Acute lymphoblastic leukaemia is the most common childhood cancer. Cytostatic agents are used in the maintenance phase of the treatment of acute lymphoblastic leukaemia, most commonly thiopurine, 6-mercaptopurine. Its metabolism is complex and involves multiple pathways of activation and deactivation. Two of the most important enzymes in its metabolic pathway are thiopurine-S-methyltransferase and inosine-5'-monophosphate dehydrogenase (IMPDH), which is one of the last enzymes in the cascade of its metabolism, leading to the formation of the active metabolites of 6-thioguanosins. Two isoenzymes, IMPDH1 and IMPDH2, have been identified in humans. Changes in their activity could lead to altered formation of the active metabolites of thiopurines, resulting in increased toxicity or reduced efficacy of thiopurine therapy. Therefore, we wanted to test whether there are genetic polymorphisms in IMPDH that have an impact on the sensitivity of lymphoblastoid cell lines and on thiopurine treatment in individuals with acute leukaemia. In a 6-mercaptopurine resistant cell line, exonic regions in IMPDH were amplified by polymerase chain reaction and subsequently sequenced. Polymorphisms rs558132944 and rs2288550 were identified in this cell line. Using pharmacogenomic databases, we then investigated and found three more polymorphisms that affect either IMPDH activity or the outcome of thiopurine therapy: rs11706052, rs2278293 and rs121912550. All five selected polymorphisms were determined by the high-resolution melting curve method in 33 lymphoblastoid cell lines and 38 patients with acute lymphoblastic leukaemia. The genotype frequency of individuals was compared with the genotype frequency in the general population and comparable results were obtained. We further tested whether genetic polymorphisms in IMPDH affect the sensitivity of lymphoblastoid cell lines and the production of 6-mercaptopurine metabolites in cells. From a previous Master's thesis, we obtained data on the sensitivity of the cells measured by the MTS assay and data on the amount of metabolites formed in the cell and the medium after exposure to 6-mercaptopurine measured by HPLC. We found that variant homozygotes for the polymorphism rs2278293 had lower sensitivity to thiopurines. The polymorphism rs2278293 produced more inactive metabolites and at the same time fewer active metabolites, so the IMPDH enzyme might be less active in these individuals. However, with the polymorphism rs2288550, we found that they have a lower formation of inactive metabolites and a higher formation of active metabolites, so these individuals could have an increased activity of the IMPDH enzyme. Polymorphisms rs2278293 and rs2288550 in the IMPDH indicate a trend towards an effect on the dose size required to achieve a therapeutic effect in patients with acute leukaemia.
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