Komponente sistema regulacije ekspresije gena za tiopurin s-metiltransferazu čoveka kao farmakogenetički markeri

Abstract

Tiopurin S-metiltransferaza (TPMT; EC 2.1.1.67) je jedan od najboÿih primera primene farmakogenetike u individualizaciji terapije. TPMT je enzim koji metabolise imunosupersivne tiopurinske lekove (6-MP), koji se koriste u lečenju kancera, autoimunih bolesti i u transpalntacionoj medicini. Aktivnost TPMT enzima je polimorfna i u beloj populacÿi je zabeležena trimodalna distibucija TPMT aktivnosti: postoje jjudi koji su slabi, umereni ili jaki „metilatori". Ipak, ne postoji jasna granica u aktivnosti TPMT izmedu ovih definisanih grupa. Pokazano je da određeni polimorfizmi u genu za TPMT utiču na aktivnost TPMT enzima. Promotor gena za TPMT čoveka je veoma polimorfan. Sadrži promenÿiv broj tandemskih ponovaka, od tri do devet, koji imaju veliki procenat GC baznih parova. Postoje tri tipa ponovaka, A, B i C. Arhitektura ovog promotorskog regionaje uvek AnBmC. U ovom radu smo ispitivali da li genotipizacija polimorfizama u genu za TPMT može biti osnova za modifikaciju tiopurinske terapije u lečenju akutne limfoidne leukemije (ALL) kod dece. Takode smo analizirali distribuciju alela i genotipova VNTR ponovaka promotora gena za TPMT u srpskoj populacÿi. Istraživali smo uticaj broja i tipa tandemskih ponovaka u promotoru na transkripcÿu gena za TPMT čoveka, koristeći eksperimente tranzÿentne transfekcÿe. Ispitivali smo i koji se transkripcioni faktori vezuju za VNTR region esejima usporene elektroforetske pokretÿivosti (EMSA) i „superšift" esejima, analizirali metilacioni status promotora, cis-regulatorne elemente u promotorskom regionu i ispitivali potencijalni uticaj 6-MP na transkripcÿu gena za TPMT. Nasa studÿaje pokazala da je genotipizacÿa TPMT neophodna i efikasna i da je osnova za modifikaciju tiopurinske terapÿe kod ALL pacÿenata, čak i kod heterozigotnih nosilaca polimorfizama u genu za TPMT. Detektovali smo 11 različitih tipova VNTR u promotoru gena za TPMT u srpskoj populacÿi. Broj ponovaka je bio od 4 do 8. Unutar promotora gena za TPMT sa istim brojem ponovaka, detektovana je različita arhitektura VNTR regiona, AnBmC, gde je n = 1-5 i m = 1-6. Detektovali smo 17 različitih TPMT VNTR genotipova u srpskoj populacÿi. Funkcionalni eseji su pokazali da najveću aktivnost TPMT promotora ima onaj sa VNTR*4b tipom (AB2C). Promotori sa 5, 6 i 7 VNTR alela su imali manje aktivnosti. Aktivnost VNTR*8 alela je bila dva

puta veća od aktivnosti VNTR*7 tipova. Utvrdili smo da postoje razlike u aktivnostima promotora između konstrukata koji su imali isti broj ali različiti tip tandemskih ponovaka. Najveća razlika detektovana je između VNTR*4 varijanti (A2BC i AB2C). Takođeje nadeno da je promotorski region uzvodno od VNTR ponovaka, na poziciji od -180 do -130, transkripcioni aktivator. Potvrdili smo da se za VNTR region direktno vezuju transkripcioni faktori Spl i Sp3. Promotor gena za TPMTje specifično odgovorio na tretman ćelija dozom od 10 pM 6-MP, i to na VNTR-zavistan način, smanjenjem ekspresije reporterskog gena za 40-50%. U ovaj odgovor uključeni su transkripcioni faktori Sp3 i KLF1. Pokazali smo da broj i tip VNTR ponovaka u promotoru gena za TPMT utiče na nivo TPMT transkripcije. Arhitektura VNTR regiona (konfiguracÿa i broj nukleotida izmedu A i C ponovaka) moduliše transkripciju gena za TPMT. VNTR region može biti odgovoran za raznolikost u aktivnosti TPMT enzima unutar tri definisane grupe metilatora. Naši rezultati pokazuju da su VNTR genotipovi koji doprinose TPMT fenotipu slabih „metilatora" zastupljeni u jednoj trećini srpske populacije. Zbog toga bi tandemski ponovci u promotoru gena za TPMT mogli biti kandidat za farmakogenetički marker. Dalja istraživanja bi potvrdila da li je ovaj region od kliničkog značaja za individuaiizaciju tiopurinske terapije.

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) represents one of the examples of pharmacogenetics applied for individualizing drug therapy. It is an enzyme that metabolizes immunosuppressive thiopurine medications, used in treatment of autoimmune diseases, cancer and in transplantation medicine. TPMT activity is polymorphic and trimodal distribution has been demonstrated in Caucasians, namely low, intermediate and high methylator groups. However, there is no clear separation between these patient groups. It was shown that certain TPMT gene polymorphisms affect TPMT enzyme activity. Human TPMT gene promoter is highly polymorphic. It contains a variable number of 3 GC-rich tandem repeats, namely A, B and C, ranging from 3 to 9 in length, but maintaining a AnBmC architecture. Here, we investigated if the TPMT genotypization could be an efficient tool for guiding of thiopurine therapy in acute lymphoblastic leukemia (ALL) patients, carriers of TPMT polymorphism. We also analyzed the promoter tandem repeat distribution of alleles and genotypes in Serbian population. We investigated the influence of number and type of promoter tandem repeats on transcription of human TPMT gene using transient transfection experiments, addressed the interaction of transcription factor binding to the

VNTRs by EMSA and supershift assays, analyzed the méthylation status of VNTR region, cis- regulatory elements in promoter region and investigated potential influence of 6-MP on

TPMT gene transcription. Our study has confirmed that TPMT genotyping is necessary and efficient for modification of thiopurine therapy in ALL patients, even if they are heterozygous carriers of TPMT polymorphism. We have detected 11 different types of VNTRs in the TPMT gene promoter in the Serbian population. Number of repeats ranged from 4 to 8. Within the TPMT promoters containing the same number of tandem repeats, different architecture of VNTRs has been determined, namely AnBmC, where n ranged from 1-5 and m ranged from 1-6. We have determined 17 different TPMT VNTR genotypes in Serbian population. Functional assays revealed that TPMT promoter with the highest activity was the one with VNTRMb type (AB2C). Promoters

with 5, 6 and 7 VNTR alleles all had successively lower activities, VNTR*8 activity was two times higher than activity of VNTR*7 types. We found differences in activity between the constructs containing the same number, but different type of tandem repeats. The most prominent difference was observed between VNTR*4 variants (A2BC and AB2C). The results of functional analysis demonstrate that the activity of TPMT promoter depends not only on the overall number of tandem repeats but also on the type of the repeat. The 5' TPMT promoter region from -180 to -130 is a transcriptional activator. We have confirmed that transcription factors Sp1 and Sp3 directly bind to VNTR region. TPMT gene promoter demonstrated a specific response to 6-MP treatment (10 pM) of the cells in a VNTR-specific manner, namely reduction of reporter gene expression from 40-50%. Transcription factors Sp3 and KLF1 are involved in this response. We have shown that number and type of VNTRs in the TPMT gene promoter determine level of TPMT transcription. VNTR architecture (distance and configuration from A to C repeats) probably spatially and in sequence specific manner, modulate TPMT transcription. VNTR region may be responsible for diversity in TPMT activity among defined groups of TPMT methylators. Our results showed that VNTR genotypes that contributed to low- methylator TPMT phenotype, comprised one third of Serbian population. For that reason promoter tandem repeats could be considered as candidate pharmacogenetic marker. Further investigation will confirm if it could be of clinical importance for individualizing thiopurine therapy.