Mutation analysis of the adenomatous polyposis coli gene.

Familial adenomatous polyposis (FAP) is a dominantly inherited syndrome characterised by the development of hundreds to thousands of adenomatous polyps throughout the colon and rectum. These usually arise during the second or third decades of life, and in the absence of surgical intervention it is inevitable that one or more will progress from a benign to a malignant state. The underlying cause of FAP is mutation of the adenomatous polyposis coli (APC) gene, a tumour suppressor gene located on chromosome 5q21-22. Analysis of the APC gene undertaken during this study has provided data on the type and distribution of APC mutations in FAP patients. Fifty two unrelated patients were investigated and mutation was detected in 35 (67%) of them. Of the different types of mutation identified deletions and insertions were the most frequently noted accounting for 85% of all mutations. Nonsense and splice consensus sequence mutations were also detected, all of which are predicted to result in premature truncation of the APC protein. Mutations were identified in most regions of the APC gene assessed but showed a marked clustering within the 5' half of exon 15. Mutations at codon 1061 and codon 1309 were found to be responsible for 10% and 19% of FAP cases respectively. In addition to analysis of APC sequence variants chromosomal rearrangements and deletions in the vicinity of the APC gene were investigated using fluorescence in situ hybridisation (FISH). Aspects of the FAP phenotype were considered and compared to the site of mutation within the APC gene. A correlation between severity of phenotype, defined by number of polyps and age of disease onset, and location of APC mutation was identified. Mutation between codons 1309 and 1464 was found to be associated with early onset of cancer and the development of thousands rather than hundreds of colorectal polyps. Interestingly the majority of patients with no family history of FAP (new mutations) were found to have mutation within this 'severe' region of APC. Presymptomatic diagnosis of FAP was performed for members of FAP families that were of uncertain gene status. This was accomplished by direct mutation detection or by linkage analysis. Additionally a strategy for preimplantation genetic diagnosis of this disorder was also developed. This involved detection of APC mutation in single cells biopsied from human preimplantation embryos at the cleavage stage. After extensive preliminary testing this method reached clinical application, but no pregnancy was achieved on this occassion.