Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics...
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Transcript of Hemoglobinopathies & Thalassemia 8/15/11 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics...
Hemoglobinopathies & Thalassemia
8/15/11
Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics
Introduction
Hemoglobin Structure and Function
HemoglobinopathiesThalassemiaSickle Cell Disease
UAB Animal Models
Red Blood Cells Contain Hemoglobin
Cooley’s Anemia is caused by the absence of b globin chains
Sickle Cell Anemia is caused by single mutation in b globin
a1
a2
b1
b2
RBC
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
Human a and b Globin Loci
5’HS: 5 4 3 2 1LCR
be dybGg AgCh 11
Ch 161 1
HS -40
Erythroid Development
>95% protein is hemoglobin
Mature Red CellBFU-ECFU-E
Pro- Basophilic Polychromatic Orthochromatic
HSC
Erythroblast
Reticulocyte
Hemoglobin content increases along erythroid differentiation
Erythropoiesis
Human Hemoglobin Switching During Development
Gestation
2e2
a2e2
22
22
22
HemoglobinHematopoiesis
Yolk Sac
HSC
HSC
Fetal Liver
Bone Marrow
AGMHSC
3 weeks
5 weeks
7 months
HbF
HbAHbA2
Globin Gene Switching.
G + A
Yh1
maj + min
Man
Mouse
% of Total Beta
Chains
100
100
Birth
Birth
% of Total Beta
Chains
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
Nature Genetics (2001) 2:245
Populations Affected by b Thalassemia
e gG & gA d & b
bh1 & ey bmaj & bmin
Hemoglobin Switching During Development
Human
Mouse Birth
Making Transgenic Mice By Pronuclear DNA Injection
GFP--Transgenic Mice
Blastocyst: Source of Embryonic Stem (ES) Cells
Homologous Recombination In Embryonic Stem Cells
Mouse -Globin Knockout
KO
-Globin Knockout
Human gA Globin Knock-in
ey bh0 bh1 bh2 bmaj bmin
hygbA
CREbAey bh0 bh1 bh2
LCR
LCR
tk
Fetal to Adult Hb Switching in Human gbA Globin Knockin Mice
0
0.2
0.4
0.6
0.8
1
9.0 9.5 10.5 14.5 16.5 18.5 N.B. Adult
Age in Days
% T
ota
l b
-Lik
e G
lob
in R
NA
0
20
40
60
80
100
eY bh0 bh1bh2LCR bA
bh1eY
bA
Cooley’s Anemia Mouse ModelKnock-In of Human g0 Globin
hygb0
CRE
ey bh0bh1bh2 bmaj bmin
LCR
b0ey bh0bh1bh2
ey bh0bh1bh2 bmaj bmin
LCR
LCR
ey bh0bh1bh2 bmaj bmin
LCR
Heterozygous Human gb0 KI Mouse ModelAnemia, Erythroid Hyperplasia, a Globin Precipitates, and Splenomegaly
Wild Type
gb0 KI
Human a2a1 Globin Knock-In
mHS-40 m m1 m2
mHS-40 m
CremHS-40 m
hyg tkh2a1
h2a1 hyg
h2a1
Humanized Cooley’s Anemia Mice Survive at Birth on Human Fetal Hemoglobin
JBC, 2009
gb0/+ gb0/gb0 gb0/gb0
Hemoglobin Switching in Humanized gHPFHb0 and gHPFHdb0 Globin Knockin Mice
0 1 2 30
20
40
60
80
100
% b
-lik
e G
lob
in C
hai
ns
Age (wks)
globin
globin
A/A
% b
-lik
e G
lob
in C
hai
ns
0
20
40
60
80
100
4 50 1 2 3
-117HPFH0/A
Age (wks)
0
20
40
60
80
100
Age (wks)
% β
-lik
e G
lob
in C
hai
ns
4 50 1 2 3
globin
-117HPFH0/A
globin globin
globin globin
Huo et al. (2010) Annals NYAS
Survival Curves of Humanized Cooley’s Anemia Mice
21/21 HPFH0/HPFH0
0
20
40
60
80
100
0 5 10 15 20 25 30 Adult
CA Mice
(n=32)
Littermate Controls(n=102)
Age (d)
% S
urv
ival
0
20
40
60
80
100
0 5 10 15 20 Adult
CA Mice(n=17)
Littermate Controls(n=66)
Age (d)
% S
urv
ival
21/21 HPFH0/HPFH0
A. B.
Sickle Hemoglobin
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
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
Sickle-Cell Anemia is a Molecular Disease
Figure 15.33 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.
Sickle-Cell Anemia is a Molecular Disease
Figure 15.33 Polymerization of Hb S.
•Sickle hemoglobin tetramers aggregate inside the red blood cell forming long polymers when deoxygenated
Sickle Hemoglobin Polymerizes
Vascular Occlusion of DeoxyHbS
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
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: 566-568
Mouse maj- and min-Globin Knock-Out
1234567y h0 h1 h2 maj min
1234567y h0 h1 h2
tkpgk/NEO
pgk/NEO
Cloned b Thalassemic Mice
Knockout-Transgenic Sickle Mouse Blood
Sickle Mouse Splenomegaly
Normal Sickle Mouse
Sickle Mouse Survival Curves
0
20
40
60
80
100
0 100 200 300 400 500 600 700 800 900 1000 1100AGE (days)
Sickle (C57Bl/6) Sickle Outbred C57Bl/6*
PE
RC
EN
T S
UR
VIV
OR
S
*C57Bl/6 data copied from Goodrick, 1975
Cell Therapy
Establish cell line from afflicted individual
Correct the mutation
Replace diseased cells by the corrected cells
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 TipFibroblasts
Mutation Correctionor
Gene Addition
Hematopoietic Stem Cells
thalassemia or Sickle mouse