Molecular Hematology

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Molecular Hematology. Galila Zaher. DNA RNA Protein. DNA & RNA. DNA RNA Double-strandedSingle-stranded 4 bases: A, C, G & T A, C, G & U Sugar: Deoxribose Sugar: Ribose Stable moleculeUnstable molecule Introns + ExonsIntrons. DNA: Building Blocks. Bases - PowerPoint PPT Presentation

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<ul><li><p>Molecular Hematology </p><p> Galila Zaher</p></li><li><p>DNA RNA Protein</p></li><li><p>DNA &amp; RNADNARNADouble-strandedSingle-stranded4 bases: A, C, G &amp; T A, C, G &amp; USugar: Deoxribose Sugar: RiboseStable moleculeUnstable moleculeIntrons + ExonsIntrons</p></li><li><p>DNA: Building BlocksPurinePyrimidineBasesPurinesAdenineGuaninePyrimidinesCytosineThymine[Uracil]Purine pair pyrimidineAdenine-ThymineGuanine-Cytosine</p></li><li><p>DNANature paper here</p></li><li><p>ChromosomesMale karyotype46:XYFemale karyotype46:XX22 pairs of autosomes + 1 pair sex chromosomes</p></li><li><p>Normal StructureSomatic cell has 46 chromosomes : diploid.Ova and sperm have 23 chromosomes :haploid.Karyotype shows chromosomes of dividing cell in numerical order.</p></li><li><p>Chromosome has two arms:Short arm = p Long arm = q.</p></li><li><p>Short and long arms meet at the Centromere.</p></li><li><p>Ends of the chromosomes are called Telomeres</p></li><li><p>Each arm is divided into regions numbered from centromere.</p></li><li><p>Each region is divided into bands.</p></li><li>Numerical AbnormalityAneuploid: Somatic cell with &gt;or 46 chromosomesHypodiploid : </li><li><p>Structural Abnormalitydel : deletion where part of chromosome is lost del(16q).Add: additional material has replaced part of a Cht: Translocation t(9; 22)inv :inversion; part of Ch runs in opposite direction.Point mutationNon sense :Result in creation of premature stop codonNormal sequence ATG CTG TGC CysMutant sequence ATG CTG TGA stopi: isochromosome is a chromosome with identical chromosome arms at each end, e.g. i(17q) has two copies of 17q joined at centromere.</p></li><li><p>Haematological MalignanciesMostly clonal disorders resulting from a genetic alteration.Tumor-Suppressor Genes : inhibit expression of tumor phenotype. When are inactivated or lost abnormal proliferation Oncogenes :Genes which can potentially induce neoplastic transformation. They include genes for growth factors, growth factor receptors, protein kinases,etc.</p></li><li><p>Normal proliferation / ApoptosisExcess proliferation / loss of ApoptosisTumour-suppressorgeneOncogeneProto-oncogeneTumour-suppressorgeneGenetics of Haematological MalignanciesMutationMutation or deletion</p></li><li><p>Clonal ProgressionActivation of OncogenesInactivation of Tumour-Suppressor GenesMalignant cells acquire new characteristics causing acceleration.Multidrug resistance (MDR) is one complication. Cells start to express a protein which actively pumps chemotherapeutic agent to outside of cells. </p></li><li><p>ThalassemiaHeterogenous group of genetic disordersMutation decrease rate of synthesis of globin chains ( or ). 0 :complete absence of chain . Common in Mediterranean.+ :partial block in chain synthesis. Noncoding introns inefficient RNA splicing ,decreased mRNA productionPromoter leading to decreased expressionTermination site production of longer, unstable mRNAPartial or total deletion of a globin gene thalassemia major :0/0, +/ +, or 0/ +</p></li><li><p>-ThalassemiasDisease manifests itself when switch chain ms after birthImbalanced synthesis low Hb production, MCV &amp; MCH Excess chains precipitate in RBC precursors in bone marrow leading to hemolysis and ineffective erythropoiesisExcess chains in circulating RBCs precipitate leading to pitting in spleen &amp; RBC survival via a chronic hemolytic process.The major cause of severe anemia is the ineffective erythropoiesis. Compensatory increase in &amp; chain synthesis Hb F&amp; A2.</p></li><li><p>Hereditary thrombophiliaPCPSATProthrombin Gene Mutation:G20210A. Factor V Leiden Mutation: R506Q.MTRFR :mutation</p></li><li><p>INHERITED RISK FACTORS Relative risk of VTE</p></li><li><p>Gene StructureSplice sites</p></li><li><p>X-linked DisordersHaemophilia A and BX-linked disordersMales affected females carriers</p></li><li><p>Queen Victoria (1819 - 1901)Queen Victoria Queen of England from 1837 to 1901 was a carrier.</p><p>Her eighth child, Leopold, had Hemophilia and suffered from frequent hemorrhage.</p></li><li><p>Descendants Eugenie, who was a carrier introduced Hemophilia into Spanish royal family .Irene married to Prince Henry of Prussia introduced the disease into the German royal family Alexandra married Russia's last czar Nicholas II introduced the disease into the Russia royal family, which ultimately played a role in the start of the Russian Revolution. </p><p>Royal Disease</p></li><li><p>Alexis, son of Nicholas and Alexandra (1904 - 1918)Alexandra gave birth to a son Alexis the long awaited heir Russian throne.Unfortunately Alexis had Hemophilia which ultimately played a role in the start of the Russian Revolution. </p></li><li><p>Victoria will be remembered as the cause of Hemophilia which spread to the Royal Family of Europe through her descendants</p></li><li><p>F8 Gene</p></li><li><p>FIX Gene</p></li><li><p>Types of MutationsMissense mutationsNonsense mutationsSplice mutationsInsertionsDeletions</p></li><li>Inversions Sever HASHA: Intron 22: ~50% cases Intron 1: </li><li><p>Haemophilia A: Intron 22 Inversion</p></li><li><p>Cytogenetic &amp;Molecular studiesKaryotype Analysis (numerical ) Immunofluorescence Staining (structural)Fluorescent in situ Hybridisation (FISH)Southern Blot AnalysisPolymerase Chain Reaction (PCR) t(9,22),hemophilia,thrombophiliaDNA Microarray Platforms</p></li><li><p>ChromosomesMale karyotype46:XYFemale karyotype46:XX22 pairs of autosomes + 1 pair sex chromosomes</p></li><li><p>Each arm is divided into regions numbered outwards from the centromere.Oncogenes :Genes which can potentially induce neoplastic transformation. They include genes for growth factors, growth factor receptors, protein kinases, signal transducers, nuclear phosphoproteins, and transcription factors. </p><p>Tumour-Suppressor GenesTumour-suppressor genes are subject to loss-of-function mutations (point mutation or deletion) and thus malignant transformation.Tumour-suppressor genes help regulate cells to pass through different phases of the cell cycle, e.g. G1 to S, S to G2 and mitosis.The amount of p53 will stop Synthesis in the cell cycle Its consists of 4 different monomers ,One gene one monomer If one of the monomers is dysfunctional abnormal protein Thus all it takes its one mutant gene for the protein to become defunctCytosol levels rise rapidly in response to DNA damaging agentsIf damage is found in the template or complementary strand then duplication stopsIf it reaches a threshold level then it induces the cell to undergo apoptosis</p><p>Du to the lack mal heirs in her family victoria became the queen at the age of 18 Victoria married her first cousin Prince Albert and had nine children Hemophilia first appear in Victorias family in her eighth child prince LeopoldPrince Leopold had highly protective life, he suffered severe hemorrhage either as a child or as an adult despite all the protection that the queen provided he died at the age of 31 secondary to ICH This couple had four daughters before Alexandra gave birth to a son Alexis he long awaited heir to the Russian throne.Unfortunately Alexis had hemophilia which ultimately played a role in the start of the Russian Revolution. </p><p>Queen Victoria will be remembered as the cause of hemophilia which spread to the royal family of Europe through her descendants 1-Karyotype Analysis(Cytogenetic studies) :Images of chromosomes are captured when cell is in metaphase.2-Immunofluorescence Staining; Can be useful for a few chromosomal abnormalities, e.g. promyelocytic leukaemia protein which normally has a punctate distribution but is diffusely scattered in acute promyelocytic leukaemia with the t(15; 17) translocation. Abnormal fusion proteins may also be detected by specific monoclonal antibodies. 3-Fluorescent in situ Hybridisation (FISH) Fluorescent-labelled genetic probes hybridise to specific parts of the genome. Can pick up extra copies of genetic material in both metaphase and interphase, e.g.trisomy 12 in CLL. Translocations can be seen by using two different probes.</p><p>4-Southern Blot Analysis Restriction enzyme digestion of DNA, gel electrophoresis and blotting to a suitable membrane. DNA fragments are hybridised to a probe complementary to the gene of interest. If the probe recognises a segment within the boundaries of a single fragment one band is identified. If the gene has been translocated to a new area in the genome a novel band of different electrophoretic mobility is seen.5-Polymerase Chain Reaction (PCR) Can identify specific translocations, e.g. t(9; 22). Can also detect clonal cells of B- or T-cell lineage by immunoglobulin or T-cell receptor (TCR) gene rearrangement analysis. Sensitivity (can detect one abnormal cell in 105106 normal cells) makes this of value in monitoring patients with minimal residual disease (MRD).6-DNA Microarray PlatformsRapid and comprehensive analysis of cellular transcription by hybridising labelled cellular mRNA to DNA probes immobilised on a solid support. Oligonucleotides or complementary DNA (cDNA) arrays are immobilised on the array and fluorescent labelled RNA from the cell sample is annealed to the DNA matrix. Can determine the mRNA expression pattern of different leukaemia subtypes.</p><p>1-Karyotype Analysis(Cytogenetic studies) :Images of chromosomes are captured when cell is in metaphase.2-Immunofluorescence Staining; Can be useful for a few chromosomal abnormalities, e.g. promyelocytic leukaemia protein which normally has a punctate distribution but is diffusely scattered in acute promyelocytic leukaemia with the t(15; 17) translocation. Abnormal fusion proteins may also be detected by specific monoclonal antibodies.Genetics :Higher incidence in siblings and twinsVirus :Clusters of leukemiaIonizing radiation :Survivors of Hiroshima and Nagasaki</p></li></ul>