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

tryan@uab.edu

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