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Analysis of APC gene mutations on Familial Adenomatous Polyposis by primer extension preamplification of microsamples of fixed paraffin-embedded tissue

The purpose of our study was to determine the usefulness of primer extension preamplification (PEP) method in analysis of APC gene mutations in FAP patients. We analyzed polyps and peripheral blood from two patients with FAP. They had the germline 5 bp deletion at the codon 1309 of the APC gene. We assumed that at least some parts some parts of polyps in two patients had the "second hit" at APC gene and other early genetic changes. The inability to detect them in DNA isolated from the whole polyp could be explained by presence of normal tissue within polyps or heterogenous genetic changes within the epithelium of a polyp. We analysed microdissections of deparaffinized tissue for mutations and LOH at the APC gene. In order to increase the quantity of target DNA available from small samples, each microdissection was first preamplified by using PEP. PCR products were analysed by RFLP method and by electrophoresis on Spreadex EL 300 gels. By using microdissection and PEP-PCR we were able to prove germline, "second hit" mutation and LOH of the APC gene. In order to confirm the accurancy of PEP method, three different pairs of primers for exon 15 of the APC gene were used – the same results were obtained. Our study showed that PEP method provided more accurate insight into different genetic changes of FAP.

FAP; PEP; APC gene mutations

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