Pharmacogenetics of 6-mercaptopurine and methotrexate in childhood acute lymphoblastic leukemia

Farmakogenomika proučava odnos između genetičkog sklopa individue i njegovog odgovora na lekove i jedan je od stubova personalizovane medicine. Dosadašnji princip lečenja da se standardna doza leka daje svim pacijentima sa istom dijagnozom po unapred utvrđenom protokolu se napušta. Za veliki broj pacijenta ta doza leka često nije efikasna i/ili sigurna za upotrebu. Cilj farmakogenomičkih studija je da identifikuju farmakogenomičke markere, varijacije u genomu koje mogu pouzdano da predvide odgovor na terapiju, što je osnov za individualizaciju terapije. Model sistemi bolesti za analizu farmakogenomičkih markera korišćeni u ovom radu su dečja akutna limfoblastna leukemija (ALL) i reumatoidni artritis (RA). Lečenje ovih bolesti uključuje imunosupresivne i citotoksične lekove 6-merkaptopurin (6-MP), metotreksat (MTX), antibiotik baktrim, antimikotik nistatin, kao i anti-TNF lekove. Genetičke varijacije koje modulišu metaboličke puteve povezane sa ovim lekovima su kandidati za farmakogenomičke markere. Cilj ove studije je da ispita učestalosti genetičkih varijanti u genima TPMT, ITPA, ABCB1, ABCC4, TYMS, MTHFR, SLC19A1, DHFR, TNF i IL-6, kao i da oceni farmakogenomički potencijal ovih varijanti u srpskoj populaciji. Biće ispitana i uloga ovih farmakogenomičkih markera kao faktora rizika za razvoj dečje ALL. Ispitaće se i uticaj terapije održavanja, gde okosnicu terapije čine lekovi 6-MP i MTX, kao i pola i uzrasta dece sa ALL na ekspresiju gena TPMT. Biće funkcionalno okarakterisane varijante u genu TPMT, potencijalni modulatori ekspresije gena TPMT, sa posebnom pažnjom na ulogu VNTR regiona u promotoru gena TPMT. U studiju je bilo uključeno 174 pedijatrijskih ALL pacijenata, 73 RA pacijenata i 104 kontrolnih zdravih ispitanika. Genetičke varijacije u svim gorepomenutim genima su određene metodama baziranim na PCR-u...Pharmacogenomics is focused on exploring the relation between the genomic signature of an individual and their drug response. It is the basis for implementation of personalized medicine. The old-fashioned therapeutic paradigm of »one protocol dose fits all patients with the same diagnosis« is getting abandoned. The standard drug dose is often not efficient and/or safe for many of patients. Pharmacogenomic studies identify pharmacogenomic markers, genomic variations that could reliably predict the drug response, which is the basis for therapy individualization. In order to analyze pharmacogenomic markers, childhood acute lymphoblastic leukemia (ALL) and rheumatoid arthritis (RA) are used as disease model systems. ALL and RA therapy protocols include cytotoxic and immunosuppressive drugs 6-mercaptopurine (6-MP) and methotrexate (MTX), antibiotic bactrim and antimycotic nystatin, as well as anti-TNF drugs. Genetic variations that modulate metabolic pathways related to these drugs are candidate pharmacogenomic markers. The aim of this study is to analyze frequencies of genetic variants in TPMT, ITPA, ABCB1, ABCC4, TYMS, MTHFR, SLC19A1, DHFR, TNF and IL-6 genes in Serbian population and to evaluate the pharmacogenomic potential of these variants. Also, the role of these pharmacogenomic markers as risk factors for development of childhood ALL will be assessed. Influence of the maintenance therapy, which includes 6-MP and MTX as most important drugs, as well as the age and gender of patients will be analyzed in regard to TPMT gene expression. Functional assays will be carried out in order to identify potential modifiers of TPMT expression with a special focus on VNTR region in promoter of TPMT gene. In this study, 174 pediatric ALL patients, 73 RA patients and 104 healthy subjects were enrolled. Genetic variants in above-mentioned genes were detected using PCR-based methodology..

Precision medicine and COVID-19: importance of host genome profiling and bioinformatics

Clinical picture and course of the disease in patients with COVID-19 vary from asymptomatic to lethal. Precision medicine could discover the cause of this phenomenon by analyzing the individual genomic profiles of the patients. We aimed to understand a host genetic component of COVID-19 focusing on variants in genes encoding proteases and genes involved in innate immunity, important for susceptibility and resistance to SARS-CoV-2 infection. Also, we wanted to identify phamracogenes and pharmacogenomics markers associated with drugs used for COVID-19 treatment in different clinical protocols in Serbia, and to compare the results with various world populations. Genotype information of 143 individuals of Serbian origin was extracted from database previously obtained using TruSight One Gene Panel (Illumina). Variants in genes encoding proteases and genes involved in innate immunity were identified and analysed in silico (PolyPhen-2, SIFT, MutPred2, Swiss-Pdb Viewer) to predict the impact of the variants to the structure and/or function of proteins. Genotype data from Serbian population was compared with European and 4 super-populations (total 2504 subjects). Data were extracted from VCF files of Phase 3 variant calls of the 1000 Genomes Project (1kGP) sample collection via Ensembl Data Slicer Tool. The level of population genetic variability at each selected loci was examined using the maximal global differences in minor allele frequencies (delta MAF) calculated by subtracting the maximum and the minimum MAF across analyzed population groups, and Fst statistics. Fisher exact test was used to measure differences in genotypes distributions between Serbian and 1kGP populations, applying Bonferoni correction. R software was utilized for genotype data manipulation and statistical calculations. Based on high alternative allele frequencies in population and the functional effect of the variants, we identified variants in genes encoding proteases and involved in the innate immunity that might be relevant for the host response to SARS-CoV-2 infection. The potential pharmacogenomics markers in pharmacogenes relevant for COVID-19 treatment were also identified. Bioinformatics tools integrated into precision medicine could contribute to better understanding of inter-individual and population-specific genetic susceptibility and resistance to the SARS-CoV-2 infection, therapy response inconsistencies, and could be applied to improve the outcome of the COVID-19 patients.Book of Abstracts: Belgrade BioInformatics Conference 202

Personalized medicine and COVID-19: the importance of genomic host profiling and bioinformatics

Novi koronavirus SARS-CoV-2, uzročnik upale pluća, sposoban je da zarazi ljude i izazove novu bolest COVID- 19 koja je preopteretila zdravstvene sisteme širom planete i izazvala globalnu ekonomsku krizu. Klinička slika i tok bolesti kod pacijenata obolelih od COVID-19 varira od asimptomatske do letalnog ishoda. Kako se radi o istom uzročniku bolesti, individualni genomski profil pacijenta krije odgovor na pitanje medicinske nauke o uzroku ovog fenomena. U radu su sumirana dosadašnja znanja o genetičkim markerima koji su odgovorni za široki spektar kliničkih slika, kao i da li se već može primeniti individualizovan pristup lečenju. Prikazane su dosada istraživane varijante u genima (sa osvrtom na populacione specifičnosti) odgovorne za predispoziciju i odgovor na SARS-CoV-2 virusnu infekciju, farmakogenetičke varijante od značaja za lekove koji se koriste u lečenju pacijenata obolelih od COVID-19, kao i nutrigenetički markeri u genima važnim za metabolizam mikronutrijenata, vitamina D, selena i cinka, koji se takođe koriste u terapiji pacijenata sa COVID-19. Udruženi napor istraživača, multidisciplinarni pristup, dostupnost modernih tehnologija koje imaju kapacitet analize celokupnih genoma, buduće sveobuhvatnije studije sa dobro okarakterisanim grupama pacijenata, kao i razvoj robusnijih bioinformatičkih alata koji koriste mašinsko učenje i napredne statističke metode, omogućiće identifikaciju novih genetičkih markera čoveka povezanih sa COVID-19, bolje razumevanje same patofiziologije bolesti, razvoj prave ciljane terapije kao i istaći značaj nutrigenomike i farmakogenomike za primenu personalizovane medicine u lečenju COVID-19.The new cause of pneumonia, coronavirus SARS-CoV-2, capable of infecting people and causing the new disease COVID-19, overloaded health systems around the planet and caused a global economic crisis. The clinical presentation and the course of the disease in COVID-19 patients vary from asymptomatic to lethal. As it is the same cause of the disease, the individual genomic profile of the patient reveals the answer to the question of medical science about the cause of this phenomenon. The paper summarizes the current knowledge about genetic markers responsible for a wide range of clinical pictures, as well as whether an individualized approach to treatment can already be applied. The variants identified so far in genes (with reference to population specifics) responsible for predisposition and response to SARS-CoV-2 viral infection, pharmacogenetic variants of importance for drugs used in the treatment of patients with COVID-19, as well as nutrigenetic markers in genes important for the metabolism of the micronutrients, vitamin D, selenium and zinc, also used in the therapy of patients with COVID-19, are presented. The combined effort of researchers, a multidisciplinary approach, the availability of modern technologies that have the capacity to analyze entire genomes, future more comprehensive studies with well-characterized patient groups, and the development of more robust bioinformatics tools using machine learning and advanced statistical methods will enable the identification of novel human genetic markers associated with COVID -19, better understanding of the pathophysiology of the disease, development of the proper targeted therapy as well as point out the importance of nutrigenomics and pharmacogenomics for the application of personalized medicine in the treatment of COVID-19

Machine Learning Modeling from Omics Data as Prospective Tool for Improvement of Inflammatory Bowel Disease Diagnosis and Clinical Classifications

Research of inflammatory bowel disease (IBD) has identified numerous molecular players involved in the disease development. Even so, the understanding of IBD is incomplete, while disease treatment is still far from the precision medicine. Reliable diagnostic and prognostic biomarkers in IBD are limited which may reduce efficient therapeutic outcomes. High-throughput technologies and artificial intelligence emerged as powerful tools in search of unrevealed molecular patterns that could give important insights into IBD pathogenesis and help to address unmet clinical needs. Machine learning, a subtype of artificial intelligence, uses complex mathematical algorithms to learn from existing data in order to predict future outcomes. The scientific community has been increasingly employing machine learning for the prediction of IBD outcomes from comprehensive patient data-clinical records, genomic, transcriptomic, proteomic, metagenomic, and other IBD relevant omics data. This review aims to present fundamental principles behind machine learning modeling and its current application in IBD research with the focus on studies that explored genomic and transcriptomic data. We described different strategies used for dealing with omics data and outlined the best-performing methods. Before being translated into clinical settings, the developed machine learning models should be tested in independent prospective studies as well as randomized controlled trials

Pharmacogenomics landscape of COVID-19 therapy response in Serbian population and comparison with worldwide populations

Uvod: Kako ne postoje odobreni terapeutici za lečenje pacijenata sa COVID-19, mogućnost upotrebe postojećih lekova je postala važna. U nedostatku vremena za testiranje farmakogenomskih markera kod pojedinaca, populaciona farmakogenomika bi mogla biti od koristi u predviđanju povećanog rizika za pojavu neželjenih reakcija i neuspeha lečenja kod pacijenata sa COVID-19. Cilj naše studije bio je identifikovanje farmakogena i farmakogenomskih markera povezanih sa lekovima koji se preporučuju za lečenje COVID-19, hlorokin/hidroksihlorokin, azitromicin, lopinavir i ritonavir, u populaciji Srbije i drugim svetskim populacijama. Metode: Podaci o genotipu 143 osobe srpskog porekla dobijeni su iz baze podataka prethodno formirane analizama genoma korišćenjem TruSight One Gene Panel (Illumina). Podaci o genotipu pojedinaca iz različitih svetskih populacija dobijeni su iz Projekta 1000 genoma. Fišerov egzaktni test korišćen je za poređenje učestalosti alela. Rezultati: Identifikovali smo 11 potencijalnih farmakogenomskih markera u 7 farmakogena značajnih za lečenje COVID-19. Na osnovu visoke alterativne učestalosti alela u populaciji Srbije i funkcionalnog efekta varijanti, ABCB1 rs1045642 i rs2032582 mogu biti značajne za smanjeni klirens lekova azitromicina, lopinavira i ritonavira, a varijanta UGT1A7 rs17868323 za hiperbilirubinemiju kod bolesnika sa COVID-19 koji se leče ritonavirom. SLCO1B1 rs4149056 je potencijalni marker odgovora na lopinavir, posebno u populaciji Italije. Naši rezultati potvrdili su da se farmakogenomski profil afričke populacije razlikuje od ostatka sveta. Zaključak: Uzimajući u obzir farmakogenomski profil specifičan za populaciju, preventivno testiranje farmakogena značajnih za lekove koji se koriste u lečenju COVID-19 moglo bi doprineti boljem razumevanju interindividualnih razlika u odgovorima na terapiju i poboljšanju ishoda lečenja pacijenata sa COVID-19.Background: Since there are no certified therapeutics to treat COVID-19 patients, drug repurposing became important. With lack of time to test individual pharmacogenomics markers, population pharmacogenomics could be helpful in predicting a higher risk of developing adverse reactions and treatment failure in COVID-19 patients. Aim of our study was to identify pharmacogenes and pharmacogenomics markers associated with drugs recommended for COVID-19 treatment, chloroquine/hydroxychloroquine, azithromycin, lopinavir and ritonavir, in population of Serbia and other world populations. Methods: Genotype information of 143 individuals of Serbian origin was extracted from database previously obtained using TruSight One Gene Panel (Illumina). Genotype data of individuals from different world populations were extracted from the 1000 Genome Project. Fisher's exact test was used for comparison of allele frequencies. Results: We have identified 11 potential pharmacogenomics markers in 7 pharmacogenes relevant for COVID-19 treatment. Based on high alternative allele frequencies in population and the functional effect of the variants, ABCB1 rs1045642 and rs2032582 could be relevant for reduced clearance of azithromycin, lopinavir and ritonavir drugs and UGT1A7 rs17868323 for hyperbilirubinemia in ritonavir treated COVID-19 patients in Serbian population. SLCO1B1 rs4149056 is a potential marker of lopinavir response, especially in Italian population. Our results confirmed that pharmacogenomics profile of African population is different from the rest of the world. Conclusions: Considering population specific pharmacogenomics landscape, preemptive testing for pharmacogenes relevant for drugs used in COVID-19 treatment could contribute to better understanding of the inconsistency in therapy response and could be applied to improve the outcome of the COVID-19 patients

The in vitro antioxidative and cytotoxic effects of selected Salvia species water extracts

The current paper presents antioxidant and cytotoxic activities and total phenolic and flavonoid content of the selected species of genus Salvia (Lamiaceae) growing wild in Macedonia (S. jurisicii Kosanin, S. amplexicaulis Lam., S. ringens Sibth. & Sm.) and Libya (S. fruticosa Mill. and S. lanigera Poir.). Crude water extracts, obtained from aerial parts, were yielded from 6.50 to 14.32%. Total phenolic content was the highest in water extracts of S. amplexicaulis and S. ringens (226.30 and 189.01 mg GAE/g, respectively), while the flavonoids were the most abundant in S. jurisicii extract (32.36 mg QE/g). Antioxidant activities of extracts were measured using DPPH, ABTS and FRAP assays. S. amplexicaulis and S. ringens extracts showed the strongest antioxidant activity, measured using DPPH (14.21 and 23.44 mu g/mL, respectively) and ABTS assays (2.91 and 2.42 mg AAE/g, respectively). In FRAP assay, S. amplexicaulis and S. fruticosa extracts exhibited strongest activity (1406.73 and 1191.51 mu mol Fe(II)/g). Water extract of S. amplexicaulis and S. ringens performed the strongest cytotoxic activity against K562 cells (151.07 and 173.06 mu g/mL, respectively). Based on these findings, it can be concluded that S. amplexicaulis and S. ringens water extracts could be considered as possible source of antioxidant and cytotoxic agents

Significance of UGT1A1*28 genotype in patients with advanced liver injury caused by chronic hepatitis C

Uvod: Hronični hepatitis C (HHC) je značajan uzročnik morbiditeta i mortaliteta u svetu. Značaj genetskih faktora u patogenezi HHC još uvek nije u potpunosti razjašnjen. Varijacije UGT1A1 gena su najčeša uzrok nasledne nekonjugovane hiperbilirubinemije - Zilberovog sindroma. Ovo je prva studija koja se bavi ispitivanjem učesta I osti TA ponavljanja u promotorskoj regiji UGT1A1 gena i analizom povezanosti UGT1A1*28 genotipa sa stepenom fibroze, viremijom i biohemijskim markerima kod pacijenata sa teškim oštećenjem jetre izazvanim HHC i virusološkim relapsom. Metode: Analizirana su TA ponavljanja u promotorskoj regiji UGT1A1 gena, kod 42 pacijenta sa teškom fibrozom i cirozom izazvanom HHC, koji postigli stabilan virusološki odgovor i 42 ispitanika u kontrolnoj grupi zdravih dobro - voljnih davalaca krvi. Pacijenti sa HHC su dodatno analizirani kliničkim pregledima, laboratorijskim analizama (hematološki, biohemijski i virusološki) i fibroskenom jetre. Rezultati: UGT1A1*28 genotip (7/7 TA ponavljanja) je bio prisutan kod 23.8% pacijenata sa HHC i 16,7% zdravih ispitanika, ali bez statistički značajne razlike (p= 0,49). Nivoi feritina i ukupnog bilirubina su bili u korelaciji sa prisustvom UGT1A1*28 pre primene antivirusne terapije, što sugeriše prediktivnu ulogu ovog genotipa. U ovoj studiji nije bilo korelacije UGT1A1*28 genotipa sa stepenom fibroze i viremijom. Prisustvo UGT1A1*28 genotipa nije uticalo na terapijske prekide i redukcije doze antivirusnih lekova, ishod lečenja niti pojavu kasnog virusološkog res lapsa kod pacijenata sa HHC i teškim oštećenjem jetre. Zaključak: Učestalost UGT1A1*28 genotipa je visoka među srpskim zdravim ispitanicima i pacijentima sa HHC infekcijom. Ispitivani genotip se ne dovodi u vezu sa nes željenim efektima ribavirina niti ima uticaj na ishod lečenja i dugoročnu prognozu pacijenata sa HHC.Background: Chronic hepatitis C (CHC) is a significant cause of liver related morbidity and mortality worldwide. The role of genetics in the host response to hepatitis C virus is not elucidated. Genetic variations in UGT1A1 gene are the most common cause of hereditary unconjugated hyperbilirubinemia-Gilbert syndrome. This is the first study investigating the association of UGT1A1 TA repeats promoter genotypes with the degree of liver injury, viremia and biochemical markers in CHC patients with advanced liver injury and late virological relapse. Methods: Genetic testing of UGT1A1 TA repeats promoter genotypes was performed in 42 CHC patients with advanced fibrosis and cirrhosis who achieved sustained virological response and 42 healthy blood donors. CHC patients were evaluated for clinical findings, laboratory tests and imaging. Results: UGT1A1*28 genotype (7/7 TA repeats) was observed in 23.8% CHC patients and 16.7% healthy controls with no significant difference in genotype frequencies (p= 0.49). Pretreatment levels of ferritin and bilirubin were associated with the presence of U G T1A1*28 genotype, indicating its potential as a predictive marker. However, in our study, there was no correlation of U G T1A 1*28 genotype with the degree of fibrosis or viremia. During antiviral treatment, dose reductions and treatment interruptions, as well as treatment success and occurrence of late virological relapse were not related to the presence of U G T1A 1*28 genotype in CHC patients with severe liver injury. Conclusions: Frequencies of U G T1A 1*28 genotype are high in both Serbian CHC patients and healthy subjects. The presence of U G T1A 1*28 genotype was not associated with ribavirin-related adverse effects and had no effect on long term outcome in CHC patients

Wilms tumor (wt)1 gene expression in children with acute leukemia in Serbia

Acute leukemias constitute the most common malignancy in childhood, accounting for 25-35% of all cancer in children. They are divided into acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Genetic susceptibility is known to play a major role in childhood leukemias. Wilms tumor (WT)1 is a zinc finger transcription factor involved in regulating the process of cell differentiation; it has been implicated in a wide range of human neoplasms. WT1 overexpression in the bone marrow at diagnosis is reported to be an independent negative prognostic factor in adults and children with AML. The aim of the present investigation was to determine the expression of WT1 in the bone marrow of children with AML and ALL in Serbia and its possible impact on patient survival. We determined bone marrow WT1 expression levels by reverse -transcription polymerase chain reaction (RT-PCR) at diagnosis in 20 children with AML and 20 children with ALL (16 B-ALL and 4 T-ALL), as well as 15 age- and sex-matched controls who were evaluated for immune thrombocytopenic purpura (ITP). For children with AML, follow-up samples were also analyzed one month after treatment initiation and at variable later timepoints of control punctures. The results were normalized based on WT1 expression in controls. We found that children with AML had significantly higher WT1 expression at diagnosis (median SD: 139.42 244.03) than those with ALL (1.18 54.37; Mann -Whitney U=82; p lt 0.01) and ITP (0.76 1.01; U=32; p lt 0.01). Patients with T-ALL had higher WT1 expression than those with B-ALL, though significance was not reached due to subgroup size; differences between AML subgroups according to the French-American-British (FAB) classification were also below the level of significance, though a tendency toward higher values in M3 and M4 leukemias was notable. There was also a tendency toward higher values in 14 children with AML who were still alive after a median follow-up of 1.5 years (181.42 192.52) than in 6 who succumbed to the disease (104.29 354.87). All children with AML who had WT1 expression 1 month after diagnosis below the fourth quartile (10 of 10) were still alive, while only 2 of 5 with 1 -month WT1 expression in the fourth quartile survived (Fisher's exact test: p=0.0952). Taken together, our results support a role for WT1 in the diagnostic workup in children with acute leukemia, although it needs to be considered in view of a complex and indvidualized context

Pharmacogenomic Markers of Methotrexate Response in the Consolidation Phase of Pediatric Acute Lymphoblastic Leukemia Treatment

Methotrexate (MTX) is one of the staples of pediatric acute lymphoblastic leukemia (ALL) treatment. MTX targets the folate metabolic pathway (FMP). Abnormal function of the enzymes in FMP, due to genetic aberrations, leads to adverse drug reactions. The aim of this study was to investigate variants in pharmacogenes involved in FMP and their association with MTX pharmacokinetics (MTX elimination profile) and toxicity in the consolidation therapy phase of pediatric ALL patients. Eleven variants in the thymidylate synthetase (TYMS), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), SLC19A1 and SLCO1B genes were analyzed in 148 patients, using PCR- and sequencing-based methodology. For the Serbian and European control groups, data on allele frequency distribution were extracted from in-house and public databases. Our results show that the A allele of SLC19A1 c.80 variant contributes to slow MTX elimination. Additionally, the AA genotype of the same variant is a predictor of MTX-related hepatotoxicity. Patients homozygous for TYMS 6bp deletion were more likely to experience gastrointestinal toxicity. No allele frequency dissimilarity was found for the analyzed variants between Serbian and European populations. Statistical modelling did not show a joint effect of analyzed variants. Our results indicate that SLC19A1 c.80 variant and TYMS 6bp deletion are the most promising pharmacogenomic markers of MTX response in pediatric ALL patients