INDIVIDUALIZED THERAPY: ROLE OF THIOPURINE S-METHYLTRANSFERASE PROTEIN AND GENETIC VARIANTS INDIVIDUALIZOVANA TERAPIJA: ULOGA PROTEINSKIH I GENETSKIH VARIJANTI TIOPURIN S-METILTRANSFERAZE

Kratak sadr`aj: Tiopurin S-metiltransferaza (TPMT: EC 2.1.1.67) jeste enzim koji metaboli{e imunosu presivne tiopurin ske lekove, koji se koriste za le~enje autoimunih bolesti, malignih oboljenja i u transplantacionoj medicini. Aktivnost enzima TPMT kod pojedinih ljudi je izrazito smanjena ili pove}ana u odnosu na normalni nivo aktivnosti. Istra`ivanja strukture i biohemijskih karakteristika proteina TPMT su uka zala na postojanje odre|enih proteinskih varijanti koje imaju izmenjenu aktivnost. Otkriveni su polimorfizmi u genu za TPMT koji daju razli~ite TPMT alozime. Smanjenoj aktivnosti enzima mo`e doprineti i manja koli~ina sintetisanog proteina, {to zavisi i od transkripcione aktivnosti promotora gena za TPMT. Polimorfizmi u samom promotoru, kao {to je pro menjiv broj tandemskih ponovaka (VNTR), mogu da moduli{u transkripciju. Primena tiopurin skih lekova kod pacije nata sa odre|enim genetskim varijan tama TPMT iza ziva te{ku hematolo{ku toksi~nost. Da bi se toksi~nost izbegla, terapija se modifikuje u skladu sa geno ti pom TPMT (farma kogenetika). Mi smo izu~avali polimor fizme u egzonima i re gu latornim elementima (promotor) gena za TPMT koji do vode do promene aktivnosti enzima TPMT u srpskoj po pu laciji. Koristili smo metodologiju baziranu na PCR i DNK sekvenciranje za detekciju genetskih varijanti TPMT. Pokazali smo da su u na{oj populaciji prisutne genetske varijante u egzonima koje ukupno daju 7,5% varijantnih alozima TPMT koji imaju smanjenu enzimsku aktivnost. Terapija za pacijente koji imaju ove farmako gene ti~ke markere je modifikovana, {to je doprinelo uspe{nijem le~enju. Funkcionalnim esejima in vitro smo pokazali da aktivnost promotora gena za TPMT, a samim tim i koli~ina sintetisanog enzima TPMT, zavisi od arhitekture (broja i tipa) VNTR u promotoru. Promotor gena za TPMT specifi~no Summary: Thiopurine S-methyltransferase (TPMT: EC 2.1.1.67) is an enzyme that metabolizes immunosuppressive thiopurine medications, used in the treatment of autoimmune diseases, cancer and in transplantation medicine. In some individuals, TPMT enzyme activity is significantly increased or decreased compared to the normal TPMT activity level. Struc tural and biochemical analyses of the TPMT protein revealed the existence of certain protein variants with altered activity. It has been shown that certain TPMT gene polymorphisms exist, that define different TPMT allozymes. Decreased TPMT enzyme activity can also be a consequence of lower protein synthesis, which depends on the promoter transcription activity. Promoter polymorphisms, such as variable number of tandem repeats (VNTR), can modulate the transcription. Admini ste ring thiopurine drugs in patients with certain genetic TPMT variants leads to severe hematologic toxicity. To avoid toxicity, therapy is being modified according to the TPMT genotype (pharma co ge netics). We investigated the polymor p hisms in exons and regu la tory elements (promoter) of the TPMT gene which affect TPMT enzyme activity in the Serbian popu lation. We used PCR-based methodology and sequen cing in the detection of genetic variants on TPMT gene. We showed that genetic variants in exons account for 7.5% of all TPMT variants with decreased enzyme activity. The therapy for patients with these pharmacogenetic markers was modified, which contributed to the efficiency of treatment. Functional assays in vitro showed that the TPMT promoter activity and, therefore, the quantity of TPMT protein synthesized, depended on the architecture of VNTRs (i.e. number and type) in the promoter. Promoter of the TPMT gene specifically responds to mercapto purine treatment of K562 cells in a VNTR-dependent man ner. Study of DNA-protein interactions