El document forma part dels materials docents programats mitjançant l'ajut del Servei de Política Lingüística de la Universitat de ValènciaOmics development is allowing to improve clinical diagnostic techniques in clinical biochemistry. Omics investigate organisms as a whole and allow to study several parameters from a holistic perspective. Through the groundbreakingly development of cytomics, metabolomics and genomics and transcriptomics we can study cells, metabolites and genes as biomarkers for the diagnostic of several pathologies through the application of biochemical and molecular biology techniques. In this seminar, a state-of-the-art review is performed, in order to evaluate the influence of omics in the molecular study of diseases

Galán Albiñana, Amparo

Repositori d'Objectes Digitals per a l'Ensenyament la Recerca i la Cultura

BIOCHEMICAL INTEGRATION AND CLINICAL BIOCHEMISTRY2ND YEAR - DEGREE IN MEDICINE 2022-23SEMINAR 4PROFESSOR:Amparo Galán Albiñanaamparo.galan@uv.esAdvanced clinical diagnostic techniques:  omics and their application to the molecular study of diseases Department ofBiochemistry and Molecular BiologyAnalogue DigitalParadigm shift in biomedical sciencesBioinformaticsDiagnosis based on individual parametersDiagnosis based on global parametersGLOBAL omicsHigh performance analytical methodsINTRODUCTION: OMICSDepartment ofBiochemistry and Molecular Biology• Omics investigate organisms as a whole, from a holistic perspective.• Omics are linked to the development of new high-performancetechnologies that arise from the collaboration across multipledisciplines (biology, computer science, chemistry, physics, engineering,etc.).• Omics generate millions of biological data that require complexprocessing and analysis.• The massive flow of data generated by the omics sciences has promotedthe emergence and development of bioinformatics, which is defined asthe discipline that applies computer science and statistics to theprocessing and analysis of biological data.INTRODUCTION: OMICSGenesmRNAsProteinsMetabolitesCellsOMICS SCIENCESGenomicsTranscriptomicsProteomicsMetabolomicsCytomicsINTRODUCTION: OMICSExplosion in the number of DNA sequences stored in public databasesINTRODUCTION: OMICSNCBIINTRODUCTION: OMICSUpdate of the NCBI’s Entrez databases repositoryCYTOMICSAnalytical method for measuring the emission of multiple fluorescence and lightscattering from cells or microscopic particles, aligned by a laminar liquidstream, when presented one at a time and at high speed in front of a light sourceof appropriate wavelength.• Fluorescence and fluorescent markers• Parameters that can be analyzed by flow cytometryWhat can be stained?Which cells can be analyzed?• Applications of flow cytometryFLOW CYTOMETRYAnalytical method that measures the simultaneous emission of multiple fluorescence and light scatteringFLOW CYTOMETRYSequentially aligned by a laminar flow and presented one at a time at high speed at an optimal point of illuminationIndividual biological cells or particles in suspensionFLOW CYTOMETRY• Extracellular proteins• Free DNA/RNA sequences• Circulating immune complexes• Individual virions • Liposomes• Isolated chromosomes• Isolated organelles• Isolated nuclei• Bacteria• Unicellular fungi• Human and animal cells• Hybridomas and cell fusions• Spheroids• Embryoid bodies• Larvae and embryos• Pluricellular organismsMolecular levelSubcellular levelCell levelSupracellular levelFLOW CYTOMETRYDIAGNOSTIC APPLICATIONCells93.73% CD44+42.53% CD44+ CD326-51.89% CD44+ CD326+6.22% CD44- CD326-0.06% CD44- CD326+CytometerNumerical information(analyzer)Purified cells(separator)Therapeutic/diagnosticdecision-makingCelltransfusionFLOW CYTOMETRY• Diagnosis and prognosis of leukemias, lymphomas, lymphoproliferative syndromes and  myelodysplastic syndrome• Absolute CD34+ cell count for auto- or allotransplantation of hematopoietic stem cells• Identification of proliferating cells and tumor cells in blood, biological fluids and complex samples• Study of cell cycle regulationMain applications: oncology and hematologyFLOW CYTOMETRYDIAGNOSTIC APPLICATIONMETABOLOMICSMetabolomics is the study of metabolites.Metabolites are the intermediates and products of metabolism. They are any molecule of less than 1 KDa (except some macromolecules: lipoproteins, albumin, etc.).Unique footprints left by specific cellular processes.Endogenous metabolites: produced by the organism.Exogenous metabolites: from external substances (drugs, poisoning, etc.).Also known as xenometabolites.The metabolome represents the collection of all the metabolites in a cell, tissue, organ, or organism that are the product of cellular processes.Biological fluids: mostly urine and plasma (non-/minimally invasive).But they also include: tissue biopsies, cells, exudates, saliva, bile salts, intestinal aspirates, and fluids (cerebrospinal, seminal, amniotic, and synovial).Metabolites have been roughly categorized as:2,500 endogenous metabolites1,200 drugs3,500 food componentsHuman metabolome data: www.hmdb.caMETABOLOMICSBIOCHEMICAL AUTOANALYZERS State-of-the-art analyzers for analyzing hundreds ofmetabolites in a few minutes Based on spectrophotometry Non-complex techniques used  Quick and precise• Enzymes• Ions: Na+/K+• Biochemical markers:Glucose, cholesterol, triglycerides, uric acid, proteins, serum albumin, creatinine• Immune assays by antibodies• Hematologic assays Erythrocyte sedimentation/coagulationParticle detection of:ROUTINE DIAGNOSTIC APPLICATIONSSamples: urine and plasma Routinely detect common metabolites and big molecules / cellsMETABOLOMICSMETHODS OF BIOMARKER DETECTION IN METABOLOMICSMass spectrometry (MS)Nuclear magnetic resonance (NMR) spectrometrySamplesUrine (by NMR):Blood/plasma (by MS and NMR):Whole tissueLipid phaseAqueous phaseIn pathophysiological processes, metabolites increase in situ. Low sample amounts (10 µg in tissue and 10 µl in fluids) high metabolite concentration and low protein concentrationmetabolic status of the organismMETABOLOMICSMetabolite detectionendogenous/exogenous originVitamin C (AA/DHAA)Vitamin B6 and derivativesTaurineAmino acidsHydroxymethylated DNA cytosinesPurine and pyrimidine basesLactulose/mannitolProstaglandinesNucleosides/nucleotidesToxicology (alkaloids, benzodiazepines, amphetamines and derivatives)Eicosanoids (polyunsaturated fatty acids)Drugs of abuseEmerging drugsDIAGNOSTIC APPLICATIONMETABOLOMICSGowda et al., Expert Rev Mol Diagn. 2008.Biomarkers: value for early diagnosis of diseases (i.e. preclinical phase of trials with therapeutic possibilities).Prognosis/treatment implicationAnalysisTargeted Non-targetedDiagnosis/prognosis:Congenital metabolic diseasesDiabetesCancerAutismCardiovascular diseasesBiomarker screening (novel or not) Congenital non-metabolic diseasesDIAGNOSTIC APPLICATIONFood and drug poisoningMETABOLOMICSMOLECULARBIOLOGY TECHNIQUESOld and new molecular diagnostic techniquesMOLECULAR DIAGNOSTICSOne molecule: one disease Set of markers that characterize a disease• Southern blot• PCR • DNA sequencing (Sanger)• Cytogenetics• Gene expression microarrays• Comparative genomics and epigenetic microarrays • Next generation sequencing: NGSPCR/qPCRPCR: endpoint PCRQualitative/semi-quantitativePolymerase chain reactionIt detects presence/expression of individual genes• Quantitative, sensitive and specific• QuickqPCR: real-time PCRMOLECULAR DIAGNOSTICSPerformed on the embryo prior to its transfer to the uterusDiseases:• Autosomal dominant inheritance: single parent• Autosomal recessive inheritance: two unaffected carrier parents• Sex-linked inheritance: an affected father or a carrier motherPreimplantation genetic diagnosisMonogenic diseases: failures or mutations in the information of a single gene that can be inherited or de novo.Mutations are detected in patients by DNA analysis and confirmed by screening offamily members.PCR/qPCRDiagnosis of monogenic diseasesDIAGNOSTIC APPLICATIONDiagnosis of infectious diseasesDetection of DNA and/or cDNA from infectious organismsFungi, bacteria, virusMOLECULAR DIAGNOSTICSArray is the equivalent to matrix.It is a solid surface containing a huge number of fixed probes (nucleic acids, proteins, metabolites, tissues…) that will be exposed to target molecules (sample).MICROARRAYSMOLECULAR DIAGNOSTICSmRNANumber of copiesMethylation Transcription factorsmRNAisoforms DNA RNASplicevariantsChIP GXaCGH CH3 miRNARNA interferenceMassive analysis of gene sequencesClassificationCarry out a high-resolution analysis of the genome to detect changes in DNA copy number associated with cancer and other diseases.Discover and monitor epigenetic changes that play fundamental roles in many cellular processes.Elucidate the role that protein-DNA interactions play in transcription, replication, modification and repair.Explore transcription at the genome-wide level through a variety of model systems.Perform a global transcriptome analysis and identify the role of isoforms (alternative splicing) in drug response and disease.Determine the microRNA profile and explore its role in gene regulation.MICROARRAYSMOLECULAR DIAGNOSTICSControl DNA marked in redPatient DNA marked in greenMICROARRAYCGH (comparative genomic hybridization)Each point corresponds to a knownsequence location in the genomeDiagnosis of diseases associated with chromosomal abnormalitiesMICROARRAYSMOLECULAR DIAGNOSTICSOn chromosomesCGH (comparative genomic hybridization)MICROARRAYSDIAGNOSTIC APPLICATIONCancerPreimplantation diagnosisMOLECULAR DIAGNOSTICSComparative resultsEXPRESSION MICROARRAYSMICROARRAYSMOLECULAR DIAGNOSTICS• Detailed molecular phenotype• Applications in oncology:Tumor stage: molecular classificationDetermination of treatment modalitiesIdentification of prognostic markers• Variation of expression profile with nutrition  Nutrigenomics• Variation of expression profile with exposure to toxic substances Toxicogenomics• Personalized medicine: patients can be treated with specific drugs based on their own expression signatures (patterns) PharmacogenomicsMICROARRAYSMOLECULAR DIAGNOSTICSDIAGNOSTIC APPLICATIONEXPRESSION MICROARRAYSWe have 3 billion (3x109) bases arranged in a unique order, with  20,000 genes regulating gene transcription to protein synthesis.NUCLEIC ACID SEQUENCINGFragments: 500 pb – 1 KbMaximum: 96 readsMOLECULAR DIAGNOSTICSSanger sequencing is a well-established method.Time-consumingExpensive for long readsThe Human Genome Sequencing ProjectNext generation sequencing (NGS)High throughputBioinformatic interpretationSequencing of 25,000 genesMOLECULAR DIAGNOSTICSSanger sequencing methodNGSHigh-throughput sequencing:“Next generation”Able to sequence a large number of copies in a single reactionSame procedureDifferent technology depending on the platformMOLECULAR DIAGNOSTICSJason M. Rizzo, and Michael J. Buck Cancer Prev Res 2012;5:887-900NGSStep 1.Sample preparationStep 2.Template generation/amplification/sequencingReads: 30-400 bpsStep 3.Data analysisData analysis is essential.Millions or trillions of data are generated.Platform software assembles the results.Sequence assembling:Known genomic sequencesDe novo sequencesProper bioinformatics analysis is essential.MOLECULAR DIAGNOSTICSLandolt et al., EMJ. 2016;1[2]:50-57NGSMOLECULAR DIAGNOSTICS Exome and targeted sequencing Whole genome sequencing (WGS) Circulating DNA Transcriptome sequencing (RNA-seq)  NGS biobankingNGSDIAGNOSTIC APPLICATIONMOLECULAR DIAGNOSTICSNGSMOLECULAR DIAGNOSTICSDIAGNOSTIC APPLICATIONWorkflow for integrating omics data into cancer research (disease) and clinical applicationsNGSDiagnosis of unknown genetic diseases• Exome sequencing to understand recessive hereditary diseases• Child developing inflammatory bowel disease• Wounds in the gut / unknown cause• 100 visits to hospital from age 4• Exome sequencing reveals mutation in XIAP gene• Bone marrow transplant as a treatment Exome and targeted sequencingCancer panels studyo 500 variants analyzed, including deletions, inversions, insertions and substitutions in 22 genes (EGFR, ALK, ERBB2, ERBB4, FGFR1, FGFR2, TP53…) o Related with different types of solid neoplasia• Presence or absence of mutations• Diagnostic factors / prognosis / biomarkers that indicate/advise against therapiesMOLECULAR DIAGNOSTICSDIAGNOSTIC APPLICATIONNGS Whole genome sequencing (WGS)• Study of all regions of the genome (SNP array) to understand recessive hereditary disorders• Patients who suffer from joint pain and show calcium deposits in their arteries on x-ray• WGS shows mutation in NT5E gene, involved in breaking down calcifications in arteriesDiagnosis of unknown genetic diseasesOncological applicationStudies of mutations, methylation, sequence fusion, SNPs, etc.MOLECULAR DIAGNOSTICSDIAGNOSTIC APPLICATIONNGS Circulating DNADetection of anomalies in the number of chromosomes99% precisionDown’s syndromeEdwards’ syndromePatau’s syndromePrenatal diagnosisDiagnosis of a great number of disorders with a genetic causeResults in 5-7 daysMOLECULAR DIAGNOSTICSDIAGNOSTIC APPLICATIONMATERNALBLOOD SAMPLEDNA EXTRACTION AND NEXT GENERATION SEQUENCINGFETALDNAANALYSISRESULTSREPORTSENSITIVITY>99.9%Identifies the presence of anomaliesWith reduced possibilities of false negativesSPECIFICITY>99.9%Excludes the presence of anomaliesWith reduced possibilities of false positivesNGS Circulating DNACirculating tumor DNALiquid biopsyMOLECULAR DIAGNOSTICSDIAGNOSTIC APPLICATIONNGS Emergence of biotechnology companies (ie. in Valencia)Great number of services offeredLow cost/priceThe techniques applied are not optimalResults based on database availabilityMetadata analysis / bioinformatics interpretationIncomplete / partially wrong resultsNon-invasive sample collectionBasic analysis techniques (from the NGS spectrum)MOLECULAR DIAGNOSTICShttp://www.labgenetics.com.es/catalogo_enfermedades_hereditarias.htmIs NGS the ultimate “personalized medicine”?• It has a predictive value of disease risk and potential for delaying or preventing the development of the disease.• It can predict adequate drug dosage, drug response or adverse effects.• It can help determine the treatment of diseases based on the patient’s genetic profile.NGSFuture perspectives Legal/political adaptations Work adaptations Social adaptationsPost-genomic era of medicineMOLECULAR DIAGNOSTICSDIAGNOSTIC APPLICATIONCONCLUSIONSThe application of omics for the diagnosis, prognosis and study of diseases has great value and potential.Cytomics is used for the diagnosis and prognosis of hematological and oncological diseases.Metabolomics develops biomarkers for the current and potential diagnosis of multiple pathologies.Molecular diagnostics has the greatest application and potential at present. CGH arrays Expression microarrays Sequencing/NGSBIOCHEMICAL INTEGRATION AND CLINICAL BIOCHEMISTRY2ND YEAR - DEGREE IN MEDICINE 2022-23SEMINAR 4PROFESSOR:Amparo Galán Albiñanaamparo.galan@uv.esAdvanced clinical diagnostic techniques:  omics and their application to the molecular study of diseases 

Advanced clinical diagnostic techniques: omics and their application to the molecular study of diseases

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Advanced clinical diagnostic techniques: omics and their application to the molecular study of diseases

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Repositori d'Objectes Digitals per a l'Ensenyament la Recerca i la Cultura