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Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph. D

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1 Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph. D
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
RBC Cooley’s Anemia is caused by the absence of b globin chains Sickle Cell Anemia is caused by single mutation in b globin

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

5 Human a and b Globin Loci
1   HS -40 Ch 16 5’HS: LCR b e d yb Gg Ag Alpha locus on Chromo 16 single embryonic zeta and two adult genes several pseudo genes Beta locus on Chromo 11 single embryonic, two fetal, two adult one pseudo gene Locus control region LCR Powerful regulatory region marked by Erythroid specific, developmentally stable, Dnase I HS Ch 11

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

7 Human Hemoglobin Switching During Development
Gestation Hematopoiesis Hemoglobin 2e2 a2e2 3 weeks 5 weeks 7 months Yolk Sac HSC HSC AGM Fetal Liver 22 HbF HbA HbA2 Primitive Hematopoiesis--YS Embryonic globins single zeta to alpha switch, alpha through to adult Definitive hematopoiesis AGM & Y.S. to fetal liver 1st switch of epsilon to gamma FL to BM 2nd switch to beta and delta HSC 22 22 Bone Marrow

8  Globin Gene Switching
. 100 G + A % of Total Beta Chains Man Birth Y Human Hb switching in the mouse Mouse definitive erythropoiesis reaches adult levels in utero 1 week before birth How would human globin genes be regulated mouse TG Mouse analysis Define sequences responsible for the high-level, erythroid-specific, and temporal expression 100 h1 maj + min % of Total Beta Chains Mouse Birth

9 Cooley’s Anemia Dr. Thomas Cooley, 1925
Thalassemia major -- homozygous b0 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 Populations Affected by b Thalassemia
Nature Genetics (2001) 2:245

11 Hemoglobin Switching During Development
Human e gG & gA d & b Mouse Birth bmaj & bmin bh1 & ey

12 Making Transgenic Mice By Pronuclear DNA Injection

13 GFP--Transgenic Mice


15 Blastocyst: Source of Embryonic Stem (ES) Cells

16 Homologous Recombination In Embryonic Stem Cells

17 Mouse -Globin Knockout

18 Human gA Globin Knock-in
ey bh0 bh1 bh2 bmaj bmin LCR tk bA hyg CRE ey bh0 bh1 bh2 bA LCR

19 Fetal to Adult Hb Switching in Human gbA Globin Knockin Mice
LCR eY bh0 bh1 bh2 bA 100 bA 80 eY bh1 60 % Total b-Like Globin RNA 40 20 9.0 9.5 10.5 14.5 16.5 18.5 N.B. Adult Age in Days

20 Cooley’s Anemia Mouse Model Knock-In of Human g0 Globin
bh0 bh1 bh2 bmaj bmin LCR ey bh0 bh1 bh2 bmaj bmin LCR b0 hyg CRE b0 ey bh0 bh1 bh2 bmaj bmin LCR

21 Heterozygous Human gb0 KI Mouse Model Anemia, Erythroid Hyperplasia, a Globin Precipitates, and Splenomegaly Wild Type gb0 KI

22 Human a2a1 Globin Knock-In
mHS-40 m m1 m2 h2a1 hyg tk mHS-40 m h2a1 hyg mHS-40 Cre m h2a1

23 Humanized Cooley’s Anemia Mice Survive at Birth on Human Fetal Hemoglobin
gb0/ gb0/gb0 gb0/gb0 JBC, 2009

24 Hemoglobin Switching in Humanized
gHPFHb0 and gHPFHdb0 Globin Knockin Mice A/A -117HPFH0/A -117HPFH0/A % b-like Globin Chains 100 % b-like Globin Chains 100 % β-like Globin Chains 100  globin 80 80  globin 80  globin 60 60 60 40 40 40  globin  globin  globin 20 20 20  globin 1 2 3 1 2 3 4 5 1 2 3 4 5 Age (wks) Age (wks) Age (wks) Huo et al. (2010) Annals NYAS

25 Survival Curves of Humanized
Cooley’s Anemia Mice A. B. 21/21 HPFH0/HPFH0 20 40 60 80 100 5 10 15 25 30 Adult CA Mice (n=32) Littermate Controls (n=102) Age (d) % Survival (n=17) (n=66) 21/21 HPFH0/HPFH0

26 Sickle Hemoglobin

27 Sickle Cell Anemia James Herrick, Chicago 1910 Hemolytic 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….

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

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
7 6 5 4 3 2 1 y h0 h1 h2 maj min pgk/NEO tk 7 6 5 4 3 2 1 y h0 h1 h2 pgk/NEO

36 Cloned b Thalassemic Mice


38 Knockout-Transgenic Sickle Mouse Blood

39 Sickle Mouse Splenomegaly
Normal Sickle Mouse

40 Sickle Mouse Survival Curves
Sickle (C57Bl/6) Sickle Outbred C57Bl/6* 100 80 PERCENT SURVIVORS 60 40 20 100 200 300 400 500 600 700 800 900 1000 1100 AGE (days) *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
 thalassemia or Sickle mouse Hematopoietic Stem Cells In vitro differentiation Transplant back into mouse Somatic cell biopsy Tail Tip Fibroblasts Reprogram to Pluripotent Stem Cell Patient Specific induced Pluripotent Stem Cells (iPS) Repair DNA lesion Mutation Correction or Gene Addition

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