Vpliv genetskih polimorfizmov rs114611199, rs17839843 in rs2518469 na aktivnost tiopurin S-metiltransferaze

Klemen, Jaka (avtor), Šmid, Alenka (mentor), Urbančič, Dunja (komentor)

Tiopurin S-metiltransferaza (TPMT) je danes klinično dobro preiskan encim, saj je v telesu odgovoren za inaktivacijo protitumornih in imunosupresivnih učinkovin, imenovanih tiopurini. Ker za tiopurine velja relativno ozko terapevtsko okno, za TPMT pa precejšnje populacijske razlike v aktivnosti, postaja vse bolj pomembno preučevanje vpliva genetskih polimorfizmov v genu TPMT na encimsko aktivnost. Klinično so dobro raziskani polimorfizmi družine TPMT*3 – TPMT*3C in TPMT*3B. Ker pa ti ne razložijo vseh odstopanj od normalne aktivnosti encima, danes iščejo nove genetske dejavnike, ki bi lahko vplivali na terapijo s tiopurini. V predhodnih študijah so identificirali potencialne genetske polimorfizme, rs114611199, rs17839843 in rs2518469, ki bi lahko povzročili spremenjeno aktivnost TPMT. V sklopu magistrske naloge smo na vzorcih 161 zdravih prostovoljcev slovenske populacije želeli proučiti pogostost prisotnosti posameznih polimorfizmov. Iz vzorcev krvi smo izolirali DNA, jih ustrezno redčili in genotipizirali z uporabo hidrolizirajočih sond. Tako smo pridobili podatke o frekvencah posameznih variantnih alelov in genotipov v slovenski populaciji ter jih primerjali z evropskimi in svetovnimi populacijami ter ugotovili odstopanja pri frekvenci variantnih alelov za rs114611199, rs17839843 in rs2518469. V vzorcih hemolizatov istih preiskovancev smo zatem določili aktivnost encima TPMT preko nastajanja produkta 6-metilmerkaptopurina z metodo tekočinske kromatografije visoke ločljivosti (HPLC). Ugotovili smo, da je razpon vrednosti aktivnosti TPMT v slovenski populaciji primerljiv z drugimi študijami, porazdelitev aktivnosti TPMT pa je kot pričakovano bimodalna. Preko primerjave aktivnosti TPMT v vzorcih s prisotnimi polimorfizmi TPMT*3 smo potrdili povezavo med genotipom teh najbolj raziskanih polimorfizmov in aktivnostjo encima. Na koncu smo ovrednotili povezavo treh slabše raziskanih polimorfizmov rs114611199, rs17839843 in rs2518469 z encimsko aktivnostjo TPMT, pri čimer smo pokazali, da imajo posamezniki, ki so variantni homozigoti za polimorfizem rs2518469 statistično pomembno znižano aktivnost TPMT. Za preostala dva polimorfizma statistično značilnih povezav z aktivnostjo TPMT nismo uspeli potrditi. Naše ugotovitve odpirajo vrata za nadaljnje raziskave tako iz znanstvenega kot kliničnega vidika.

• Jezik: Slovenski jezik
• Ključne besede: tiopurin S-metiltransferaza (TPMT), 6-merkaptopurin, polimorfizmi TPMT, aktivnost TPMT
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-165905
• Datum objave v RUL: 13.12.2024

KLEMEN, Jaka, 2024, Vpliv genetskih polimorfizmov rs114611199, rs17839843 in rs2518469 na aktivnost tiopurin S-metiltransferaze [na spletu]. Magistrsko delo. [Dostopano 21 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=165905

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The influence of rs114611199, rs17839843 and rs2518469 genetic polymorphisms on the thiopurine S-methyltransferase activity

Izvleček

Thiopurine S-methyltransferase (TPMT), a well-studied enzyme in clinically setting, is responsible for inactivation of anti-tumor and imunosuppresive group of drugs, called thiopurines. Due to the narrow therapeutic window of thiopurines and significant population differences in TPMT activity, studying genetic polymorphisms in the TPMT gene is increasingly important. Clinically significant polymorphisms include TPMT*3C and TPMT*3B. However, these do not account for all variations that affect enzyme activity, provoking the search for new genetic factors that could affect thiopurine therapy. Previously, new genetic polymorphisms, including rs114611199, rs17839843, and rs2518469, were identified that could alter TPMT activity. In this study, we analyzed the frequency of these polymorphisms in 161 healthy Slovenian donnors. DNA was isolated from blood samples, diluted, and genotyped using hydrolyzing probes. We compared the allele and genotype frequencies with European and global populations, finding deviations in the allele frequencies of rs114611199 and rs17839843 in Slovenian population. Next, we measured TPMT enzyme activity via production of 6-methylmercaptopurine in hemolysates from the same individuals using high-performance liquid chromatography (HPLC). Results indicated that TPMT activity levels in the Slovenian population were comparable to other studies and they were showing the expected bimodal distribution. We confirmed the correlation between TPMT*3 genotypes and enzyme activity. Additionally, we evaluated the connection of the less-studied polymorphisms rs114611199, rs17839843, and rs2518469 with TPMT activity. We found that individuals who are variant homozygotes for rs2518469 have significantly reduced TPMT activity. No statistically significant correlations were found for the other two polymorphisms. Our findings pave the way for further research from both scientific and clinical perspectives.

• Ključne besede: thiopurine S-methyltransferase polymorphisms, TPMT activity