1. The Molecular Basis of Hemoglobin
Disorders and the Sickle
Hemoglobinopathies
Babette B. Weksler MD
April 12, 2012
No conflict of interest disclosure
2. Hemoglobin Genes and Products
Hgb is a tetramer of the products of two
genes, alpha globin and non-alpha globin.
2 identical a chain genes on chromosome 16p
g, d, b chain genes clustered on chromosome 11p
Hemoglobin Genotype Amount
Hb A a2b2 96%
Hb A2a2d2 3%
Hb F a2g2 1%
If a-chain synthesis is decreased, see Hb Barts = g4 or Hb H =b4
3. Two Human Globin Gene Clusters
Exist on Different Chromosomes
EMBRYONIC FETAL ADULT
α2ε2 δ2ε2 δ2γ2 α2γ2 α2δ2 α2β2
Gower 1 Gower 2 F A2 A
Portland
5. Some Human Hemoglobins
Hemoglobin Structure Percentage in Adult
Adult Hemoglobins:
A a 2b 2
96.5
A2 a2d2 2.5
F a2g2 <1.0
Embryonic Hemoglobins:
Gower 1 z 2e 2 0
Gower 2 a 2e 2 0
Portland z 2g 2 0
H b4 0
Bart’s g4 0
6. Oxyhemoglobin A
- heterotetramer:
a 2 b2
- a chain: 141 AAs
- b chain: 146 AAs
- chains: weak, Van
der Waals forces
- covalently-linked
heme group via His
- no intra- or
interchain –SS- bonds
9. Hemoglobin Dissociation
Shift
The affinity of Hgb
sick patient
for O2 is regulated
by temperature,
pH, and
bisphosphoglycerate
(BPG, or 2,3 DPG)
10. Hemoglobin Abnormalities
1. Quantitative defects: imbalance of chain
synthesis thalassemia syndromes
2. Qualitative defects: additions, substitutions or
deletions of amino acids e.g. sickle cell disease
or altered oxygen affinity, stability
3. Failure to silence genes: hereditary persistence
of fetal hemoglobin (HPFH)
11. Case Presentation
History:
20 yo Afro-American male
10 year H/O leg ulcers, otitis media,
pneumonia, and attacks of abdominal pain
with jaundice
1 prior episode joint swelling and pain
Presented with fever, cough and anemia
Physical Exam:
pallor, scleral icterus, round and oval scars over
lower extremities
bilateral rales, dullness both bases
cardiomegaly with soft systolic murmur
12. Case Presentation, continued
Urinalysis: trace albumin; few granular casts
Peripheral blood: WBCs 15,250/mm3
72 P, 1 Band, 15L, 7 M, 5 Eo
RBCs 2,800,000/mm3
74 nRBCs/100 WBC
13. Case Presentation, continued.
This was the first description of
sickle cell anemia, 100 yrs ago in
a dental student from Granada
who presented with pneumonia to
Cook County Hospital in Chicago
in 1909.
James R. Herrick “Peculiar
elongation and sickle-shaped red
blood corpuscles in a case of
severe anemia.” Arch Int Med
6:517, 1910.
“…some change in the composition
of the corpuscle itself may be
the determining factor”
15. Itano, Singer, Wells, and Pauling - 1949
demonstated abnormal electrophoretic
mobility of Hb S and predicted molecular
charge alteration
16. Linus Pauling’s 1949 Prediction of
Molecular Basis of Sickling
"Let us propose that there is a surface region on the . . . sickle cell
anemia hemoglobin molecule which is absent in the normal molecule
and which has a configuration complementary to a different region of
the surface of the hemoglobin molecule. . . Under the appropriate
conditions [as in low oxygen or air pressure], then, the sickle cell
anemia hemoglobin molecules might be capable of interacting with one
another at these sites sufficiently to cause at least a partial
alignment of the molecules within the cell, resulting in the
erythrocyte's . . . membrane's being distorted to accomodate the
now relatively rigid structures within its confines.”
Longitudinal “liquid Cross section of
crystal” crystal stack
23. Sickle Cell Disease - Clinical
SS disease in 0.15% African Americans (89,000 in US)
60% SS/SBOthal, 40% SC/SB+thal at birth
Sickle trait (SA) in 8% in US, up to 50% in Africa
Also SCD in other Mediterranean peoples: e.g. Sicily
Greece, Arabian peninsula, India
Chronic hemolytic state with short rbc survival
Vascular obstruction, inflammation, hypercoagulability
End organ damage: splenic
infarction, cardiomyopathy, bone
infarction, stroke, renal dysfunction, skin
ulcers, pulmonary hypertension, retinopathy, gallstones
24. Natural History of Sickle Cell Disease
1960’s Disease of childhood
1973 Median age at death = 14 yr (1/6 cases>50)
1990 Survival >20 yr of 85%, if SC 95%
1994* Median age at death 42 for men, 48 for women
2001 Median age at death 53 for men, 58 for women
Main causes of death: organ failure 18%, stroke
22%, acute event 32%
* when hydroxyurea became available
25. Bone infarcts and marrow expansion in SCD
Fishbone vertebrae Infarction of humeral head
30. Modulating Factors in Sickle Cell Disease
High Hb F (interferes with sickling)
Other non-S hemoglobins
Beta thalassemia trait/ alpha thalassemia trait
Beta globin haplotype: Senegal>Benin>Bantu>Cameroon
(correlates with Hb F levels and to X-linked F-cell
production locus)
31. a-Thalassemia: 1 or 2 Gene Deletion
if combined with SS, ameliorates phenotype
by decreasing MCH
32. Sickle/Beta+ Thal has milder phenotype due to low
MCV, residual Hb A and lower Hb S content but
Sickle/Beta-zero Thal is as severe as SS
33. Hereditary Persistence of Hb F
Two types:
Homogeneous: similar level Hb F in all rbc
Hb F may be 30-35% of total Hb
Heterogeneous: variable level of Hb in rbc
but high percentage F cells (lower overall
percentage of Hb F)
Normal O2 delivery
34. Hb S Haplotype Distribution
Senegal haplotype has
mildest disease, Hb F
CAR has most severe
disease and lowest Hb F
(Bantu)
Benin is in between
Asian and Arab
haplotypes are
associated with milder
disease
35. Hemoglobin O Arab
Beta globin mutation B121glut-->121lys
HbO Arab/Hb A is normal
Hb O Arab/Bthal is mildly anemic,
mild microcytosis, splenomegaly
HbO Arab/Hb S is severely anemic
similar to Hb SS
Rare hemoglobinopathy
36. Hemoglobin Percentages in Sickle Cell
and Hb C Syndromes
Syndrome % Hb A Hb S Hb F Hb A2 HbC
Sickle Cell Anemia 0 75-95 2-25 <3.5 0
Sickle-Beta0 Thal 0 80-92 2-15 3.5-7 0
Sickle-Beta+ Thal 5-30 65-90 2-10 3.5-6 0
Sickle-HbC 0 45-50 1-5 ND 45-50
Sickle Cell Trait 50-60 35-45 <2 <3.5 0
Hb C Trait 50-60 0 <2 <3.5 35-45
Hb CC Disease 0 0 <2 <3.5 98
38. Hemoglobin C
Single mutation b 6glulys Origin in Ghana
Forms crystals that dissolve on deoxygenation
RBC lose K+, tend to dehydrate
Target cells, desiccocytes, low MCV
Trait is asymptomatic, Hb CC mild hemolysis
Hb level 8g to normal, splenomegaly in 33%
Hyperviscosity may occur if Hb high
Retinopathy is common
Hb SC is more clinically severe than Hb CC
but less than SS
39. Hemoglobin SC Disease
% Hb S is greater than in sickle trait, so more severe
Crises less frequent than SS, Hb level higher
and ranges from ~8 g/dL to normal.
RBC lifespan longer than in SS
RBC dehydration
High rate of aseptic necrosis and retinal disease
Splenomegaly common
Anemia, crises common in pregnancy
Responds to hydroxyurea
40. Recent Hemoglobinopathy Cases at NYPH
Acute hepatic sequestration in SCD
Total splenic infarct (Sickle/B+ thal), hemoperitoneum
Pulmonary hypertension with repeated PEs
Hyperbilirubinemia in Sickle/B+ thal with Gilbert’s
syndrome
Mycoplasma pneumonia
Sudden death after severe pulmonary HT
Repeated SC crises in pregnancy
Avascular necrosis of hips in SS treated with THR
Iron unloading in SCD with concomitant HepC
Bloodless orthopedic surgery in SS Jehovah’s witness
using hydrea +Epo
Chemotherapy of lymphoma in SS patient with HIV
Exchange Tx for Acute Chest Syndrome
41. Complications of Sickle Cell Disease in
the Older Patient
Cardiomyopathy
Pulmonary hypertension
Nephropathy
Bone marrow failure
Aseptic necrosis of joints: hips, knees,
shoulders
Retinopathy
42. Survival in SCD Patients with Pulmonary Hypertension
Mehari, JAMA 307:1256, 2012
43. Predictors of Mortality in Sickle Cell
Disease
Persistent high WBC and platelet count
Acute chest syndrome
Pulmonary hypertension
Renal impairment
Hb < 7 g/dL
44. Coagulopathy in SCD
Activated vascular endothelium = prothrombotic
Decreased vascular NO favors platelet activation
Prothrombotic microparticles from RBC and platelets
Increased blood tissue factor level
VWF activating factor increased (Activated A1 domain
that binds platelets)
Total VWF increased
High WBC and positive surface PS also favor thrombosis
Pulmonary hypertension favors silent pulmonary emboli
45. Mainstays of Therapy in SCD
Analgesics
Fluid, Oxygen
Blood
Immunizations
Penicillin
Hydroxyurea
46. Newer Therapeutic Approaches in Sickle
Cell Disease
Newborn Screening vs antenatal diagnosis
Transfusion in children at stroke risk
Hydroxyurea or Hydroxyurea + Epo
Oral iron chelation (Exjade = deferasirox)
Nitric Oxide (NO), Butyrate/ Arginine (NO inducers)
Clotrimazole/senicapoc (Gardos channel inhibitors)
Sildenafil
Bone Marrow Transplantation
47. Hydroxyurea in Sickle Cell Disease
Raises Hb F --- decreases Hb S
polymerization in rbc
Decreases sickling, crises, ACS
Decreases inflammatory cytokines
Lowers WBC
Induces eNOS-cGMP pathway in endothelium
Reduces procoagulant state of endothelium
Decreases stroke incidence in SCD children
48. Hydroxyurea
• Inhibits ribonucleotide reductase,
thereby favoring synthesis of more
slowly dividing red cell progenitors,
which contain high levels of Hgb F
• Its metabolism may also result in
production of nitric oxide (NO)
• Improved RBC survival, and reduced
sickling
• Orally active
• 60% of patients respond with
decreased pain, pulmonary events,
hospitalization
Platt,O. N Engl J Med 358: 1362,
2008
52. Transfusion
High level matching to avoid alloimmunization
Exchange vs simple transfusion
Blood viscosity
Pre-operative transfusion
goal to reduce Hb S to 30%
Hb ~ 10
Phlebotomy
Iron overload management
53. Iron Chelation: Deferasirox (Exjade)
Oral, T1/2 8-16 hrs, iron excreted in stool
Use at 20-30 mg/kg/d
Effective in thalassemia and sickle cell
if transfusion need is not very high
Adverse effects:
GI symptoms, rash, increased Cr, rarely
ALT
Need longterm compliance to unload Fe
Very expensive
54. Is Gene Therapy Feasible for SCD?
Need for safe and effective vector
Requirement for LCR as well as coding
region for human bA-globin gene
Harvest sufficient HSC from recipient
Stable expansion of transduced HSC
55. Reprogramming Somatic Cells
to Treat Sickle Cell Disease
Inducible
pluripotent
stem cells
HOXB4
required
for
engraftmen
Hanna et al (Jaenisch) Science t
(5 retroviruses + electroporation) 318:1920,
2007.
56. Allogeneic Hematopoietic SCT for Sickle
Cell Disease
Hsieh et al NEJM 361:2309 Dec 10, 2009
9/10 Adults (16-45) successfully transplanted from HLA-
matched sibling; all alive p 15-54 mo
Conditioning with alemtuzumab, TBI x1, sirolimus
No GVHD
Chimerism >91% myeloid, 53% T-cell
Post-SCT Hb mean 12.6 g/dL, bili 0.7, LDH 186
No strokes post SCT, stable PH, stable renal Fxn.
Adverse events: 1 CMV reactivation, arthralgias,
sirolimus-related pneumonitis. 1 patient needed second
SCT (successful)
57. Gene Therapy for SCD with Autologous SC
Pilot study MSKCC-NYPH-NY Blood Center
P.I. Michel Sadelain starting 2012
Harvest autologous BMSC with plerixafor
establish safety and efficacy of harvest
Ex-vivo gene therapy with normal beta- or
gamma-globin genes at MSKCC in GMP facility
Demonstrate normal rbc progeny result
Reinfusion of transduced SC into donor after
BM ablation
58. Tailored Lentiviral Vectors May Permit High
Stable Expression of Normal Beta-Globin in
Thalassemic and SCD Mice and Human SC
S. Rivella’s lab found adding an ankyrin insulator
to lentiviral vectors containing the normal human
beta-globin gene improves transcription of beta-
globin mRNA and increases hemoglobin
production.
Can predict how many copies of vector would give
optimal production of Hb in individual recipients
by simple test.
One patient with HbE-Beta thal has been
transplanted successfully.
Breda et al Plos One 7:e32345 Mar 2012
59. Need for in vitro tests predictive of blood
rheologic behavior in SCD
mouse models have many limits
SCD clinical phenotype is highly variable although
molecular defect is the same
Can in vitro testing of blood predict severe vs mild
disease?
Can in vitro testing predict effects of physiologic
changes or pharmacologic interventions?
60. In Vitro Microfluidic Model of Microvasculature
Brightfield
Red= EC membrane + nuclei
Human HUVEC or MVECs seeded 36 hr on fibronectin in channels
Tsai M et al JCI 122:408 2012
61. Endothelialized Microfluidic Channels Express NO and
VE-Cadherin under Physiologic Shear Flow Conditions
Brightfield NO
VE-cadherin (EC junctions)
Tsai M JCI 122:408 2012
62. Decreased flow
and occlusion due
to activation with
TNFa
Maximal effects with
TNF treatment of both
blood and EC
63. Microfluidics Shows Beneficial Effect of Hydrea on
Flow Velocity and Vasoocclusion in Sickle Cell Disease
Whole blood
flow velocity
Percent occlusion
of microchannels
Tsai M, JCI 122:408
2012
64. Fig. 1 Microfluidic device for studying sickle cell blood flow conductance.
Wood D K et al. Sci Transl Med 2012;4:123ra26-123ra26
Can study flow, occlusion and effects of oxygenation
Published by AAAS
65. Rheodynamics of Severe Sickle Phenotype and Benign
Sickle Phenotype Blood Samples
Benign Sickle Sample Severe Sickle Sample
Wood DK Sci Trans Med 4:123ra26 2012
66. Blood Conductance Decrease after Deoxygenation
Reflects Decrease in Flow Velocity
Normal Hb S A
Hb S S Hb S S
Hb S S Hb S S
Wood et al Science Translat. Med. 4:123ra26, 2012
67. Rate of Blood Conductance Decrease is Modulated
by 5-Hydroxymethylfurfural that Increases Hb
Oxygen Affinity
5-HMF improves phenotype in mouse model of sickle cell disease
Wood DK, Sci Trans Med 4:123ra26 2012
68. New Handles on Hemoglobinopathies
Microfluidic devices using whole blood flowing
through 3D channels lined with fibronectin or
endothelial cells can measure behavior of rbc,
cytokines, leukocytes/platelets under physiologic
flow conditions in small blood samples.
Effects of oxygen concentration, cytokine
activation of cells, or drugs can be evaluated
Predictions of rheologic behavior of blood cells and
their interactions with each other and the vessel
wall in vivo can be made that correlate with clinical
situations and might guide management of individual
patients
