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Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics

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Presentation on theme: "Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics"— Presentation transcript:

1 Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics

2 Introduction Hemoglobin Structure and Function Hemoglobinopathies Thalassemia Sickle Cell Disease UAB Animal Models

3 Red Blood Cells Contain Hemoglobin Cooleys Anemia is caused by the absence of globin chains Sickle Cell Anemia is caused by single mutation in globin RBC

4 Globin Gene Regulation Globin genes are expressed at high levels Expressed specifically in erythroid cells Individual globin genes are temporally regulated during development and -like globin genes are coordinately regulated for balanced expression/synthesis

5 Human and Globin Loci 5HS: LCR G A Ch 11 Ch HS -40

6 Erythroid Development >95% protein is hemoglobin Mature Red Cell BFU-E CFU-E Pro-BasophilicPolychromaticOrthochromatic HSC Erythroblast Reticulocyte Hemoglobin content increases along erythroid differentiation Erythropoiesis

7 Human Hemoglobin Switching During Development Gestation HemoglobinHematopoiesis Yolk Sac HSC Fetal Liver Bone Marrow AGM HSC 3 weeks 5 weeks 7 months HbF HbA HbA 2

8 Globin Gene Switching. G + A Y h1 maj + min Man Mouse % of Total Beta Chains 100 Birth % of Total Beta Chains

9 Cooleys Anemia Dr. Thomas Cooley, 1925 Thalassemia major -- homozygous 0 thalassemia Age of onset is one year of age--severe anemia Erythroid hyperplasia, ineffective hematopoiesis, and hemolysis Blood tranfusion dependent Hepatosplenomegaly, skeletal deformities, retarded growth, iron overlod, liver and heart disease Lifelong transfusions and iron chelation therapy Can be cured by allogeneic bone marrow transplantation

10 Nature Genetics (2001) 2:245 Populations Affected by Thalassemia

11 G & A & h1 & y maj & min Hemoglobin Switching During Development Human Mouse Birth

12 Making Transgenic Mice By Pronuclear DNA Injection

13 GFP--Transgenic Mice

14

15 Blastocyst: Source of Embryonic Stem (ES) Cells

16 Homologous Recombination In Embryonic Stem Cells

17 Mouse -Globin Knockout KO -Globin Knockout

18 Human A Globin Knock-in y h0 h1 h2 maj min hyg A CRE A y h0 h1 h2 LCR tk

19 Fetal to Adult Hb Switching in Human A Globin Knockin Mice N.B.Adult Age in Days % Total -Like Globin RNA Y h0 h1 h2 LCR A h1 Y A

20 Cooley s Anemia Mouse Model Knock-In of Human 0 Globin hyg 0 CRE y h0 h1 h2 maj min LCR 0 y h0 h1 h2 y h0 h1 h2 maj min LCR y h0 h1 h2 maj min LCR

21 Heterozygous Human 0 KI Mouse Model Anemia, Erythroid Hyperplasia, Globin Precipitates, and Splenomegaly Wild Type 0 KI

22 Human 2 1 Globin Knock-In mHS-40 m m 1m 2 mHS-40 m Cre mHS-40 m hyg tk h 2 1 hyg h 2 1

23 Humanized Cooleys Anemia Mice Survive at Birth on Human Fetal Hemoglobin JBC, /+ 0 / 0 0 / 0

24 Hemoglobin Switching in Humanized HPFH 0 and HPFH 0 Globin Knockin Mice % -like Globin Chains Age (wks) globin A / A % -like Globin Chains HPFH 0 / A Age (wks) Age (wks) % β -like Globin Chains globin -117HPFH 0 / A globin Huo et al. (2010) Annals NYAS

25 Survival Curves of Humanized Cooleys Anemia Mice A.B.

26 Sickle Hemoglobin

27 Sickle Cell Anemia Vascular occlusion causes tissue injury and pain Frequent Infections - Prophylactic antibiotics til age six Stroke and brain injury Splenic sequestration Acute chest syndrome Polyuria, Kidney failure, Priapism Leg ulcers Frequent Blood Transfusions, Iron Overload Can be cured by bone marrow transplantation, but…. James Herrick, Chicago 1910 Hemolytic Anemia

28 Sickle-Cell Anemia is a Molecular Disease Sickle-cell anemia patients have abnormally-shaped red blood cells The erythrocytes are crescent-shaped instead of disc-shaped The sickle cells pass less freely through the capillaries, impairing circulation and causing tissue damage A single amino acid substitution in the β-chains of Hb causes sickle-cell anemia Glu at position 6 of the β-chains is replaced by Val As a result, Hb S molecules aggregate into long, chainlike polymeric structures

29 Sickle-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.

30 Sickle-Cell Anemia is a Molecular Disease Figure Polymerization of Hb S.

31 Sickle hemoglobin tetramers aggregate inside the red blood cell forming long polymers when deoxygenated Sickle Hemoglobin Polymerizes

32 Vascular Occlusion of DeoxyHbS

33 Mouse Model of Sickle Cell Disease Produce a mouse that synthesizes high levels of human sickle hemoglobin--Transgenic Mouse Produce a mouse that synthesizes no endogenous mouse hemoglobin--Knockout Mouse Knockout-Transgenic Sickle Cell Mouse

34 First Generation Animal Model of SCD Transgenic model High level expression and synthesis of human HbS Sickle poymer formation under hypoxic conditions Little in vivo pathology under normoxic conditions Science 247:

35 Mouse maj - and min -Globin Knock-Out y h0 h1 h2 maj min y h0 h1 h2 tk pgk/NEO

36 Cloned Thalassemic Mice

37

38 Knockout-Transgenic Sickle Mouse Blood

39 Sickle Mouse Splenomegaly Normal Sickle Mouse

40 Sickle Mouse Survival Curves AGE (days) Sickle (C57Bl/6)Sickle OutbredC57Bl/6* PERCENT SURVIVORS *C57Bl/6 data copied from Goodrick, 1975

41 Cell Therapy Establish cell line from afflicted individual Correct the mutation Replace diseased cells by the corrected cells

42 Cell Therapy For Hemoglobinopathies Somatic cell biopsy Reprogram to Pluripotent Stem Cell Patient Specific induced Pluripotent Stem Cells (iPS) Repair DNA lesion In vitro differentiation Transplant back into mouse Tail Tip Fibroblasts Mutation Correction or Gene Addition Hematopoietic Stem Cells thalassemia or Sickle mouse


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