Presentation on theme: "Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph. D"— Presentation transcript:
1Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph. D Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics
2Introduction Hemoglobin Structure and Function Hemoglobinopathies ThalassemiaSickle Cell DiseaseUAB Animal Models
3Red Blood Cells Contain Hemoglobin RBCCooley’s Anemia is caused by the absence of b globin chainsSickle Cell Anemia is caused by single mutation in b globin
4Globin Gene Regulation Globin genes are expressed at high levelsExpressed specifically in erythroid cellsIndividual globin genes are temporally regulated during developmenta and b-like globin genes are coordinately regulated for balanced expression/synthesis
5Human a and b Globin Loci 1HS -40Ch 165’HS:LCRbedybGgAgAlpha locus on Chromo 16 single embryonic zeta and two adult genes several pseudo genesBeta locus on Chromo 11 single embryonic, two fetal, two adult one pseudo geneLocus control region LCR Powerful regulatory region marked by Erythroid specific, developmentally stable, Dnase I HSCh 11
6Erythroid Development ErythropoiesisMature Red CellBFU-ECFU-EPro-BasophilicPolychromaticOrthochromaticHSCErythroblastReticulocyte>95% protein is hemoglobinHemoglobin content increases along erythroid differentiation6
7Human Hemoglobin Switching During Development GestationHematopoiesisHemoglobin2e2a2e23 weeks5 weeks7 monthsYolk SacHSCHSCAGMFetal Liver22HbFHbAHbA2Primitive Hematopoiesis--YS Embryonic globins single zeta to alpha switch, alpha through to adultDefinitive hematopoiesis AGM & Y.S. to fetal liver 1st switch of epsilon to gammaFL to BM 2nd switch to beta and deltaHSC2222Bone Marrow
8 Globin Gene Switching .100G + A% of Total Beta ChainsManBirthYHuman Hb switching in the mouseMouse definitive erythropoiesis reaches adult levels in utero 1 week before birthHow would human globin genes be regulated mouseTG Mouse analysis Define sequences responsible for the high-level, erythroid-specific, and temporal expression100h1maj + min% of Total Beta ChainsMouseBirth
9Cooley’s Anemia Dr. Thomas Cooley, 1925 Thalassemia major -- homozygous b0 thalassemiaAge of onset is one year of age--severe anemiaErythroid hyperplasia, ineffective hematopoiesis, and hemolysisBlood tranfusion dependentHepatosplenomegaly, skeletal deformities, retarded growth, iron overlod, liver and heart diseaseLifelong transfusions and iron chelation therapyCan be cured by allogeneic bone marrow transplantation
10Populations Affected by b Thalassemia Nature Genetics (2001) 2:245
11Hemoglobin Switching During Development HumanegG & gAd & bMouseBirthbmaj & bminbh1 & ey
12Making Transgenic Mice By Pronuclear DNA Injection
27Sickle Cell Anemia James Herrick, Chicago 1910 Hemolytic Anemia Vascular occlusion causes tissue injury and painFrequent Infections - Prophylactic antibiotics til age sixStroke and brain injurySplenic sequestrationAcute chest syndromePolyuria, Kidney failure, PriapismLeg ulcersFrequent Blood Transfusions, Iron OverloadCan be cured by bone marrow transplantation, but….
28Sickle-Cell Anemia is a Molecular Disease Sickle-cell anemia patients have abnormally-shaped red blood cellsThe erythrocytes are crescent-shaped instead of disc-shapedThe sickle cells pass less freely through the capillaries, impairing circulation and causing tissue damageA single amino acid substitution in the β-chains of Hb causes sickle-cell anemiaGlu at position 6 of the β-chains is replaced by ValAs a result, Hb S molecules aggregate into long, chainlike polymeric structures
29Sickle-Cell Anemia is a Molecular Disease Figure The polymerization of Hb S molecules arises because Val replaces His on the surface of β-chains. The “block” extending from Hb S below represents the Val side chains. These can insert into hydrophobic pockets in neighboring Hb S molecules.
30Sickle-Cell Anemia is a Molecular Disease Figure Polymerization of Hb S.
31Sickle Hemoglobin Polymerizes •Sickle hemoglobin tetramers aggregate inside the red blood cell forming long polymers when deoxygenated
33Mouse Model of Sickle Cell Disease Produce a mouse that synthesizes high levels of human sickle hemoglobin--Transgenic MouseProduce a mouse that synthesizes no endogenous mouse hemoglobin--Knockout MouseKnockout-Transgenic Sickle Cell Mouse
34First Generation Animal Model of SCD Transgenic modelHigh level expression and synthesis of human HbSSickle poymer formation under hypoxic conditionsLittle in vivo pathology under normoxic conditionsScience 247:
35Mouse maj- and min-Globin Knock-Out 7654321yh0h1h2majminpgk/NEOtk7654321yh0h1h2pgk/NEO