[Implementation of direct sequencing as a method of ABL gene mutations analysis in patients with chronic myeloid leukemia treated with tyrosine kinase inhibitor]. / Opracowanie i wdrozenie bezposredniego sekwencjonowania jako metody analizy mutacji genu ABL u chorych na przewlekla bialaczke szpikowa oporna na leczenie inhibitorami kinaz tyrozynowych.

ABSTRACT

Chronic Myeloid Leukemia (CML), belonging to mieloproliferative syndromes, is one of the myeloproliferative clonal hyperplasia. It is caused by the Philadelphia chromosome resulting from the reciprocal translocation, t(9;22) between the long arms of chromosomes 9 and 22. This results in the production of fusion BCR-ABL transcript and chimeric protein--tyrosine kinase activity. This protein leads to increased proliferation, resistance to apoptosis, and worse adhesion of CML cells. Molecular analysis are very important in the era treatment of CML by tyrosine kinase inhibitors (TKI). Constant monitoring of the level of BCR-ABL transcript aimed at monitoring response to medical treatment as well as early detection of resistance to TKI therapy. The most common causes of resistance are point mutations ABL kinase domain of the BCR-ABL gene. In this aim, the biological material used (peripheral blood) derived from 58 patients of the Department of Hematology, Jagiellonian University Collegium Medicum. The isolated RNA was performed in successive stages RT-PCR, RQ-PCR to a semi-nested PCR. In order to detect point mutations ABL kinase domain technique used direct sequencing of the product obtained in response to a semi-nested PCR. Using this technique allow in do not only a rapid detection of point mutations but also identification of its position in the ABL domain, type of mutation (e.g., T3151), as well as nucleotide and the amino acid substitution. The most common point mutations detected were T3151 and M244V.