MULTIPLEX PCR ANALYSIS AND GENOTYPE-PHENOTYPE CORRELATIONS OF FREQUENT APC MUTATIONS

Germline mutations of the adenomatous polyposis coli (APC) gene tend t o cluster in discrete regions. Some of these mutations occur frequentl y in familial adenomatous polyposis coli (FAP) patients, and strategie s for genetic diagnosis of the disease should include simple methods f or their detection, We studied a total of 48 FAP affected or ''at-risk '' members from 31 unrelated FAP pedigrees. Unrelated patients were an alyzed using heteroduplex analysis on agarose minigels (HAAM) and mult iplex allele-specific PCR. This novel strategy readily and reliably de tected the three frequently occurring APC deletions at codons 1061, 10 68, and 1309, allowing identification of mutant alleles in nine unrela ted patients. A targeted mutational analysis, based on HAAM and amplif ication refractory mutation system (ARMS), allowed the rapid identific ation of 11 additional subjects with germline deletions, among relativ es of the patients in whom mutations had been detected by multiplex PC R and HAAM. The use of two independent PCR-based tests, employing dist inct sets of primers, reduces the possibility that artifacts occurring during DNA amplification may interfere with the diagnostic evaluation , The analysis of genotype-phenotype correlations provided evidence fo r heterogeneity with regard to the extent of colonic and extracolonic manifestations of the disease in subjects bearing identical mutations. However, the consistent association of the deletion at codon 1309 wit h more severe colonic disease than that observed in patients with muta tions at codons 1061 and 1068, supports a correlation between mutation site and penetrance of FAP. (C) 1995 Wiley-Liss, Inc.