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Proučevanje vloge polimorfizmov v genu za inozin-5’-monofosfat dehidrogenazo pri odzivu na tiopurine
ID Hlupić, Ines (Author), ID Mlinarič-Raščan, Irena (Mentor) More about this mentor... This link opens in a new window, ID Urbančič, Dunja (Comentor)

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Abstract
Akutna limfoblastna levkemija je najpogostejše rakavo obolenje pri otrocih. V vzdrževalni fazi zdravljenja akutne limfoblastne levkemije se uporabljajo citostatične zdravilne učinkovine tiopurini, med njimi v največji meri 6-merkaptopurin. Njegov metabolizem je kompleksen in zajema več poti aktivacije in deaktivacije. Dva izmed pomembnejših encimov v njegovi metabolni poti sta tiopurin-S-metiltransferaza in inozin-5'-monofosfat dehidrogenaza (IMPDH). Slednji je predstavnik encimov v kaskadni reakciji nastanka aktivnih metabolitov 6-tiogvanozinov. Pri ljudeh sta identificirana dva izoencima, IMPDH1 in IMPDH2. Spremembe v njuni aktivnosti bi lahko vodile do spremenjenega nastanka aktivnih metabolitov tiopurinov in posledično do večje toksičnosti ali zmanjšane učinkovitosti zdravljenja s tiopurini. Zato smo želeli preveriti, ali obstajajo genetski polimorfizmi v IMPDH, ki imajo vpliv na občutljivost limfoblastoidnih celičnih linij in na zdravljenje posameznikov z akutno limfoblastno levkemijo s tiopurini. Pri celični liniji, rezistentni na 6-merkaptopurin, smo pomnožili eksonske regije v IMPDH s pomočjo verižne reakcije s polimerazo ter nato le-te sekvencirali. V tej celični liniji smo identificirali polimorfizma rs558132944 in rs2288550. S pomočjo farmakogenomskih baz podatkov smo nato preiskali in našli še tri polimorfizme, ki vplivajo bodisi na aktivnost IMPDH ali na izid terapije s tiopurini: rs11706052, rs2278293 in rs121912550. Vseh pet izbranih polimorfizmov smo določili z metodo talilne krivulje visoke ločljivosti v 33 limfoblastoidnih celičnih linijah in 38 pacientih z akutno limfoblastno levkemijo. Frekvenco genotipov posameznikov smo primerjali s frekvenco genotipov v splošni populaciji in dobili primerljive rezultate. Nadalje smo preverili, ali genetski polimorfizmi v IMPDH vplivajo na občutljivost limfoblastoidnih celičnih linij in nastanek metabolitov 6-merkaptopurina v celicah. Iz predhodne magistrske naloge smo pridobili podatke o občutljivosti celic, ki je bila izmerjena s testom MTS, ter podatke o količini nastalih metabolitov v celici in mediju po izpostavitvi 6-merkaptopurinu, izmerjenih z metodo HPLC. Ugotovili smo, da so variantni homozigoti pri polimorfizmu rs2278293 imeli manjšo občutljivost na tiopurine. Pri polimorfizmu rs2278293 je nastalo več neaktivnih metabolitov in hkrati manj aktivnih metabolitov, torej bi encim IMPDH pri teh posameznikih lahko bil manj aktiven. Pri polimorfizmu rs2288550 pa smo ugotovili, da imajo manjši nastanek neaktivnih metabolitov in hkrati večji nastanek aktivnih, tako bi lahko ti posamezniki imeli povečano aktivnost encima IMPDH. Pri polimorfizmih rs2288550 in rs2278293 smo opazili trend, da vplivata na velikost odmerka, ki je potreben za doseganje terapevtskega učinka pri bolnikih z akutno limfoblastno levkemijo.

Language:Slovenian
Keywords:Akutna limfoblastna levkemija, tiopurini, inozin-5'-monofosfat dehidrogenaza, genetski polimorfizmi, verižna reakcija s polimerazo.
Work type:Master's thesis/paper
Organization:FFA - Faculty of Pharmacy
Year:2023
PID:20.500.12556/RUL-152290 This link opens in a new window
Publication date in RUL:17.11.2023
Views:1023
Downloads:121
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Secondary language

Language:English
Title:Studying the role of polymorphisms in inosine-5’-monophosphate dehydrogenase gene on thiopurine response
Abstract:
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.

Keywords:Acute lymphoblastic leukemia, thiopurines, genetic polymorphism, inosine-5’-monophosphate dehydrogenase, the polymerase chain reaction.

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