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 analyse s of the adenomatous polyposis coli (APC) gene encompassing exons 1-15 revealed a complex pattern of products that were due to alternative s plicing 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 tra nslation. This observation is of particular relevance for the diagnost ic protein truncation test (PTT), since this assay will pick up mRNA v ariants derived from physiological splice events, e.g., skipping of ex ons 9, 10A and 14. In vitro-translated proteins of reduced molecular w eight were therefore detectable in healthy individuals. We extended th is observation to the PTT of cDNA encompassing APC exons 1-14 of famil ial adenomatous polyposis patients. Knowledge of the normal polypeptid e 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 cha nges of the isoform pattern. Western immune blot analysis on protein e xtracts of Epstein-Barr virus-immortalized lymphocytes of the same pat ients revealed that mutations accessible to the RT-PCR PTT yield intra cellularly undetectable APC proteins.