Detection of Ki-ras mutations by PCR and differential hybridization and of p53 mutations by SSCP analysis in endoscopically obtained lavage solution from patients with long-standing ulcerative colitis.

Abstract

The goal of this study was the early detection of malignant transformation in patients with long-standing ulcerative colitis; therefore, mutations of the Ki-ras and p53 gene were analyzed in lavage solution and biopsies obtained at surveillance colonoscopy. DNA was isolated from 14 patients (nine female, five male) with a history of pancolitis for more than 10 yr. Exon 1 of the Ki-ras gene and exons 5-8 of the p53 gene were amplified via polymerase chain reaction. Mutations of the p53 gene were detected via single-strand conformation polymorphism analysis; point mutations of the Ki-ras gene were hybridized on dot blots with oligonucleotides marked with digoxigenin. Wild-type Ki-ras and wild-type p53 were detected in all cases of ulcerative colitis and in four of seven control patients. In two ulcerative colitis patients, a mutation was found in the Ki-ras gene (Gly --> Asp 12 and Gly --> Val 12), and in one patient, a mutation in exon 5 of the p53 gene. Mutations were found only in the lavage fluid, whereas random biopsies were negative. From colonic lavage fluid, it is possible to extract DNA of sufficient quantity and quality for polymerase chain reaction-based amplification and subsequent analysis via single-strand conformation polymorphism or hybridization. Mutations were found in three of 14 patients with long-standing ulcerative colitis but were not found in controls. The method may be useful for the screening of such patients.