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Analiza farmakogenetskih dejavnikov pri bolnikih z akutno limfoblastno levkemijo na terapiji s tiopurini
ID Vrevc Žlajpah, Jaka (Author), ID Karas Kuželički, Nataša (Mentor) More about this mentor... This link opens in a new window, ID Šmid, Alenka (Comentor)

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Abstract
Akutna limfoblastna levkemija predstavlja 80 % vseh primerov levkemij otroške populacije. Bolniki med najdaljšo, vzdrževalno fazo zdravljenja prejemajo dnevne odmerke 6-merkaptopurina in tedenske odmerke metotreksata, ki so izračunani glede na telesno površino pacientov in se prilagajajo glede na koncentracije levkocitov ter pojavljanje hudih neželenih učinkov. Posamezniki se na tako predpisano terapijo odzivajo precej raznoliko. Za pojasnitev tovrstne interindividualne variabilnosti v odzivu na tiopurinsko terapijo smo v študiji na slovenski pediatrični populaciji preučevali vpliv potencialnih genetskih in drugih dejavnikov na varnost in učinkovitost vzdrževalne terapije akutne limfoblastne levkemije. Kandidatni geni so bili nosilci zapisa za encime v metabolizmu 6-merkaptopurina, čezmembranskem transportu metotreksata, v folatnem in metioninskem ciklu. Potek vzdrževalne terapije smo ovrednotili z izračunom razmerja med prejetim in izračunanim odmerkom 6-merkaptopurina, tako na dnevni ravni kot na ravni kumulativnih odmerkov med vzdrževalno terapijo. Pred uvedbo Bonferroni korekcije za multiplo testiranje sta bila variantna alela v ABCC2 (rs717620) in MTHFD1 (rs2236225) po dominantnem modelu povezana s prejemanjem višjih odmerkov od izračunanih. ABCC2 (rs717620) je ostal značilen dejavnik poteka tiopurinske terapije tudi po pretvorbi razmerja na ravni kumulativnih odmerkov v kategorično spremenljivko (fenotipsko slabi presnavljalci pod 0,9; med 0,9 in 1,1 fenotipsko normalni presnavljalci ter nad 1,1 fenotipsko hitri presnavljalci). V genu TPMT, ki je glavni farmakogenetski dejavnik tiopurinske terapije, najpogostejših klinično pomembnih alelov TPMT*3C, TPMT*3A in TPMT*2 nismo našli. S sekvenciranjem naslednje generacije smo med 6 različicami, ki so že poročane v farmakogenetski bazi PharmGKB, našli eno sinonimno različico (rs17839843), ki bi lahko bila povezana s povečano encimsko aktivnostjo. Dodatno smo identificirali še 4 intronske različice, ki še niso bile poročane v bazi PharmGKB, in sta jih vsaj 2 napovedna modela v bioinformacijskem orodju PredictSNP označila kot škodljive (rs114611199, rs146407521, rs2518469 in rs1024721139). Nadalje smo prvič v slovenski populaciji bolnikov z akutno limfoblastno levkemijo določali prisotnost različic v genu NUDT15 in določili 4 polimorfizme v odsekih, ki vključujejo ekson 1 in 3 (rs45465203, rs116855232, rs61746486, rs116855232). Po analizi z napovednimi modeli in spletnimi bazami podatkov sta se kot najpomembnejši izkazali intronski različici rs45465203 in drugačnosmiselna različica rs116855232. Frekvenci variantnih alelov rs61746486 in rs116855232 sta za faktor 10 višji od tistih, poročanih v literaturi za splošno evropsko populacijo. Med negenetskimi dejavniki je s potekom vzdrževalne tiopurinske terapije povezana razvrstitev v skupino tveganja, kjer skupini s standardnim in srednjim tveganjem prejmeta odmerke značilno bližje predvidenim kot v skupini z visokim tveganjem. Na 15 preiskovancih smo razvili preliminarni napovedni model za vrednost razmerja med prejetimi in izračunanimi kumulativnimi odmerki 6-merkaptopurina, ki po metodi izbora najprimernejšega modela AIC vsebuje spremenljivke skupina tveganja IR in HR, genotip MTHFD1 (rs2236225) po dominantnem modelu in prilagojeno vsoto variantnih alelov TPMT. Ugotovitve in opažanja v magistrski nalogi so lahko smernice za nadaljnje raziskave na večjem vzorcu bolnikov.

Language:Slovenian
Keywords:akutna limfoblastna levkemija, farmakogenetika tiopurinske terapije, TPMT, NUDT15, ABCC2, MTHFD1
Work type:Master's thesis/paper
Organization:FFA - Faculty of Pharmacy
Year:2023
PID:20.500.12556/RUL-150198 This link opens in a new window
Publication date in RUL:15.09.2023
Views:545
Downloads:5
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Secondary language

Language:English
Title:Analysis of pharmacogenetic factors in patients with acute lypmhoblastic leukemia undergoing thiopurine therapy
Abstract:
Acute lymphoblastic leukemia represents 80 % of all leukemias in the pediatric population. During maintenance treatment, which is the longest treatment phase, patients receive 6-mercaptopurine daily and methotrexate weekly. The doses are calculated based on body surface area and adjusted accordingly to leukocyte levels and occurrence of severe adverse events. However, there is large interindividual variation in the response to the thus prescribed therapy. To explain these interindividual variations we conducted a study on Slovenian pediatric population undergoing thiopurine therapy, investigating genetic factors which might influence the safety profile and efficacy of thiopurine-based treatment. Candidate genes included in the study were selected from those encoding proteins in 6-mercaptopurine metabolism, methotrexate transmembrane transport, folate, and methionine cycles. The course of maintenance therapy was evaluated by calculating the ratio between administered and calculated dose of 6-mercaptopurine, both daily and cumulatively through the course of whole maintenance treatment. Variants in ABCC2 (rs717620) and MTHFD1 (rs2236225, dominant model) were significantly associated with higher doses than calculated, both on daily and cumulative basis (before Bonferroni correction was applied). ABCC2 (rs717620) remained statistically significant after we categorized the cumulative ratio to three distinct groups based on clinical utility of these values (poor metabolizer phenotype bellow 0,9; fast metabolizer phenotype above 1,1: normal metabolizer phenotype between 0,9 and 1,1). We did not detect the most common clinically relevant TPMT variants (TPMT*2, TPMT*3A, TPMT*3C). Using next-generation sequencing, we did however find a synonymous variant that had previously been associated with increased enzymatic activity in the pharmacogenomic PharmGKB database (rs17839843). Additionally, we described 4 intronic variants in the TPMT gene that had previously not been reported in the PharmGKB database and were annotated as deleterious by at least 2 prediction models, included in the bioinformatic prediction tool PredictSNP (rs114611199, rs146407521, rs2518469, rs1024721139). To our knowledge, we were the first to genotype samples from a Slovenian population for the presence of variants in exons 1 and 3 of NUDT15. We detected 4 variants (rs45465203, rs116855232, rs61746486, rs116855232), among which intronic variant rs45465203 and missense variant rs116855232 appear to have the biggest potential to influence enzymatic activity. The frequencies of variant alleles rs61746486 and rs116855232 are 10-times higher than those reported in the literature for the European population, suggesting the need for further research on a larger Slovenian sample. Among non-genetic factors, the categorization into risk groups is associated with the course of maintenance thiopurine therapy, with the standard and medium-risk groups receiving doses closer to the predicted ones compared to the high-risk group. From data of 15 patients, included in TPMT sequencing analysis, we developed a preliminary model for the prediction of ratio between administered and predicted cumulative doses of 6-mercaptopurine during maintenance therapy using AIC model selection method. It includes the following variables: stratification to intermediate and high risk groups, MTHFD1 genotype (dominant model) and the adjusted sum of TPMT variant alleles, which, in spite of its limitations, confirms most of study conclusions. Observations made in this thesis can serve as guidelines for future research on a larger sample.

Keywords:pharmacogenetics of thiopurine-based therapy, acute lymphoblastic leukemia, TPMT, NUDT15, ABCC2, MTHFD1

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