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Multiplex PCR analysis and genotype–phenotype correlations of frequent APC mutations
Author(s) -
Cama Alessandro,
Palmirotta Raffaele,
Curia Maria Cristina,
Esposito Diana L.,
Ranieri Annalisa,
Ficari Ferdinando,
Valanzano Rosa,
Battista Pasquale,
Modesti Andrea,
Tonelli Francesco,
MarianiCostantini Renato
Publication year - 1995
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.1380050208
Subject(s) - biology , genetics , phenotype , genotype , multiplex polymerase chain reaction , genotype phenotype distinction , multiplex , gene , polymerase chain reaction
Germline mutations of the adenomatous polyposis coli (APC) gene tend to cluster in discrete regions, Some of these mutations occur frequently in familial adenomatous polyposis coli (FAP) patients, and strategies for genetic diagnosis of the disease should include simple methods for their detection. We studied a total of 48 FAP‐affected or “at‐risk” members from 31 unrelated FAP pedigrees. Unrelated patients were analyzed using heteroduplex analysis on agarose minigels (HAAM) and multiplex allele‐specific PCR. This novel strategy readily and reliably detected the three frequently occurring APC deletions at codons 1061, 1068, and 1309, allowing identification of mutant alleles in nine unrelated patients. A targeted mutational analysis, based on HAAM and amplification refractory mutation system (ARMS), allowed the rapid identification of 11 additional subjects with germline deletions, among relatives of the patients in whom mutations had been detected by multiplex PCR and HAAM. The use of two independent PCR‐based tests, employing distinct 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 for 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 with more severe colonic disease than that observed in patients with mutations at codons 1061 and 1068, supports a correlation between mutation site and penetrance of FAP. © Wiley‐Liss, Inc.