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..

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

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

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

Pharmacogenomic and Pharmacotranscriptomic Profiling of Childhood Acute Lymphoblastic Leukemia: Paving the Way to Personalized Treatment

Personalized medicine is focused on research disciplines which contribute to the individualization of therapy, like pharmacogenomics and pharmacotranscriptomics. Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. It is one of the pediatric malignancies with the highest cure rate, but still a lethal outcome due to therapy accounts for 1-3% of deaths. Further improvement of treatment protocols is needed through the implementation of pharmacogenomics and pharmacotranscriptomics. Emerging high-throughput technologies, including microarrays and next-generation sequencing, have provided an enormous amount of molecular data with the potential to be implemented in childhood ALL treatment protocols. In the current review, we summarized the contribution of these novel technologies to the pharmacogenomics and pharmacotranscriptomics of childhood ALL. We have presented data on molecular markers responsible for the efficacy, side effects, and toxicity of the drugs commonly used for childhood ALL treatment, i.e., glucocorticoids, vincristine, asparaginase, anthracyclines, thiopurines, and methotrexate. Big data was generated using high-throughput technologies, but their implementation in clinical practice is poor. Research efforts should be focused on data analysis and designing prediction models using machine learning algorithms. Bioinformatics tools and the implementation of artificial i Lack of association of the CEP72 rs924607 TT genotype with intelligence are expected to open the door wide for personalized medicine in the clinical practice of childhood ALL

The pharmacogenomics of vincristine-induced peripheral neuropathy in pediatric acute lymphoblastic leukemia patients in Serbia: A single center experience

Uvod/Cilj Vinkristin je jedan od ključnih lekova u protokolima lečenja dečje akutne limfoblastne leukemije (ALL). Vinkristin dovodi do destabilizacije mikrotubula, čime se ćelija zaustavlja u metafazi i indukuje apoptoza. Takođe dovodi do degradacije aksona i poremećaja aksonskog transporta, uzrokujući vinkristinom indukovanu perifernu neuropatiju (VIPN). Cilj ove studije bio je da istraži povezanost pet varijanti u farmakogenima uključenim u metabolizam vinkristina kod dece obolele od ALL koja su razvila VIPN, u Srbiji. Takođe, cilj nam je bio da otkrijemo kandidate za nove farmakogenomske markere VIPN-a u srpskoj populaciji. Metode Detekcija varijanti gena CYP3A5, CEP72, ACTG1, MIR3117 i MIR4481 izvedena je metodologijom zasnovanom na PCR-u i sekvenciranju. Statističkim metodama je ispitana njihova asocijacija sa VIPN-om kod 56 pedijatrijskih bolesnika obolelih od ALL. Urađena je i populaciona vinkristin farmakogenomska analiza 17 farmakogena iz postojećih podataka dobijenih sekvenciranjem nove generacije u srpskoj populaciji. Podaci o distribuciji frekvencija alela za evropsko stanovništvo preuzeti su iz javnih baza podataka. Rezultati Tokom lečenja, 17,86% bolesnika je razvilo VIPN. Asocijativne analize pokazale su da nijedna genetička varijanta nije bila povezana sa VIPN-om u našoj studiji. Naše populaciono farmakogenomsko istraživanje nije otkrilo validne farmakovarijante za VIPN. Naši rezultati ne preporučuju preventivno farmakogenetičko ispitivanje vinkristina u Srbiji. Zaključak Potreban je sveobuhvatniji pristup kako bi se identifikovao panel gena kojim bi se mogao objasniti razvoj VIPN-a posle primene vinkristina kod pedijatrijskih bolesnika obolelih od ALL. Bolje osmišljene studije asocijacija na nivou genoma (GWAS) i robusniji alati koji koriste veštačku inteligenciju doveli bi do dizajniranja panela farmakogena za preventivno testiranje predispozicije za razvoj VIPN-a, doprinoseći individualizaciji i unapređenju terapije dece obolele od ALL.Introduction/Objective Vincristine (VCR) is one of the key drugs in current treatment protocols for pediatric acute lymphoblastic leukemia (ALL). By destabilizing microtubules, VCR arrests cells in metaphase, inducing apoptosis of malignant cells. VCR also causes axonal degradation and impairment of axonal transport, which leads to VCR-induced peripheral neuropathy (VIPN). This study aimed to investigate the association of five variants in pharmacogenes involved in VCR metabolism with VIPN in Serbian ALL children. We also wanted to discover candidate pharmacogenomic markers of VIPN in Serbian population. Methods PCR and sequencing-based methodology was used to detect variants in CYP3A5, CEP72, ACTG1, MIR3117, and MIR4481 genes. Statistical analyses were performed for investigating their association with VIPN in 56 pediatric ALL patients. Population VCR pharmacogenomics analysis of 17 pharmacogenes from in-house next-generation sequencing data was also done. Data on allele frequency distribution for the European population were extracted from public databases. Results During the treatment, 17.86% of patients developed VIPN. Association analyses have shown that none of the genetic variants contributed to the occurrence of VIPN in our study. Population pharmacogenomics study did not reveal valid candidate pharmacovariants for VIPN. Our results suggested that pre-emptive pharmacogenetic testing for VCR is not applicable presently. Conclusion More comprehensive approaches are needed to identify the panel of genes that could explain the VIPN development after VCR administration in ALL patients. Utilizing better designed genome-wide association studies and more robust artificial intelligence-based tools would provide a panel of pharmacogenes for pre-emptive tests of VIPN to individualize therapy for ALL in children

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

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 Košanin, 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 μg/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 µmol Fe(II)/g). Water extract of S. amplexicaulis and S. ringens performed the strongest cytotoxic activity against K562 cells (151.07 and 173.06 μg/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