Determination of Rare Genomic Variants Leading to Hematological Malignancies Using Next-Generation Sequencing

Rare hematological malignancies are a heterogeneous group of the disease. Malignant transformation of a hematopoietic stem cell is a result of gradual accumulation of mutations in a number of genes involved in basic cellular processes. One of the main goals in the study of hematological malignancies is the definition of genomic markers crucial for the development of the disease, as well as for recognition of multiple entities characterized by distinct prognosis and outcome of the disease. Application of new highthroughput technologies has enabled better insight into genomic landscape of hematological malignancies. The most important achievement of genome-based medicine is more precise classification of patients with hematological malignancies, based on newly discovered molecular markers, and molecular–targeted therapy, tailored to genomic profile of a disease. In our studies we applied amplicon based next generation sequencing (NGS) approach, using TruSeq Amplicon Cancer Panel (Illumina, Inc) for analysis of 48 cancer-related genes in primary diffuse large B-cell lymphoma of central nervous system (DLBCL CNS), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Our findings strongly suggested that the TP53 and ATM genes were involved in the molecular pathophysiology of primary DLBCL CNS. Mutations in the PTEN and SMO genes affect survival of the patients. Additionally, we found that AML and ALL contain small number of genetic alterations, contrary to lymphomas. While protein-changing variants were found in tyrosine kinase genes, genes encoding tyrosine kinase associated proteins (JAK3, ABL1, GNAQ, and EGFR) and in the methylation and histone modifying genes (IDH1, IDH2, and SMARCB1) in patients with AML, the mutations detected in ALL patients are related to key signaling pathways, primarily on Ras/RTK cascade. Application of next-generation sequencing technology resulted in the information about genetic profile of each patient. Individual genetic profiling leads to highly specific personalized therapy of hematological malignancies

Individualized therapy: Role of thiopurine S-methyltransferase protein and genetic variants

Tiopurin S-metiltransferaza (TPMT: EC 2.1.1.67) jeste enzim koji metaboliše imunosupresivne tiopurinske lekove, koji se koriste za lečenje autoimunih bolesti, malignih oboljenja i u transplantacionoj medicini. Aktivnost enzima TPMT kod pojedinih ljudi je izrazito smanjena ili povećana u odnosu na normalni nivo aktivnosti. Istraživanja strukture i biohemijskih karakteristika proteina TPMT su ukazala na postojanje određenih proteinskih varijanti koje imaju izmenjenu aktivnost. Otkriveni su polimorfizmi u genu za TPMT koji daju različite TPMT alozime. Smanjenoj aktivnosti enzima može doprineti i manja količina sintetisanog proteina, što zavisi i od transkripcione aktivnosti promotora gena za TPMT. Polimorfizmi u samom promotoru, kao što je promenjiv broj tandemskih ponovaka (VNTR), mogu da modulišu transkripciju. Primena tiopurinskih lekova kod pacijenata sa određenim genetskim varijantama TPMT izaziva tešku hematološku toksičnost. Da bi se toksičnost izbegla, terapija se modifikuje u skladu sa genotipom TPMT (farmakogenetika). Mi smo izučavali polimorfizme u egzonima i regulatornim elementima (promotor) gena za TPMT koji dovode do promene aktivnosti enzima TPMT u srpskoj populaciji. Koristili smo metodologiju baziranu na PCR i DNK sekvenciranje za detekciju genetskih varijanti TPMT. Pokazali smo da su u našoj 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 farmakogenetičke markere je modifikovana, što je doprinelo uspešnijem lečenju. Funkcionalnim esejima in vitro smo pokazali da aktivnost promotora gena za TPMT, a samim tim i količina sintetisanog enzima TPMT, zavisi od arhitekture (broja i tipa) VNTR u promotoru. Promotor gena za TPMT specifično odgovara na tretman ćelija K562 tiopurinom zavisno od tipa VNTR. Izučavanje interakcija DNK i proteina je otkrilo da transkripcioni faktori Sp1 i Sp3 interaguju sa VNTR. Naša istraživanja ukazuju na to da bi region VNTR u promotoru gena za TPMT mogao postati novi farmakogenetički marker od kliničkog značaja za individualizaciju tiopurinske terapije.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. Structural 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. Administering 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 (pharmacogenetics). We investigated the polymorphisms in exons and regulatory elements (promoter) of the TPMT gene which affect TPMT enzyme activity in the Serbian population. We used PCR-based methodology and sequencing 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 mercaptopurine treatment of K562 cells in a VNTR-dependent manner. Study of DNA-protein interactions revealed that Sp1 and Sp3 transcription factors interact with VNTRs. Our research pointed out that the VNTR promoter region of the TPMT gene could become a new pharmacogenetic marker, clinically significant for the individualization of thiopurine therapy

Pharmacogenomics and pharmacotranscriptomics of acute leukemia in children: a path to personalized medicine

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 implementation of pharmacogenomics and pharmacotranscriptomics. Emerging high-throughput technologies, microarrays and next-generation sequencing, have provided an enormous amount of molecular data with potential to be implemented in childhood ALL treatment protocols. In the current review, we summarized the contribution of these novel technologies to pharmacogenomics and pharmacotranscriptomics of childhood ALL. We have presented data on molecular markers responsible for efficacy, side effects and toxicity of the drugs commonly used for childhood ALL treatment, i.e., glucocorticoid drugs, vincristine, asparaginase, anthracyclines, thiopurines and methotrexate. Big data was generated using high-throughput technologies, but their implementation in clinical practice is poor. Research efforts have to be focused on data analysis and designing prediction model using machine learning algorithms. Bioinformatics tools and implementation of artificial intelligence are expected to open the door wide for personalized medicine in clinical practice of childhood ALL.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202

Precision medicine and COVID-19: Importance of host genome profiling

Introduction: The clinical picture and the course of the disease in COVID-19 patients, caused by coronavirus SARS-CoV-2, vary from asymptomatic to fatal outcome. As the same agent cause the disease, the individual genomic profile of the patient could contribute to better understanding of this phenomenon. The current knowledge about genetic markers responsible for a wide range of clinical pictures, as well as possible application of individualized treatment, will be presented. Methods: Variantsin genesresponsible for predisposition and response to SARS-CoV-2 infection, pharmacogenetic variantsrelated to drugs used in the treatment of COVID-19, nutrigenetic markersin genes relevant for the metabolism of the micronutrients(vitamin D,selenium and zinc) were investigated using GWAS, PCR and sequencing. Genotype data were extracted from database previously obtained using TruSight One Gene Panel (Illumina). Results: Eleven pharmacogenomics markers in 7 pharmacogenes relevant for COVID-19 treatment and 10 variants affecting the structure and/or function of proteinsimportant forsusceptibility and resistance to SARS-CoV-2 infection were identified. Several variants in genes related to micronutrients were associated with severe COVID-19. Moreover, GWAS detected a significant genetic signal associated with COVID-19 related pneumonia. Conclusion: Multidisciplinary approach, modern sequencing technologies, comprehensive studies with well-characterized patients’groups, as well as the design of robust bioinformatics tools, enable identification of novel human genetic markers associated with COVID-19. Newly gained knowledge will empower the development of the targeted therapy, as well as the implementation of nutrigenomics/pharmacogenomics, leading to the application of precision medicine in the treatment of COVID-19 patients

Molecular genetic markers as a basis for personalized medicine

Genetika i genomika su danas potpuno integrisane u medicinsku praksu. Personalizovana medicina, poznata i kao medicina zasnovana na genomu, koristi znanja o genetičkoj osnovi bolesti da bi se individualizovalo lečenje svakog pacijenta. Veliki broj genetičkih varijanti, molekularno-genetičkih markera, već se koristi u kliničkoj praksi za dijagnozu, prognozu i praćenje bolesti (monogenska nasledna oboljenja, fuzioni geni i rearanžmani u pedijatrijskim i adultnim leukemijama) i presimptomatsku procenu rizika od obolevanja (BRCA1/2 za kancer dojke). Osim toga, primena farmakogenomike u kliničkoj praksi značajno je doprinela individualizaciji terapije u skladu sa genotipom i profilom ekspresije gena pacijenta. Genetičko testiranje za nekoliko farmakogenomičkih markera (TPMT, UGT1A1, CYP2C9, VKORC1) obavezno je ili se preporučuje pre započinjanja terapije. Najvažniji doprinos medicine zasnovane na genomu je ciljana molekularna terapija, prilagođena genetskom profilu bolesti. Testiranje genetičkih varijanti u malignim oboljenjima (BCR-ABL, PML/RARa, RAS, BCL-2, KIT, PDGFR, EGF) doprinosi tačnijoj stratifikaciji različitih kancera i adekvatnom izboru terapije. Krajnji cilj medicinske nauke je da primeni gensku terapiju koja bi eliminisala uzrok bolesti ili prevenirala bolest, ciljajući genetički defekt koji leži u osnovi bolesti. Tehnologija koja prati gensku terapiju veoma se brzo razvija i već se uspešno primenjuje. Iako je medicina oduvek suštinski bila "personalizovana", prilagođena svakom pacijentu, personalizovana medicina danas koristi modernu tehnologiju i znanja iz oblasti molekularne genetike i genomike, omogućujući stepen personalizacije koji vodi ka značajnom napretku medicinske prakse.Nowadays, genetics and genomics are fully integrated into medical practice. Personalized medicine, also called genome-based medicine, uses the knowledge of the genetic basis of disease to individualize treatment for each patient. A number of genetic variants, molecular genetic markers, are already in use in medical practice for the diagnosis, prognosis and follow-up of diseases (monogenic hereditary disorders, fusion genes and rearrangements in pediatric and adult leukemia) and presymptomatic risk assessment (BRCA 1/2 for breast cancer). Additionally, the application of pharmacogenomics in clinical practice has significantly contributed to the individualization of therapy in accordance with the patient's genotype and gene expression profile. Genetic testing for several pharmacogenomic markers (TPMT, UGT1A1, CYP2C9, VKORC1) is mandatory or recommended prior to the initiation of therapy. The most important achievement of genome-based medicine is molecular-targeted therapy, tailored to the genetic profile of a disease. Testing for gene variants in cancer (BCR-ABL, PML/RARa, RAS, BCL-2) is part of the recommended evaluation for different cancers, in order to achieve better management of the disease. The ultimate goal of medical science is to develop gene therapy which will fight or prevent a disease by targeting the disease-causing genetic defect. Gene therapy technology is rapidly developing, and has already been used with success. Although medicine has always been essentially "personal" to each patient, personalized medicine today uses modern technology and knowledge in the field of molecular genetics and genomics, enabling a level of personalization which leads to significant improvement in health care

Components of human thiopurine S-methyltransferase gene expression regulation system as pharmacogenetic markers

Tiopurin S-metiltransferaza (TPMT; EC 2.1.1.67) je jedan od najboÿih primera primene farmakogenetike u individualizaciji terapije. TPMT je enzim koji metabolise imunosupersivne tiopurinske lekove (6-MP), koji se koriste u lečenju kancera, autoimunih bolesti i u transpalntacionoj medicini. Aktivnost TPMT enzima je polimorfna i u beloj populacÿi je zabeležena trimodalna distibucija TPMT aktivnosti: postoje jjudi koji su slabi, umereni ili jaki „metilatori". Ipak, ne postoji jasna granica u aktivnosti TPMT izmedu ovih definisanih grupa. Pokazano je da određeni polimorfizmi u genu za TPMT utiču na aktivnost TPMT enzima. Promotor gena za TPMT čoveka je veoma polimorfan. Sadrži promenÿiv broj tandemskih ponovaka, od tri do devet, koji imaju veliki procenat GC baznih parova. Postoje tri tipa ponovaka, A, B i C. Arhitektura ovog promotorskog regionaje uvek AnBmC. U ovom radu smo ispitivali da li genotipizacija polimorfizama u genu za TPMT može biti osnova za modifikaciju tiopurinske terapije u lečenju akutne limfoidne leukemije (ALL) kod dece. Takode smo analizirali distribuciju alela i genotipova VNTR ponovaka promotora gena za TPMT u srpskoj populacÿi. Istraživali smo uticaj broja i tipa tandemskih ponovaka u promotoru na transkripcÿu gena za TPMT čoveka, koristeći eksperimente tranzÿentne transfekcÿe. Ispitivali smo i koji se transkripcioni faktori vezuju za VNTR region esejima usporene elektroforetske pokretÿivosti (EMSA) i „superšift" esejima, analizirali metilacioni status promotora, cis-regulatorne elemente u promotorskom regionu i ispitivali potencijalni uticaj 6-MP na transkripcÿu gena za TPMT. Nasa studÿaje pokazala da je genotipizacÿa TPMT neophodna i efikasna i da je osnova za modifikaciju tiopurinske terapÿe kod ALL pacÿenata, čak i kod heterozigotnih nosilaca polimorfizama u genu za TPMT. Detektovali smo 11 različitih tipova VNTR u promotoru gena za TPMT u srpskoj populacÿi. Broj ponovaka je bio od 4 do 8. Unutar promotora gena za TPMT sa istim brojem ponovaka, detektovana je različita arhitektura VNTR regiona, AnBmC, gde je n = 1-5 i m = 1-6. Detektovali smo 17 različitih TPMT VNTR genotipova u srpskoj populacÿi. Funkcionalni eseji su pokazali da najveću aktivnost TPMT promotora ima onaj sa VNTR*4b tipom (AB2C). Promotori sa 5, 6 i 7 VNTR alela su imali manje aktivnosti. Aktivnost VNTR*8 alela je bila dva puta veća od aktivnosti VNTR*7 tipova. Utvrdili smo da postoje razlike u aktivnostima promotora između konstrukata koji su imali isti broj ali različiti tip tandemskih ponovaka. Najveća razlika detektovana je između VNTR*4 varijanti (A2BC i AB2C). Takođeje nadeno da je promotorski region uzvodno od VNTR ponovaka, na poziciji od -180 do -130, transkripcioni aktivator. Potvrdili smo da se za VNTR region direktno vezuju transkripcioni faktori Spl i Sp3. Promotor gena za TPMTje specifično odgovorio na tretman ćelija dozom od 10 pM 6-MP, i to na VNTR-zavistan način, smanjenjem ekspresije reporterskog gena za 40-50%. U ovaj odgovor uključeni su transkripcioni faktori Sp3 i KLF1. Pokazali smo da broj i tip VNTR ponovaka u promotoru gena za TPMT utiče na nivo TPMT transkripcije. Arhitektura VNTR regiona (konfiguracÿa i broj nukleotida izmedu A i C ponovaka) moduliše transkripciju gena za TPMT. VNTR region može biti odgovoran za raznolikost u aktivnosti TPMT enzima unutar tri definisane grupe metilatora. Naši rezultati pokazuju da su VNTR genotipovi koji doprinose TPMT fenotipu slabih „metilatora" zastupljeni u jednoj trećini srpske populacije. Zbog toga bi tandemski ponovci u promotoru gena za TPMT mogli biti kandidat za farmakogenetički marker. Dalja istraživanja bi potvrdila da li je ovaj region od kliničkog značaja za individuaiizaciju tiopurinske terapije.Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) represents one of the examples of pharmacogenetics applied for individualizing drug therapy. It is an enzyme that metabolizes immunosuppressive thiopurine medications, used in treatment of autoimmune diseases, cancer and in transplantation medicine. TPMT activity is polymorphic and trimodal distribution has been demonstrated in Caucasians, namely low, intermediate and high methylator groups. However, there is no clear separation between these patient groups. It was shown that certain TPMT gene polymorphisms affect TPMT enzyme activity. Human TPMT gene promoter is highly polymorphic. It contains a variable number of 3 GC-rich tandem repeats, namely A, B and C, ranging from 3 to 9 in length, but maintaining a AnBmC architecture. Here, we investigated if the TPMT genotypization could be an efficient tool for guiding of thiopurine therapy in acute lymphoblastic leukemia (ALL) patients, carriers of TPMT polymorphism. We also analyzed the promoter tandem repeat distribution of alleles and genotypes in Serbian population. We investigated the influence of number and type of promoter tandem repeats on transcription of human TPMT gene using transient transfection experiments, addressed the interaction of transcription factor binding to the VNTRs by EMSA and supershift assays, analyzed the méthylation status of VNTR region, cis- regulatory elements in promoter region and investigated potential influence of 6-MP on TPMT gene transcription. Our study has confirmed that TPMT genotyping is necessary and efficient for modification of thiopurine therapy in ALL patients, even if they are heterozygous carriers of TPMT polymorphism. We have detected 11 different types of VNTRs in the TPMT gene promoter in the Serbian population. Number of repeats ranged from 4 to 8. Within the TPMT promoters containing the same number of tandem repeats, different architecture of VNTRs has been determined, namely AnBmC, where n ranged from 1-5 and m ranged from 1-6. We have determined 17 different TPMT VNTR genotypes in Serbian population. Functional assays revealed that TPMT promoter with the highest activity was the one with VNTRMb type (AB2C). Promoters with 5, 6 and 7 VNTR alleles all had successively lower activities, VNTR*8 activity was two times higher than activity of VNTR*7 types. We found differences in activity between the constructs containing the same number, but different type of tandem repeats. The most prominent difference was observed between VNTR*4 variants (A2BC and AB2C). The results of functional analysis demonstrate that the activity of TPMT promoter depends not only on the overall number of tandem repeats but also on the type of the repeat. The 5' TPMT promoter region from -180 to -130 is a transcriptional activator. We have confirmed that transcription factors Sp1 and Sp3 directly bind to VNTR region. TPMT gene promoter demonstrated a specific response to 6-MP treatment (10 pM) of the cells in a VNTR-specific manner, namely reduction of reporter gene expression from 40-50%. Transcription factors Sp3 and KLF1 are involved in this response. We have shown that number and type of VNTRs in the TPMT gene promoter determine level of TPMT transcription. VNTR architecture (distance and configuration from A to C repeats) probably spatially and in sequence specific manner, modulate TPMT transcription. VNTR region may be responsible for diversity in TPMT activity among defined groups of TPMT methylators. Our results showed that VNTR genotypes that contributed to low- methylator TPMT phenotype, comprised one third of Serbian population. For that reason promoter tandem repeats could be considered as candidate pharmacogenetic marker. Further investigation will confirm if it could be of clinical importance for individualizing thiopurine therapy

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

Application of Next-Generation Sequencing Technology and Establishment of Biobanks

Institute of Molecular Genetics and Genetic Engineering has become widely recognized as an expert centre for rare diseases (RD). It is the first institution is Serbia that applied NGS methodology in research and diagnostics of RD. We are also the institution with RD biobank collections containing DNA, RNA, mononuclear cells and tissue samples from over 2000 of patients affected with 50 different diseases. An accurate diagnosis was provided to over than 100 RD patients who were undiagnosed for years. We used Clinical-Exome Sequencing TruSightOne Gene Panel (4813 clinicaly-relevant genes), Illumina MiSeq instrument and Illumina VariantStudio. For monogenic diseases, filtration and prioritization of variants were performed according to “in-house” pipeline, using virtual gene panels. Variants were analyzed by various in silico softwares and classified according to ACMG guidelines. Variants selected by these criteria were confirmed by conventional Sanger sequencing and parents’ samples were analyzed whenever available. Furthermore, novel variants in DNAI1, MUT, PAH, PCCB, SLC37A4, SPAG16 and SPAG17 genes were functionally characterized in adequate in vitro systems such as immortalized patients’ fibroblasts or CRISPR /Cas9 edited commercial cell lines. Clinical-exome sequencing enabled diagnosis of more than 50 different diagnosis (hematological, metabolic, endocrinological, pulmonary, immunological, orthopedic, dermatological, ophthalmological, cardiological, epileptic encephalopathies etc.). It was particularly important for genetically heterogeneous diseases, such as glycogen storage diseases, branched-chain organic acidurias, primary ciliary dyskinesia, MODY or mitochondriopathies. Moreover, different diseases with overlapping clinical manifestations were accurately diagnosed. Also, we used TruSeq-Amplicon Cancer Panel to analyse different childhood and adult rare hematological malignancies. Besides studying diagnostic and prognostic malignancy markers, we designed “in-house” virtual pharamocogenomic panel, and performed association studies of pharmacogenomic markers and the course and outcome of rare hematological malignancies, resulting in recommendations for therapeutic modalities in accordance with genomic profile of the patien

Genetic basis of otosclerosis

Uvod Otoskleroza je poremećaj koštane kapsule lavirinta i slušnih koščica uva, koji dovodi do gubitka sluha zbog nemogućnosti provođenja zvuka. Genetički uzrok otoskleroze je nepoznat. Cilj ovog rada je bio da se sačini sveobuhvatni pregled savremenih saznanja o genetičkoj osnovi otoskleroze. Metode Za prikaz podataka o genetici otoskleroze korišćen je narativni pregled literature. Rezultati Genetika otoskleroze nije mnogo izučavana i literaturni podaci o genetičkoj osnovi otoskleroze su oskudni. Međutim, u novije vreme, proširuju se znanja o genetičkoj osnovi otoskleroze. Ovde je prikazan pregled znanja o asocijaciji genetičkih markera i otoskleroze, koja su rezultat analiza genetičke povezanosti, kao i asocijativnih studija gena kandidata i sveobuhvatnih analiza genoma. Zaključak Otoskleroza zbog svoje kompleksnosti nije bolest čija će genetička osnova biti lako rasvetljena. Analize omika i bioinformatika će doprineti razumevanju genetičke osnove otoskleroze.Introduction Otosclerosis is a disorder of the bone labyrinth and stapes resulting in conductive hearing loss. The genetic basis of otosclerosis still remains unknown. We aimed at reporting a comprehensive review of up-to-date knowledge on genetic basis of otosclerosis. Methods Narrative literature review was undertaken to summarize the data about genetics of otosclerosis. Results Genetics of otosclerosis has not been studied extensively and the literature on this topic is scarce. However, knowledge of genetic basis of otosclerosis is recently increasing. We have presented an overview of the knowledge of association of genetic markers with otosclerosis, gained from linkage analyses, candidate-gene studies, and modern high-throughput genomic studies. Conclusion Due to its complex pathophysiology, otosclerosis is not a disease whose genetic base will be easily understood. Multiple omics analysis and bioinformatics will lead to elucidation of genetic basis of otosclerosis