APC MUTATION ANALYSIS BY CHEMICAL CLEAVAGE OF MISMATCH AND A PROTEIN TRUNCATION ASSAY IN FAMILIAL ADENOMATOUS POLYPOSIS

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 e xhaustively the entire coding region of APC for causative mutations in ten patients with classical FAP from Scottish kindreds shown to be li nked to 5q markers. Chemical cleavage of mismatch analysis was employe d as the initial screening technique. Mutations were confirmed by dire ct DNA sequencing and shown to generate a premature stop codon by an i n vitro protein synthesis assay. Mutations resulting in a premature st op codon either by base substitution or by frameshift were identified in nine families. Although the remaining kindred was linked to intrage nic APC markers with a lodscore of 1.69 at Z(max) = 0.0, further analy sis of DNA, RNA and chromosome spreads from the proband failed to dete ct any abnormality. This was despite employing single-strand conformat ion polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencin g, reverse transcription-polymerase chain reaction (RT-PCR) analysis f or splicing defects, a protein truncation test encompassing the entire APC gene and fluorescent in situ hybridisation chromosome analysis (F ISH). These data show that 90% of these FAP kindreds had APC mutations detectable by chemical cleavage of mismatch and that none of the nume rous other techniques employed could detect the mutation in the remain ing kindred. This study shows the value of screening the APC vitro pro tein truncation test.