Hémoglobinopathies

Jacques Elion

Module de Médecine MoléculaireAnnée universitaire 2012-13

Faculté de MédecineInstitut national de la santé et de la recherche médicale

Hemoglobinopathies :

• pathologie monogénique la plus commune au monde

• transmission récessive(rare cas transmission dominante)

• Hétérogénité clinique : du porteurasymptomatique à la mort foetale in utéro

Tokyo 99

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Globin Gene Organization

Expression of Different Globins

Classification:Abnormal hemoglobins: structural abnormalitiesThalassemias: synthesis defects

the difference is not in geneticsPrimary defects are similar (point mutations, deletions…)Some mutations give rise to a mixed defect

the difference is in pathophysiology:The protein can be functionally normal/abnormalThe protein is quantitatively deficient/absent

Hb A = 2 2

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1.the thalassemias

decreased synthesis of the or globin chains

- and -thalassemias

- two types (i.e. ° or +-thal)

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1.the thalassemias

- -thalassemias - -thalassemias - thalassemias with an abnormal Hb

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Pathophysiology of Pathophysiology of --thalassemiathalassemia

/ chain disequilibrium

Excess of -chains

Inclusion bodies,ineffective erythropoiesis, hemolysis

Anemia

Proteolysis

Persistence of fetal hemoglobin- cell selection- absolute increase of HbF ?

Bone Iron Jaundice Infectiondisease loading

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CCTCACCC

CCACACCC

CCAATATAAA

-106 -91 -76 -31

ATG

NullNullMutationsMutations

(FS,NS)

CapCapSiteSite

Initiation Initiation CodonCodon

CleavageCleavagemutationsmutations

AATAAA

Stop Stop CodonCodon

SpliceSpliceMutationsMutations

Mutations Mutations PromotorPromotor

--thalassemias: thalassemias: point mutations are the most common causepoint mutations are the most common cause

Cao A, Moi P.

Regulation of the globin genes.Pediatr Res. 2002 Apr;51(4):415-21.

Rarely, -thalassemias result from deletions

- of the structural genes

- of major regulatory regions

-thalassaemia major:Cooley’s anemiaa life-threatening transfusion-dependent condition

more than 200 -thal mutations described to date

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-thalassaemia intermedia:a mild transfusion-independent condition mayresult from mild mutations

D.J. Weatherall, Nature Reviews Genetics, 2001, 2/245-255 InsermU665

Allelic combination* participates to clinical heterogeneity*compound heterozygozity

D.J. Weatherall, Nature Reviews Genetics, 2001, 2/245-255

Mild -thalalleles are indicated in bold

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1.the thalassemias

- -thalassemias - -thalassemias - thalassemias with an abnormal Hb

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--thalassemiasthalassemias

FunctionalFunctional--genesgenes

44

33

22

22

11

00

normalnormal

HeterozygousHeterozygous ++ --thalthal

HomozygousHomozygous ++--thalthal

HeterozygousHeterozygous 00--thalthal

Hb H Hb H diseasedisease

HydropsHydrops fetalisfetaliswithwith Hb BartHb Bart’’ss

Most commonly results from deletions InsermU665

Hydrops Fetalis

Chui DH, Fucharoen S, Chan V.

Blood. 2003 Feb 1;101(3):791-800.

In most cases, -thalassemias result from large deletions

+

+

0

0

0

(-)+ defects result from unequal crossing-over

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Geographical distribution of -thalassemia

D.J. Weatherall, Nature Reviews Genetics, 2001, 2/245-255Inserm

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3. abnormal hemoglobins

- classification - common Hb variants - sickle cell disease

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Central cavity:2,3-DPG bindingabnormal affinity

11 contacts:unstable Hbs

2

1

2

12 contacts:abnormal affinity

Heme pocket:Hb Mabnormal affinityunstable Hbs

Surface:No functional consequencesEXCEPT Hb S

Classification of hemoglobin variants

1

Haemoglobin S = Sickle Cell Disease

Other frequent haemoglobin variants such as HbC or HbE in the homozygous state result in mild haemolyticanaemia or are asymptomatic

BUT

compound heterozygosities such as- S/C- E/-thal

produce moderate to severe phenotypes

OBVIOUSLY such associations are most oftenencountered in populations in which the two types of defect coexist

Frequent Haemoglobin variants

Original geographic distribution of HbS

Hb S

Malaria

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HbS in the “New World”

Original geographic distribution of frequenthaemoglobin variants

HbC

HbE

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Geographic distribution of haemoglobin E

gene frequency

<.02

.02-.10

> .10

2nd most commonvariant after HbS

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Haemoglobin E

HbE heterozygotes are asymptomatic- with normal Hb, but microcytosis (average MCV 73 fl)- HbE is expressed at 30-35%

HbE homozygotes are also asymptomatic- Hb is normal or slightly decreased- microcytosis (average MCV 67 fl)

HbE/-thal compound heterozygotes present withan often severe thalassemic syndrome

HbE results from a mixed defect which associates a Hb variant and a -thalassaemia determinant

Haemoglobin E defect

HBB:c.G79AHBB p.Glu26Lys

normal splicing

alternative splicing

part of the mRNA precursor is abnormally spliced, resulting in a +-thal allele

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Geographic distribution of haemoglobin C

gene frequency

.01

.02

.04

.08

.10

3rd mostcommon variant

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5 ' 3 'LCR G A

Chromosome 11

S Glu

chaîne de globine S

l

polymérisation

GTG codon 6Gène SA

Val

oxy HbS desoxy HbS

globule rouge

Falciformationdéformationrigidificationfragilisation

Vaso-occlusion

Anémiehémolytique Inserm

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HbS

Hb S : Drépanocytose

HbS2S2

deoxyHbS polymer

S

S

S

S

S

S

S

85 Phe88 Leu

6 Val

6 Val

85 Phe88 Leu

6 Val

B

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Globules rouges normaux et falciformés

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H2O2

-O2

Hb S

O2

protéinebande 3

spectrine

ankirine

corps de Heinz

Libération devésicules

*OH

IgG

OH

Fe

protéinebande 3

OH

hémichromes

Fe+3

Microenvironnementoxydant

Distorsion

polymères

Ca+2

Canal GardosK+

K+Cl-

CotransportK-Cl

Activation des transport ioniques et déshydratation Inserm

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Steinberg M.H., N Engl J Med 1999; 340:1021-1030

Drépanocytose :

Le schéma physiopathologique classique est insuffisant pour expliquer la crise vasoocclusive car le délai à la polymérisation de la désoxyHbS est supérieur au temps de passage dans la microcirculation

Toute cause de ralentissement circulatoire peut constituer un facteur déclenchant

…. tonus vasculaire…. adhérence cellulaire

Drépanocytose

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-nouvelles données physiopathologiques :

- adhérence des GR à l’endothélium

- activation cellulaire et rôle de l’hypoxie

- environnement pro-inflammatoire

- dysfonctionnement vasculaire

- un seul médicament actif : hydroxyurée (1995)

Mohandas & Evans, Blood 1984

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Adhérence des GR drépanocytairesà l’endothélium :

Trois questions:

- quel territoire vasculaire ?- quels globules rouges ?- quels mécanismes moléculaires ?

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Kaul et al,Proc Natl Acad Sci USA 1989

Vessel diameter (m)

Adherent red blood cells/100m2

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Adhesion des GR drépanocytairesà l’endothélium :

Trois questions:

- quel territoire vasculaire ?

veinules post-capillaires- quels globules rouges ?- quels mécanismes moléculaire ?

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Adhesion des GR drépanocytairesà l’endothélium :

Trois questions:

- quel territoire vasculaire ?- quels globules rouges ?

Gr jeunes : "réticulocytes de stress"(cellules vieillies)

- quels mécanismes moléculaires ?Inserm

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Les réticulocytes de stress expriment des protéinesd’adhérence normalement présentes sur les précurseursérythroïdes dans la moelle

Elion & LabieHématologie 1998

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Matrice sous-endothéliale

IgG

FcR

GR vieilli

laminine

VLA4VCAM1

Reticulocyte de stress

CD36

CD36

TSP

GP1b/2b3a-like

HMW vWF

GP1b-likecellules endothéliales

BCAM/Lu

Erythropoièse accrue

Oxydationdéshydratation

Activation endothéliale

plaquettes

Flux sanguin

Les acteurs de l’adhérence cellulaire dans la drépanocytose

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Les acteurs de l’adhésion cellulaire dans la drépanocytose

- réticulocytes de stress +++- GR matures- GR vieillis

- cellules endothéliales activées- structures sous-endothéliales

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In Sickle Cell Disease

abnormal cell adhesion to the endothelium plays a major in targetting vaso-occlusion

from JP Cartron SCADHESION project C Le Van Kim, PL Tharaux, J Elion

….but so does the vascular toneInserm

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Tonus vasculaire

-

Deux médiateurs produits par l ’endothélium sont élevés dans la drépanocytose :

Endothéline-1 vasoconstricteur

NO vasodilatateurmais l’hyperproduction de NO est «futile»car destruction du NO par l’hémolyse

vasoconstrictionInserm

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blood

subendothelium

Interactions between the endothelium and circulatingblood cells play a central role in vascular biology

EC

RBCs

EC

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Barrier between blood and the subendothelial structuresCrucial role in controlling

vascular tonethrombogenesisvascular remodeling

Numerous secretory propertiesanti- / pro-coagulant proteinsvasodilators (NO) / vasoconstrictors (Et-1)growth factors

Expressing membrane receptorscell adhesion

Activated by various stimulihypoxiainflammatory cytokines

The vascular endothelium

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Barrier between blood and the subendothelial structuresCrucial role in controlling

vascular tonethrombogenesisvascular remodeling

Numerous secretory propertiesanti- / pro-coagulant proteinsvasodilators (NO) / vasoconstrictors (Et-1)growth factors

Expressing membrane receptorscell adhesion

Activated by various stimulihypoxiainflammatory cytokines

The vascular endothelium

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Endothelium-derived nitric oxyde (NO)is a major actor of vascular biology

- vasodilation- inhibition of platelet activation- endothelial expression of adhesion molecules

NO activates the soluble guanylate cyclase (sGC) in smoothmuscle cells vasodilation

Haemoglobin is the most potent NO scavengerparadox at NO working as a diffusible effector and

its proximity to large amounts of Hb in the RBC

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but physiologically, physical and dynamical barriersprevent NO destruction by Hb

NO release in hypoxic tissues InsermUMR 665

Gladwin et al. Nature Medicine, 2003

but physiologically, physical and dynamical barriersprevent NO destruction by Hband rather Hb in the RBC acts as a NO transporter

NO release in hypoxic tissues

Gladwin et al. Nature Medicine, 2003

Nitrites may act as a second important NO transporter

Paracrine vasodilation

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Haemolytic anaemiasNO reacts 1,000 times more rapidly with free Hb than with RBCs

NO scavenging is complete

Common vascular complicationsPulmonary hypertension, cutaneaous ulceration, acute and chronicrenal failure

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Kato et al Blood Reviews, 2007, 21:37-47

Kato et al Blood Reviews, 2007, 21:37-47

Two overlaping sub-phenotypes in SCD?

Therapeutic implications

NO production

NO destruction

NO response

Kyle Mack and Kato Int J Biochem Cell Biol, 2006, 38:1237-43

4. Genetic modifyers

- in sickle cell disease - in -thalassemia

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SICKLE CELL ANEMIA

Alleviating genetic factors

• -thalassemia• high output of fetal hemoglobin (HbF)• lowered expression of the S gène (India)

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- SCD onset parallels extinction of HbF expression during development

- High HbF levels are statistically associated with a milderclinical expression of the disease

- HbF prevents growth of the deoxyHbS polymer

FetalFetal hemoglobinhemoglobin and SCDand SCD

rationale for the treatment of severe forms of SCD by hydroxyurea

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Propensity for maintaining high HbF levels is under genetic control

- at the cellular level (F cell number) - at the molecular level (HbF/F cell)

polygenic modulationdeterminants within the -Gb cluster (HPFH is rare)polymorphisms = RFLPs, microsatellites

FetalFetal hemoglobinhemoglobin

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3. Genetic modifyers

- in sickle cell disease - in -thalassemia

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Excess of -globin chainsis the pathophysiological hallmark of -thalassaemias

Secondary modifiers -chain excess mild phenotypes

- -thalassaemia- increased fetal -chain production (high HbF)

linked to the -cluster (chr 11)unlinked (chr X: FCP locus?, chr 6: HBS1L-MYB,

chr 2: BCL11A, chr 19: KLF1)

-chain excess severe phenotype- -globin gene expansion

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