Hemoglobinopathies

• Defined as diseases that result from a genetically

determined abnormality of the structure or synthesis of the
hemoglobin molecule.
• The abnormality is associated with globin chains while the
heme synthesis is normal.

• It can be a qualitative defect in the globin chain (structural

abnormalities) such as sickle cell anemia or a quantitative
defect in the globin synthesis ( thalassemia).
 The Qualitative Defect
 The qualitative (structural) defect result from genetic mutations in
the coding region of the globin gene.

 Resulting in amino acid deletion or substitution in the globin protein

chain.

 Cause structural variation in one of the globin chain classes

(structural hemoglobin variants).

 Clinical expression varies depending on the nature and site of the

defect, in addition to the class of globin chain involved ( α , β , γ
or δ ).
Any of the globin chain classes can be affected, however, the number of identified β-
chain mutations exceeds the number identified of α, γ or δ chains.

Why….???
 γ -chain in Hb F is expressed to significant level only during fetal
development, therefore, variants Hb F are unlikely to be detectable after
3-6 months of age.
 δ -Chain in hemoglobin A2 which is usually with a concentration less
than 3% of the total hemoglobin in adult, accordingly, Hb A2 variants
are also unlikely to cause clinical complications.
 α -chain gene is duplicated on each of chromosome no. 16, thus ,
mutation in a single α-locus leads to production of only small amount of
abnormal Hb.
 β-gene present in a single copy on each of chromosome no. 11.
 Nomenclature
• The first Hb discovered was called Hb S due to sickle-
shaped erythrocytes.

• Hb M was given to variant that form methemoglobin.

• Hb F was given to fetal hemoglobin

• Other Hb variants were discovered and given a successive

letters in an alphabetical order beginning with C.

• Letter A was already in use.

Letter B was not used to avoid confusion with ABO blood
group system.
Standardized Hb nomenclature

It includes:
The mutated chain.
 The position of the affected amino acid.
 The helical position of the mutation.
 The amino acid substitution.

Hb S is designated by: β6(A3)Glu → Val

The mutation is in beta chain, amino acid in the sixth position of A3 helix position.
The amino acid glutamic acid is being substituted by valine
 Cont…

If the individual is homozygous for the gene coding to the globin chain (for example β-chain), no Hb A is produced and the term disease or anemia is used.
(Sickle cell anemia)

If one of the genes coding for the chain (β-chain) is normal and the other is coded for structural variant , both Hb A and the abnormal Hb are produced and the term trait is
used to describe the heterozygous disorder.
(Sickle cell trait)
Most structural variants result in no clinical or hematological abnormality.

Mutations result in clinical manifestation if the amino acid substitution occurs at a critical site within the molecule that leads to an alteration in the
hemoglobin molecule's:
1- Solubility.
2- Function (oxygen affinity).
3- Stability.
 Altered solubility
If a non-polar amino acid is substituted a polar
one near the molecule's surface, the solubility
of Hb molecule can be affected.

Hemoglobin S and C are examples of this type

of substitution.
 Altered function
Amino acid substitution may cause:

- Methemoglobin Hb M ( cannot bind oxygen ).

- Impairment in oxygen binding site (mutation near the

heme pocket).

- Impairment in 2,3-DPG binding site.

 Altered stability
Amino acid substitutions that reduce the stability of Hb
tetramer result in unstable Hb.
This mutation usually disrupt hydrogen bonding or
hydrophobic interaction which hold the tetramer together.

disruption of tetramer integrity.

Hb denatures, aggregates and precipitates as Heinz bodies.

Congenital Heinz Body Hemolytic Anemia

• Most common symptomatic Hb variant.
• Non-polar valine is substituted for polar glutamic acid at the
sixth position in the A3 helix of β-chain.
β6(A3)Glu → Val

• This substitution occurs at the surface of molecule leading to:

 Alteration in the solubility.
 Tendency of Hb S to polymerize into rigid aggregates forming
crescent shape. Polymerization occurs upon deoxygenation.

Polymerization is reversible upon reoxygenation

Sickling delay time: time of deoxygenation required for
formation of significant amount of Hb S polymers.

• Depends on:
1. Hypoxia
2. Acidity
3. Hyper-tonicity
4. Hb composition (proportion of Hb A , Hb S ,
Hb A2 and Hb F) ….. WHY ???
The spleen and kidneys provide a sufficiently hypoxic, acidic and hypertonic
microenvironment to promote Hb S polymerization and sickling
 Irreversible Sickled Cells (ISC)
Repeated cycles of sickling result in:

*Decouple the lipid bilayer from the membrane skeleton

*Leaky and rigid membrane
*Impaired cell deformability

ISC whether oxygenated or deoxygenated

• About 10 – 50% of circulating erythrocytes in
sickle cell anemia are ISC.
• ISC has relatively high MCHC.
• ISC are removed in spleen and liver by
phagocytosis.
• ISC account most if not all of the sickle forms
on a peripheral blood smear…. WHY ???
 At peripheral blood

* From 10-50% of circulating erythrocytes in sickle cell anemia are

ISC.
* ISC account for most if not all of sickle form in peripheral blood
smear.
 Oxygen affinity of Hb S

 Hb S has a decreased oxygen affinity

 Release more oxygen to tissue
 Increase the concentration of deoxygenated Hb S,
thus promote the formation of sickle cells.
RBCs destruction:
The primary cause of anemia in sickle cell disease is extravascular hemolysis.

Change in RBCs membrane, decrease deformability and formation of Hb S polymers.

Enhance the removal of RBCs by splenic macrophages.

 Clinical finding

Clinical signs appear at about 6 months after the

concentration of Hb S predominates over Hb F.

-Anemia
-Vaso-occlusive crisis
-Overwhelming infections
-Acute splenic sequestration
 Anemia
Moderate to severe anemia characterized by:

• Cardiac overload due to an attempt to compensate for the tissue

oxygen deficit cardiac enlargement
• Hyper-plastic bone marrow
• Folate deficiency can occur due to increase erythrocytes turnover.

Aplastic crisis (temporary cessation of erythropoiesis from few days to 2

weeks) may develop following viral, bacterial or mycoplasmal
infections.
Aplastic crisis leads to an acute worsening of the anemia. Aplastic crisis
can occur in normal individuals but with no clinical symptoms ……
WHY ???
 Vaso-occlusive crisis

-Blockage
-Necrosis
-Organ dysfunction
(Spleen, Kidney, Retina)

 Auto-splenectomy
 Arterioles of eye (blindness)
 Chronic leg ulcers without any known injury
• Erythrocytes at the blockage site release more
oxygen to provide enough oxygenation to the
tissue, as a result, more deoxyhemoglobin S
are formed and expanding the blockaded site.

• Although splenomegaly is present in early

childhood, repeated splenic infarctions result
in organ damage and autosplenectomy.
 Bacterial infection
• Increase susceptibility to infection especially
from encapsulated bacteria
• Not fully understood but could be due to
dysfunction of spleen.
• Significant impairment of adherence of
neutrophil to vascular endothelium.
 Acute splenic sequestration
 Sudden splenic pooling of sickled red blood
cells.
 Massive decrease in erythrocytes mass within
few hours.
 Thrombocytopenia can also occur. ….WHY

** Sequestration crisis **
Enlarged spleen
Lab. Finding at peripheral blood
- Normocytic, normochromic anemia
- Marked reticulocytosis (10-15%)
- Hb 7-10g/dl
- Hematocrit 20-30%
(Calculation is more reliable than centrifugation)….WHY ???
- Anisocytosis, poikilocytosis(sickle+target cells)
- RDW increased with the presence of anisocytosis
and reticylocytosis.
- Splenic dysfunction (poikilocytosis +Howell-Jolly
bodies).
Sickle cells and Target cells
Howell-Jolly bodies
 Bone marrow
 Hyperplasia reflecting the attempt of bone
marrow to compensate for hemolysis

 Not usually performed because it does not give

a definitive diagnostic information.
Hb electrophoresis in Sickle Cell Anemia

85-95% Hb S
Hb F is usually not more than 15%
Hb A2 is normal

Newborn
60-80% Hb F
The remainder is Hb S
 Solubility test
- Screening test for Hb S
- Hb S relatively insoluble when combined with
reducing agent (sodium dithionite)
(Whole blood+ lysing agent+ reducing agent)
Elevated plasma proteins (remind we use whole blood) can give false
positive result.

Sickling test cab be performed (slide).

What is sickling test ????
 Other non-specific finding
o Increase bilirubin
o Decrease osmotic fragility (target cells)

Not specific tests but can be used to evaluate the

complicating conditions
 Therapy
*Transfusion of packed erythrocytes
- aplastic crisis and splenic sequestration
- decrease the production of new Hb S containing erythrocytes by
patient's bone marrow
 Disadvantages: blood-borne disease, alloimmunization and iron overload.

*Pharmacologic agents (hydroxyurea-HU)

- Reduce intracellular sickling by increasing the level of Hb F
- Decrease the chance of vaso-occlusive crisis

*Hematopoietic stem cell transplantation

*Gene therapy
 Sickle cell trait
• Heterozygous state (βA/βS)
• One normal β gene and another for βS gene
• Hb A and other non-S hemoglobins interfere with the process of Hb S
polymerization. Polymerization occurs only under very low oxygen
situation.

50-65% Hb A and 35-45% Hb S

Normal Hb F and Hb A2
If Hb A less than 50% compound heterozygous

Clinical symptoms are occasionally if the person exposed to extreme and

prolonged hypoxia.

Importance of diagnosis …..????

Hemoglobin C disease
 Hemoglobin C disease
*The first case was discovered in heterozygous state with Hb S
*Non-polar Lysine substituted for glutamic acid at the sixth
position of A3 helix in β-chain.
β6(A3)Glu → Lys

-Decrease in solubility
-Intra-erythrocytic crystals formation of Hb C

At spleen: hypertonic environment, thus dehydration and crystals

formation enhanced
Erythrocytes become rigid and destroyed in the spleen
Decreased life span as few as 30-55 days.
Hb C crystal
 At Hb C disease (βC/βC):

• variable hemolysis results in mild to moderate anemia

• Hb 9-12 g/dL
• Hematocrit 27-35%
• Slight to moderate reticulocytosis
• Target cells
• Osmotic fragility decreased (target cells)
• The spleen is most often enlarged ( in contrast to sickle cell anemia)

 At Hb electrophoresis:
more than 90% HbC
increased level of Hb F (not more than 10%)
 At Hb C trait (βC/βA):

• Asymptomatic
• No hematological abnormalities except that target cells in
blood smear

60-70 % is Hb A
30-40 % Hb C

• Lower level of Hb A is found when Hb C is associated with

β-thalassemia (βC/β thal). Depending on the β-thalassemia
gene inheritance (β+ or βº).
 (βC/β thal)
Clinical and hematological features depending on β thal gene interacting
with Hb C

Hb C/ β+thal
Mild anemia
65-80 % Hb C
2-5% Hb F
The remainder is Hb A

Hb C/ βº thal
More severe anemia
Absence of Hb A
Only Hb C, Hb A2 and Hb F ranging 3-10%
 HB S/C disease (βS/βC)
• One β-gene is coded for βS chain and the other for βC
chain (double heterozygous state for Hb S and Hb C)
• Hb A is absence
• Less severe than homozygous Hb S but more severe than
homozygous Hb C
• % of Hb S greater than Hb C (negative charge). The cell
does not form αβC dimers as readily as αβS dimers.
• Both sickling and crystals formation contribute to the
pathophysiology of the disease
Cont…..

-Mild to moderate normocytic normochromic anemia

-Hb is 10-13 g/dl
-Vaso-occlusive crisis can develop (less frequently than sickle cell anemia)

At blood film
-Large number of target cells( up to 85%)
-Anisocytosis and poikilocytosis

Hb electrophoresis
Hb S greater than Hb C
Hb F can increase up to 7%
No Hb A
 Hemoglobin D
β121(GH4)Glu → Gln

• Hb D molecules do not sickle

• Have normal solubility properties
• Both homozygous and heterozygous are asymptomatic
• No hematological abnormalities but occasionally there
is an increase in target cells and decrease osmotic
fragility.
• Compound heterozygous of Hb D with Hb S can exist
but very rare.
 Hemoglobin E
β26(B8)Glu → Lys

-Nucleotide substitution creates improper mRNA

processing, thus, synthesis of abnormal β-chain
decreased
-Hb E trait and disease have some thalassemia-like
characteristics including an increase ratio of alpha to
non-alpha chain synthesis and alpha chain excess.
 Hb E/Thalassemia
• Compound heterozygosity of Hb E and β-thalassemia
• Moderate to severe anemia similar to thalassemia.
• The most severe type is βE/βº-thal
• Moderate anemia if Hb E/ β+thal (βE/β+thal)

Microcytic, hypochromic anemia

MUTATIONS THAT AFFECT
HEMOGLOBIN STABILITY
Unstable Hb variants include a mutation in globin
chain that is:

1. Replacement of non-polar by polar a.a. at the

interior of the molecule.

2. Mutation at inter-subunit contacts creating a

tendency to dissociate into monomers.
 Peripheral blood

 Normocytic, normochromic.

 Increase in reticulocytes.

 Bite cell.

 Heinz bodies (not specific <G6PD>).

Bite Cell
MUTATIONS THAT ALTERED
OXYGEN AFFINITY
 Decreased oxygen affinity
 Favor the deoxygenated (Tense) conformation or
destabilize the oxygenated (Relax) form.
 Impairment in oxygen binding site
 Most low oxygen affinity Hb possess enough affinity to
become fully saturated in the lung, however, at tissue
deliver higher than normal amounts of oxygen.

-Oxygen requirement can be met at lower hematocrit.

-Reticulocytes ????
 Increased oxygen affinity
 Mutation that favor (R) conformation or destabilize
the (T) form.
 Few substitution affect 2,3-BPG binding site.
 Bind oxygen more readily than normal but retain
more oxygen at tissue.

-Hypoxia stimulates EPO release.

-Increase RBCs count and in hematocrit.
-Hb levels are increased to about 19g/dL.
Cont….

ruddy face due to erythrocytosis.

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