcolm G. Dunlop, J. M. Horn, M. Wright |
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| Rok vydání: | 1994 |
| Předmět: |
Adult
Cancer Research Genes APC Adolescent Transcription Genetic Adenomatous polyposis coli RNA Splicing DNA Mutational Analysis Molecular Sequence Data Polymerase Chain Reaction Frameshift mutation Familial adenomatous polyposis medicine Humans Coding region Gene In Situ Hybridization Fluorescence Polymorphism Single-Stranded Conformational Genetics Base Sequence biology Homozygote RNA-Directed DNA Polymerase Single-strand conformation polymorphism Sequence Analysis DNA Middle Aged medicine.disease Molecular biology Adenomatous Polyposis Coli Oncology biology.protein Mutation testing Research Article Heteroduplex |
| Zdroj: | British Journal of Cancer |
| ISSN: | 1532-1827 0007-0920 |
| DOI: | 10.1038/bjc.1994.408 |
| Popis: | Overall, the causative APC mutation has been identified in only 30% of the patients with familial adenomatous polyposis (FAP) who have been included in studies reported in the literature. In order to determine the true frequency of detectable APC mutations, we set out to search exhaustively the entire coding region of APC for causative mutations in ten patients with classical FAP from Scottish kindreds shown to be linked to 5q markers. Chemical cleavage of mismatch analysis was employed as the initial screening technique. Mutations were confirmed by direct DNA sequencing and shown to generate a premature stop codon by an in vitro protein synthesis assay. Mutations resulting in a premature stop codon either by base substitution or by frameshift were identified in nine families. Although the remaining kindred was linked to intragenic APC markers with a lodscore of 1.69 at Zmax = 0.0, further analysis of DNA, RNA and chromosome spreads from the proband failed to detect any abnormality. This was despite employing single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencing, reverse transcription-polymerase chain reaction (RT-PCR) analysis for splicing defects, a protein truncation test encompassing the entire APC gene and fluorescent in situ hybridisation chromosome analysis (FISH). These data show that 90% of these FAP kindreds had APC mutations detectable by chemical cleavage of mismatch and that none of the numerous other techniques employed could detect the mutation in the remaining kindred. This study shows the value of screening the APC gene using a combination of chemical cleavage of mismatch analysis and an in vitro protein truncation test. Images Figure 1 Figure 2 Figure 3 |
| Databáze: | OpenAIRE |
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