Multiple products in the protein truncation test due to alternative splicing in the adenomatous polyposis coli (APC) gene

Reverse transcription-polymerase chain reaction (RT-PCR)-based analyses of the adenomatous polyposis coli (APC) gene encompassing exons 1-15 revealed a complex pattern of products that were due to alternative splicing of exons 9, 10A and 14. The multiplicity of polypeptide chains obtained from T7-promoter-directed in vitro translation of the RT-PCR product pool was confirmed immunochemically to correspond to the mRNA isoforms, but not to represent products of internal initiation of translation. This observation is of particular relevance for the diagnostic protein truncation test (PTT), since this assay will pick up mRNA variants derived from physiological splice events, e.g., skipping of exons 9, 10A and 14. In vitro-translated proteins of reduced molecular weight were therefore detectable in healthy individuals. We extended this observation to the PTT of cDNA encompassing APC exons 1-14 of familial adenomatous polyposis patients. Knowledge of the normal polypeptide pattern seen in the diagnostic in vitro translation assay allowed us not only to identify translational stop mutations, but even to detect a splice acceptor mutation of exon 14 as a result of quantitative changes of the isoform pattern. Western immune blot analysis on protein extracts of Epstein-Barr virus-immortalized lymphocytes of the same patients revealed that mutations accessible to the RT-PCR PTT yield intracellularly undetectable APC proteins.