Dr.S.Chakravarty MD
Specific Learning Objectives
• At the end of today’s lecture you should be
able to –
– Enumerate the various hemoglobinopathies with
their associated genetic defects .
– Describe the molecular basis and the basis of
laboratory diagnosis of Sickle cell anaemia , alpha
and beta thalassemia in detail.
α
β
β
α
25%
25%
0.5%
1.5%
48%
α
α
γ
δ
β
α
α
γ
δ
β
25%
25%
0.5%
1.5%
48%
Chromosome 16
Chromosome 11
α
β
α
γ
α
δ
β
α
γ α
δ
α
HbA
HbF
HbA2
98%
~1%
<3.5%
Steps in globin
chain synthesis:
1.Transcription
2.Modification of
mRNA precursor by
splicing
3.Translation by
ribosomes & further
modifications (i.e.
glycosylation)
• An inherited mutation of the globin genes
leading to a qualitative or quantitative
abnormality of globin synthesis.
• Hereditary disorders that can result in
moderate to severe anemia
•
•
•
•
•
Structural hemoglobinopathies
Thallasemias
Thallasemic Hb variants
Hereditory persistance of fetal Hemoglobin
Acquired hemoglobinopathies
10
1. Structural Hemoglobinopathies
a) Altered Hb polymerization
b) Altered affinity
c) Unstable Hb Variants.
11
• Mutation :
of Beta chain.
is replaced by
at 6th position
• Polymerizes reversibly when deoxygenated to form a
gelatinous network of fibrous polymers that stiffen the
erythrocyte membrane.
• The abnormal hemoglobin is less soluble under decreasing
oxygen concentrations and polymerize into crystals that
distort the red blood cells into a sickle shape.
A number of factors may precipitate a sickle cell
crisis, these include:
•Hypoxia
•Acidosis
•Dehydration
•Infections
• Severe exercise
•Increase physical / physiological demand (Pregnancy, physical
exercise)
Hb S is less soluble in acidosis and dehydration
Hydrophobic valine causes stickiness
Lippincott’s Illustrated Biochemistry
1. Hemolysis Anaemia
2. Occlusion of blood vessels by sickled
red cellsSEVERE PAIN( DUE TO TISSUE ANOXIA)
Site of Sickling
Clinical Features
Management
Bone
Painful crises
Pain relief and
hydration.
Hydroxyurea
Lung
Acute chest syndrome
Transfusion
regimen, pain
relief and
hydration
Brain
Stroke
Transfusion
regimen.
Heart
Myocardial infarction
Transfusion
regimen, pain
relief and
hydration
Spleen
Acute splenic sequestration:
Transfusion,
pain relief and
hydration
Spleen
Hyposplenism:
Pneumovax
• Complete Hemogram  Anemia
• Sickling test :- Blood smear prepared after
adding reducing agent sodium dithionite
See under microscope (NOT SPECIFIC )
• Solubility test :- Hemolysate is prepared
in the presence of reducing agent
.Opalescence is suggestive of sickle cells.
(NOT SPECIFIC )
• Hb electrophoresis- CONFIRMATORY
• Southern Blot -CONFIRMATORY
• @ pH 8.6 Glutamic acid carboxy
group is –ve charged
• Lack of this charge of HbS makes it
less negatively charged and
decreases the electrophoretic
mobility towards positive pole.
• However at acidic pH (citrate
buffer)HbS moves faster than Hb A
Lippincott’s Illustrated Biochemistry
• Repeated Blood transfusions- MAINSTAY
• IRON OVERLOAD is a problem
• CHELATION therapy with Desferrioxamine
• CHRONIC HEINZ BODY ANAEMIAS -Unstable
Hb variants have a tendency to denature. They
tend to form molecular aggregates called
Heinz Bodies within cells hemolysis
– Hb Köln (Hb β 98valmet)
– Hb Poole – γ chain unstable variant
• INCREASED O2 AFFINITY
– Hb Chesapeake
– erythrocytosis (ODC shifts to left )
• DECREASED O2 AFFINITY
– Hb Kansas
– Cyanosis
• Hb M– Most HbM are produced by substitution of Tyr for proximal/Distal
His in the haem pocket of the alpha or ß-chainsThis results in
facilitated oxidation of the hemoglobin to yield excess methemoglobin
which leads to cyanosis.
– Hb M Hyde Park, β92His→Tyr; Hb MBoston, α58His→Tyr; Hb
MSaskatoon, β63His→Tyr; Hb MMilwaukee-1, β67Val→Glu.
• Hereditary disorders that can result in
moderate to severe anemia
• Basic defect is reduced production of
globin chains.
•Found most frequently in the Mediterranean,
Africa, Western and Southeast Asia, India and
Burma
•Distribution parallels that of Plasmodium
falciparum
Hb D
Symbolism
Alpha Thalassemia
• Greek letter used to designate globin
chain:

Symbolism
Alpha Thalassemia
/ : Indicates division between genes
inherited from both parents:
/
• Each chromosome 16 carries 2 genes. Therefore the
total complement of  genes in an individual is 4
Symbolism
Alpha Thalassemia
- : Indicates a gene deletion:
-/
Classification & Terminology
Alpha Thalassemia
• Normal
/
• Silent carrier
-/
• Minor
-/-
--/
• Hb H disease
--/-
• Barts hydrops fetalis
--/--
Symbolism
Other Thalassemia
• Greek letter used to designate globin
chain:

Symbolism
Other Thalassemia
+
: Indicates diminished, but some
production of globin chain by gene:
+
Symbolism
Other Thalassemia
0
:Indicates no production of globin chain by
gene:
0
Classification & Terminology
Beta Thalassemia
• Normal
/
• Minor
/0
/+
• Intermedia
0/+
• Major
0/0
+/+
Some mutations lie within promoter regions and typically lead to reduced
globin gene transcription.
In some cases a single-nucleotide change in one of the exons leads to the
formation of a termination, or "stop" codon, which interrupts translation of βglobin messenger RNA (mRNA) and completely prevents the synthesis of βglobin. Such alleles are designated β0.
 Mutations that lead to aberrant mRNA processing are the most
common cause of β-thalassemia. Most of these affect introns, but some
have been located within exons. If the mutation alters the normal splice
junctions, splicing does not occur, and all of the mRNA formed is abnormal.
Unspliced mRNA is degraded within the nucleus, and no β-globin is made
Abnormal associations of otherwise
normal subunits.
• With severe α-thalassemia, the β-globin subunits begin to
associate into groups of four (tetramers) due to the paucity of
potential α -chain partners.
• These tetramers of b-globin subunits are functionally inactive
and do not transport oxygen. No comparable tetramers of
alpha globin subunits form with severe beta-thalassemia.
• Alpha subunits are rapidly degraded in the absence of a
partner from the beta-globin gene cluster (gamma, delta, beta
globin subunits).
β-Thalassemias
Thalassemia major
β-thalassemia trait
Homozygous or
Severe, requires
compound
blood transfusions
heterozygous (β0/β0, regularly
β0/β+, or β+/β+)
Defects in
transcription,
processing, or
translation of mRNA,
Asymptomatic, with resulting in absent
mild microcytic
(β0) or decreased
anemia, or
(β+) synthesis of βmicrocytosis without globin
anemia
β/β+or β/β0
α-Thalassemias
Hydrops fetalis
--/--
Fatal in utero
HbH disease
--/-α
Moderately severe
anemia
α-thalassemia trait
--/αα(Asian) or
-α/--α(African)
Similar to βthalassemia trait
Silent carrier
-α/αα
Asymptomatic,
normal red cells
Gene deletions
spanning one or
both α-globin loci
Excessive RBC BREAKDOWN
• Anaemia
• Bone changes (hair on end)
• Ethnicity: Mediterranean, Africa, Southeast
Asia
• Hypo-Micro, Poikilocytosis
• NRBC’s, reticulocytosis, basophilic stippling
• Siderocytes (with repeated transfusions)
Thalassemia Blood Smears
X-ray of scull
in Thalassemia:
“Hair-on-end”
MRI showing marked
widening of the diploic space
containing alternating bands
(arrows) of hypointense
trabeculae and hyperintense
marrow.
Beta thalassemia major
Male 18 years
HEPATOSPLENOMEGALY
OVERALL DECREASED GROWTH
• Hb Lepore:  fusion seen in some types
of  thalassemia
• Hb Constant Spring
•  chain with 31 additional amino acids
• --/cs
• Hereditary persistence of fetal hemoglobin
(HPFH) This is usually caused by
mutations in the β-globin gene.
• Beneficial to patients with sickle cell or thalassemia.
• Hb H
• 4 tetramer and γ4 tetramers
• Associated with --/- thalassemia
• Hb Barts & hydrops fetalis
•
•
•
•
Barts is a 4 tetramer
Associated with --/-Lethal
High concentrations are capable of sickling
• Peripheral smear –
• Severe cases present with
• Microcytosis
• Hypochromia
• Poikilocytosis
• Hb Electrophoresis
• DNA studies (PCR + S.blot)
• Time of presentation
• Related to degree of severity
• Usually in first few years of life
• Untreated severe  thalassemia
• --/--: Prenatal or perinatal death
• --/- & --/cs: Normal life span with chronic
hemolytic anemia
• Untreated  thalassemia
• Major: Death in first or second decade of life
• Intermedia: Usually normal life span
• Minor: Normal life span
• Repeated Blood transfusions - MAINSTAY
• Repeated blood transfusions leading to Iron
excess and Hemosiderosis.
• Pigment stones in Gall Bladder and CBD are
more common in such patients.
POINT
Hb
MUTATION
POSITION
AMINO ACID
SUBSTITUTION
Hb S
β6
Glu- Val
Hb C
β6
Glu-Lys
Homozygotes – CC Mild anaemia
Heteozygous – AC no disease
Double heterozygous SC –Moderate disease
Hb E
β26
Glu-Lys
Heterozygous – Asymptomatic
Homozygous – Mild disease
Hb D
(Punjab or Los
Angeles)
β121
Glu-Gln
HbSD – severe disease
Hb O
(Arab)
β121
Glu-Lys
Homozygous –mild anaemia
Hb G (Philadelphia)
Hb Lepore
α68
Glu-Lys
δ(1-87) β(116-146)
HbG Philadelphia is an α variant, often associated
with deletions of the nonaffected a genes. With no
deletions, there is approximately 20% HbG, with one
deletion about 30% G is present, and with two about
40% is present.
Lepore is the product of the indicated crossover
during meiosis.(NOT A MUTATION)
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C
Normal
S
F
A+
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C
Normal
Hb SS
S
F
A
+
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C
Normal
Hb SS
Hb AS
S
F
A
+
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C
Normal
Hb SS
Hb AS
Hb SC
Hb CC
S
F
A
+
Can you identify which person has
severe sickle cell anemia and who is
heterozygous for the condition ?
• In general on alkaline electrophoresis in order
of increasing mobility are hemoglobins
A2, E=O=C, G=D=S=Lepore, F, A, K, J, Bart's, N, I, and H.
• In general on acid electrophoresis in order of
increasing mobility are hemoglobins
F,A=D=G=E=O=Lepore, S, and C
• Test partners of heterozygous or
affected individuals
• Antenatal diagnosis from DNA
obtained by chorionic villus
sampling, or by amniocentesis
MCQ 1
An Asian child has severe anemia with prominence of the
forehead (frontal bossing) and cheeks. The red cell hemoglobin
concentration is dramatically decreased, and it contains only
beta-globin chains with virtual deficiency of alpha-globin chains.
Which of the following mechanisms is the most likely
explanation?
A. A transcription factor regulating the alpha-globin gene is
mutated
B. A regulatory sequence element has been mutated adjacent
to an alpha-globin gene
C. A transcription factor regulating the beta-globin gene is
mutated
D. A transcription factor regulating the alpha and beta -globin
genes is deficient
E. A deletion has occurred surrounding an alpha-globin gene
67
MCQ 2
HbC disease is caused by a single amino acid substitution
(lysine instead of Glutamic acid) at position 6 in the bete-globin
chain of the hemoglobin molecule. Patients homozygous for HbC
have a mild chronic hemolytic anemia. HbS disease generally
causes a more severe condition compared to HbC disease
because HbS disease:
A. Impairs oxygen binding to the heme moiety
B. Impairs proper folding of the alpha-helix in the beta-globin
chain
C. Allows hydrophobic interaction among hemoglobin molecules
D. Impairs beta-globin interaction with 2,3-bisphosphoglycerate
E. Stabilizes iron moiety at ferric state (fe3+).
MCQ 3
An infant to a greek immigrant appears healthy at
birth but develops transfusion dependent hemolytic
anemia by the age of 6 months. his erythrocytes
contain insoluble aggregates of hemoglobin
subunits. The child developed normally in utero
because at that time he produced high quantities
of:
A. Alpha globin
B. Beta globin
C. Gamma globin
D. Delta globin
E. Epsilon globin
69
MCQ 4
which one of the following statements concerning the
ability of acidosis to precipitate a crisis in sickle cell
disease is correct?
A. acidosis influences the shape of hemoglobin
B. Acidosis decreases the solubility of Hb S
C. Acidosis favours the conversion of hemoglobin from
the taut to the relaxed conformation
D. Acidosis shifts the oxygen-dissociation curve to the
left
E. Acidosis decreases the ability of 2,3-BPG to bind to
Hemoglobin.