THALASSEMIA

Dr. Prabha Panthi

Department of Pathology Chitwan Medical
College
 Learning
Objectives

1. To define thalassemia
2. To elaborate pathogenesis of thalassemia
3. To classify thalassemia
4. To enlist morphology of thalassemia
5. To explain complications of thalassemia
Normal Adult
Haemoglobin:
Hemoglobin:
- Molecular weight: 64,500
Dalton
- Composed of Haeme (iron
and protoporphyrin) and
globin

Globin consists of four (two

pairs of ) polypeptide chains α
and β.
One haem group is bound to
each polypeptide.
 Embryonic life hemoglobin:
Hb Gower I, II and Portland

Newborn hemoglobin : HbF

80%, HbA 20%

Adult hemoglobin: HbA 97%,

HbA2 2.5% and HbF 0.5%

Variants of normal
hemoglobin
- After fetal development, synthesis of zeta and epsilon chains is
replaced by alpha and gamma chain respectively.

- Similarly, after birth, production of gamma chains switches to beta

and delta chains.
Hemoglobin genes….
Tetramer, composed of a pair of α and a pair of β
polypeptide chains.

•The two α chains in HbA are encoded by an identical

pair of α-globin genes on chromosome 16.

•While the two β chains are encoded by a single β-

globin gene on chromosome 11.
In humans, autosomal chromosomes occur
in pairs.
Each members of chromosome 16 has two Whereas, there is only one β globin
α gene loci (a specific physical portion of gene locus on chromosome 11 and
gene on chromosome), hence total four α therefore β genes are two in number.
genes
 Thalassemia
syndrome
Definition
- Heterogenous group of disorders caused by
inherited
mutations that decrease the synthesis of either the
α- globin orβ- globin chains that compose adult Hb,
HbA (α2β2)l

This leads to anemia, tissue hypoxia, and red cell

hemolysis due to imbalance in globin chain
synthesis.
 Classification of Thalassemia
• β-thalassemia:
Caused by deficient synthesis of β
chains.
• α-thalassemia:
Caused by deficient synthesis of α
chains

Hematologic consequences of diminished synthesis of one globin chain stem not only from
hemoglobin deficiency but also from a relative excess of the other globin chain,
particularly in β-thalassemia
Classification of
thalasssemia
 Thalassemia syndromes are endemic in the
Mediterranean basin (indeed, thalassa means
“sea ” in Greek)

Other location:
Middle East,
tropical Africa, the
Indian
subcontinent, and
Asia.
Beta Thalassemia

• β-thalassemia are caused by mutations that

diminish the synthesis of β-globin chains.

• Characterized by excess of unpaired alpha-

chain.
Molecular
pathogenesis

Causative mutation falls into two

categories:

1. β 0 mutation: Absent of β-globin synthesis

2. β+ mutation: Reduced (but detectable) β-globin

synthesis
 Steps in synthesis of globin
Involves 3 steps: Transcription, processing of RNA and
translation.
1. Transcription:
- Synthesis of single strand of
RNA (DNA copied) from DNA
template by enzyme RNA
polymerase.
-RNA polymerase reads DNA strand
in a 5’ to 3’ direction and build RNA
molecule until chain termination
sequence is encountered
- RNA thus formed is called pre-
mRNA which is edited to form mRNA
that carries information for protein
synthesis
 Normal transcription and splicing
Pre-mRNA thus formed at
first is then "edited" to
produce the desired mRNA
molecule in a process
called RNA splicing

Pre-mRNA contains introns which

are not required for protein
synthesis hence, pre-mRNA is
chopped up to remove the
introns and create messenger
RNA (mRNA), a process which is
called RNA splicing
 Steps in synthesis of
globin……
2. Processing of mRNA:
• mRNA molecule is capped, poly-A tail is added and introns are
removed

• Capping at 5’ end for initiation of translation

• Adding of poly-A tail at 3’ end (AAUAAA sequence at 3’ end)

consisting of about 150 adenyle acid residues
- for transcript stability and its transport to the
cytoplasm

• Excision of introns and joining together of exons before its

transport to the cytoplasm from the nucleus
- Guided by the presence of GT dinucleotide at the exon-
intron boundary and AG at the intron-exon boundary by initially
forming a lariat structure
Transcription and translation
 Steps in synthesis of
globin……
3. Translation:
Formed mRNA in transcription is
transported out of the nucleus, into
the cytoplasm, to the ribosome (the
cell's protein synthesis factory).

mRNA is not directly involved in

protein synthesis but needs
assistance of transfer RNA (tRNA), a
process which is called translation.
Hence, globin protein is formed.
 Beta Thalassemia:
Molecular
pathogenesis…….
1. Splicing mutations: most common cause of β+ thalassemia.
- Mostly mutations lie within introns, and few in exons.

- Some mutations destroy normal RNA splice junctions and

completely prevent the production of normal β-globin mRNA,
resulting in β0-thalassemia.

- Others create an “ectopic” splice site within an intron and

hence, normal splice sites remain, both normal and abnormal
splicing occurs resulting in formation of normal β-globin hence
in β+ thalassemia.

Splice site mutation

Beta Thalassemia:
Molecular pathogenesis…….
3. Chain terminator mutations.
• Most common cause of β0-
2. Promoter region mutation:
thalassemia.
• Two subtypes of mutations: most
• Promoter region enhances
common type creates a new stop
transcription.
codon within an exon;
- the other introduces small
- Mutation causes decreased insertions or
transcription by 75% to 80% hence deletions that shift the mRNA
decreased globin synthesis resulting reading
β+ Thalassemia frames (frameshift mutations).

• Both block translation and prevent

the synthesis of any functional β-
 Transcription ends when the RNA polymerase
enzyme reaches a triplet of bases that is read as
a "stop" signal.

There are three stop codons, TAG, TGA, and

TAA
(UAG, UGA, and UAA on mRNA)

How to remember?
UAG: U Are Gone
UGA: U Go Away
UAA: U Are Away
1. Promoter region enhances transcription: Mutation causes
decreased transcript tion followed by decreased globin synthesis-
β+ Thalassemia
Point mutation
2. Chain terminator mutation: Formation of stop codon: no further is the major
translation- β0 mutation (Frameshift mutation or non-sense cause for Beta
mutation) thalassemia

3. Splicing mutation: mutation in intron(β +) , Mutation in exon(β 0)

 Unpaired α chains precipitate within red
Pathogenesis: cell precursors, forming insoluble inclusions
Impaired β-globin synthesis causing membrane damage in most of the
results in anemia by two precursor red cells which undergo
mechanisms: apoptosis.

- The deficit in HbA synthesis In severe β-thalassemia, about 70% to 85%

produces “under of red cell precursors suffer this fate,
hemoglobinized” hypochromic, leading to ineffective erythropoiesis.
microcytic red cells with sub-
normal oxygen transport
capacity. Red cells that are released from the marrow
also contain inclusions and have membrane
- Diminished survival of red cells damage, leaving theme prone to splenic
and their precursors, which sequestration and extravascular
results from the imbalance in α- hemolysis.
and β-globin synthesis
Pathogenesis….
.
PATHOGENESIS:
Types of Beta Thalassemia
Clinical classification of β-thalassemia:
- Based on the severity of the anemia, which depends on
genetic defect (β+ or β0) and the gene dosage
(homozygous or heterozygous)
 1. β-Thalassemia Major
• Genotype: Homozygous state, two abnormal β- thalassemia alleles
(β+/β+,β+/β0, or β0/β0)

• Most common in mediterranean countries

• Major red cell hemoglobin is HbF, which is markedly elevated, and

HbA2 increased, normal or low.

• Severe form of thalassemia, completely lacks HbA

• Also k/a Cooley‘s anemia,

• Transfusion dependent (In untransfused patients, hemoglobin levels are 3 to 6

 Erythropoietic drive in
response to severe
uncompensated anemia,
massive erythroid
hyperplasia in the marrow
and extensive extra -
medullary hematopoiesis
occurs.

The expanding mass

of red cell precursors erodes
the bony cortex, impairs
bone
growth, and produces
skeletal abnormalities as
Pathogenesis of β-thalassemia major: aggregates of unpaired α- well.
globin chains (alpha Tetramer), a hallmark of the disease.
 Clinical features

- Anemia
- Features of extra-medullary
hematopoiesis
- Hemolytic features
- Increased erythropoiesis
- Skeletal deformities
•Anemia manifests only after 6-9 months of birth
(Because HbF switches to Hb A only after 6
Hepato-splenomegaly
 Clinical
features

Related to
marrow
expansion
Chipmunk /thalassemia Hair on end appearance/ crew
facies cut appearance

Hair on
end
Crew cut hairstyle
 (Hemochromatosis)

Blood transfusions:
Double-edged sword,
diminishing the
anemia and its
attendant
complications, but also
adding to the systemic
iron overload.
 Morphology: Peripheral Smear

• Severe red cell abnormalities

• Marked variation size(anisocytosis) and shape (Poikilocytosis)

• Microcytic hypochromic RBCs, Target cells +, fragmented cells,

• Increased reticulocytes ( but not as effective as anemia severity)

• Nucleated red cell precursors (normoblasts) in the peripheral blood

as a result of “stress” erythropoiesis and abnormal release from sites
of extramedullary hematopoiesis.
 Target cells

Nucleated RBCs
-Confirmatory Diagnosis:
Hemoglobin electrophoresis: :Lack of HbA and
presence of HbF

-Prenatal diagnosis is possible by molecular

analysis of DNA

Treatment:
- Heavily transfused patients: must be treated with iron chelators
to prevent or reduce this complication and increase survival rate
(3rd decades)

-Hematopoietic stem cell transplantation is the only therapy to

cure.
Complications
• Untreated children: Growth retardation and die at an early age from
the effects of anemia.

• Who survives long enough: Chipmunk facies

(cheekbones and other facial bony prominences are enlarged and
distorted).

• Hepato-splenomegaly: due to extramedullary hematopoiesis

• Secondary hemochromatosis (iron overload due to blood transfusion):

- Diabetes, Liver cirrhosis, Heart failure and arrhythmia

Cardiac disease resulting from progressive iron overload and secondary hemochromatosis is
an important cause of death, particularly in heavily transfused patients.
 2.Beta- Thalassemia Minor/ β-
thalassemia trait:

• Much more common than Thalassemia-major

• Heterozygous carrier with one β-thalassemia gene
(mutated) and one normal gene.
• Genotype: (β+/β or β0/β),

• Asymptomatic and mild anemia

• PBS: Microcytic hypochromic RBCs, Target cells

• Hb electrophoresis: Increase of Hb A2 to 4% to 8% of
 3. β- Thalassemia
Intermedia

• Milder form of homozygous(β+/ β+) or (β+/β0) or

unusual form of heterozygous variant (two defective
β-globin along with an α- globin genes

• Less imbalance between β and α globin and less

severe form
Alpha-
Thalassemia
 Definition
•Inherited deletion that result in reduced or absent α-globin chain

• Normally 4 α-globin genes

• Severity depends upon deletion of number of globin genes

A variety of molecular lesions give rise to α-thalassemia, but gene deletion is

the most common cause of reduced α-chain synthesis.
 Consequences of alpha
thalassemia
Anemia results from two underlying mechanism :
- Lack of adequate hemoglobin, and

- Presence of excess unpaired globin chains (β, γ, and δ):

varies in type at different ages.

 In newborns with α-thalassemia, excess unpaired γ-globin

chains form γ4 tetramers known as hemoglobin Barts
(Hydrops Fetalis),

 In older children and adults excess β-globin chains form β4

tetramers known as HbH.
Classification of Alpha-
Thalassemia
1. Silent carrier state:

- Caused by deletion of single α-globin

genes

- Asymptomatic,

- Slight microcytosis
2. α- Thalassemia Trait:

• Caused by deletion of two α-globin gene from single

chromosome(α/ α -/-) Or deletion of α- globin gene from each of
two chromosome( α /- α /-)

• Clinical features are similar to beta- thalassemia trait

- Microcytosis, minimal or no anemia,

- No abnormal physical signs

- HbA2 levels are normal or low.

3. Hemoglobin H disease:

• Caused by deletion of three Alpha-globin genes

• Most common in asian

• With only one normal α-globin gene, synthesis of α chain is markedly

reduced, and tetramers of β-globin (Known as HbH form).

• HbH has high affinity for oxygen and not good in oxygen
delivery and
leads to tissue hypoxia
Clinical manifestation

• HbH is prone to oxidation, that causes it to precipitate and form

intracellular inclusion that causes red cell sequestration and
phagocytosis in spleen.

• Results in moderately severe anemia, resembling Beta

thalassemia intermedia.
 Diagnosis
• Microcytic hypochromic RBCs in Peripheral
blood smear

• Supravital stain for reticulocyte:

Golf ball like reticulocytes.
4. Hydrops fetalis:
- Most severe form of alpha thalassemia

- Caused by deletion of all 4 Alpha genes

- Formation of Hb Bart(Tetramer of γ4)

- Has high affinity for oxygen hence leading to inadequate deliver

of oxygen to tissue lead to tissue hypoxia

- Survival in early development is due to the expression of ζ chains,

an embryonic globin that pairs with γ chains to form a functional
ζ2γ2 Hb tetramer.
 • Signs of fetal distress are evident after third
trimester in utero

• Early gestation period has embryonic Hb which constitute α2 ζ

2.

• Later in fetal life formation of fetal Hb gamma has nothing to

pair and forms tetramer: ɣ4 tetramer, also known as Hb
Bart
Clinical
manifestation
• Signs of fetal distress
• Usually death in utero
• Fetus show severe pallor, edema, hepatospleenomegaly
• Features similar to clinical features of hemolytic disease of
newborn.

•Treatment:
- Hematopoetic stem cell transplantation
Clinical
manifestation
SUMMARY
1. Promoter region enhances transcription: Mutation causes decreased transcrip
tion followed by decreased globin synthesis- β+ Thalassemia

2. Chain terminator mutation: Formation of stop codon: no further translation-

β0 mutation (Frameshift mutation or non-sense mutation)

3. Splicing mutation: mutation in intron(β +) , Mutation in exon(β 0)

Thank
you….