Hemoglobin is composed of four globin chains that each bind to a heme group. The genes that encode for the different types of globin chains are located on chromosomes 11 and 16. Mutations in these genes can result in disorders called thalassemias, where there is imbalanced production of the globin chains. In β-thalassemia major, there is little to no production of the β-globin chains. This causes unpaired α-globin chains to precipitate and destroy red blood cell precursors in the bone marrow. Patients with β-thalassemia major suffer severe anemia starting in infancy and require regular blood transfusions to survive, but this leads to iron overload in tissues

1. HAEMOGLOBIN AND THE INHERITED
DISORDERS OF GLOBIN SYNTHESIS
Guvera Vasireddy, Pathology, OMC

3. Hemoglobin molecule
• Each complex consists of :
• Four polypeptide chains, non-covalently bound
• Four heme complexes with iron bound
• Four O2 binding sites

4. The structure, genetic control and
synthesis of haemoglobin
• Different haemoglobins are synthesised in the embryo,
fetus and adult, each adapted to their particular oxygen
requirements.
• They all have a tetrameric structure made up of two
different pairs (one α - like and one β - like) of globin
chains,
• Each attached to one haem molecule, the moiety
responsible for the reversible binding and transfer of
oxygen.

5. Globin Chains
• Alpha Globin:141 amino acids - Coded for on Chromosome 16
Found in normal adult hemoglobin, A1 and A2
• Beta Globin: 146 amino acids - Coded for on Chromosome 11,
found in Hb A1
• Delta Globin – gene located on Chromosome 11
Found in Hemoglobin A2--small amounts in all adults
• Gamma Globin – gene located on chromosome 11
Found in Fetal Hemoglobin
• Zeta Globin – gene located on chromosome 16
Found in embryonic hemoglobin

7. Location of different globin genes

8. Genetic control of human hemoglobin
• The main globin gene clusters are located on
chromosomes 11 and 16.
• At each stage of development, different genes in these
clusters are activated or repressed.
• The different globin chains directed by individual genes
are synthesized independently and combine in random
fashion.

9. Genetic control
of human
hemoglobin.
The main globin gene
clusters are located
on chromosomes 11
and 16.
At each stage of
development,
different genes in
these clusters are
activated or
repressed.
The different globin
chains directed by
individual genes are
synthesized
independently and
combine in random
fashion as indicated
by the arrows

11. Hemoglobin types
• Hemoglobin Type
• Hb A1—92%---------
• Hb A2—2.5%--------
• Hb F — <1%---------
• Hb H ------------------
• Bart’s Hgb--------------
• Hb S--------------------
• Hb C-------------------
Globin Chains
a2b2
a2d2
a2g2
b4
g4
a2b26
gluval
a2b26
glulys

12. Diagnostic investigations
• complete blood count, peripheral blood film examination,
• hemoglobin analysis by electrophoresis or high
performance liquid chromatography (HPLC),
• confirmed by molecular genetic analysis.
• Both a- and b-globin gene mutational analysis should be
conducted to confirm the diagnosis and determine co-
inheritance patterns that may help predict clinical
phenotype.
• Parents should be tested if no prior testing has been
performed.

13. Hemoglobin Electrophoresis (Identifying Thalassemia
and hemoglobinopathy)
Alkaline (Cellulose Acetate) pH 8.6:
• All Hemoglobin molecules have a negative charge, and
migrate towards the anode proportional to their net
negative charge.
• Amino acid substitutions in hemoglobin variants alter net
charge and mobility.
Acid (Citrate agar) pH 6.2:
• Hemoglobin molecules separate based on charge
differences and their ability to combine with the agar.
• Used to differentiate Hemoglobin variants that migrate
together on the cellulose gel (i.e. HbS from HbD and
HbG, HbC from HbE).

14. High-Performance Liquid Chromatography (HPLC)
• Weak cation exchange column. The ionic strength of the
eluting solution is gradually increased and causes the
various Hemoglobin molecules to have a particular
retention time.
• Amino acid substitutions will alter the retention time
relative to HbA.
• There is some analogy between retention time and
pattern on alkaline electrophoresis.

15. DNA analysis
Several molecular techniques are available.
• DNA from white blood cells, amniocytes, or chorionic tissue may be
utilized for diagnosis of various a- and b-globin chain abnormalities.
• Typically, deletional mutations causing a-thalassemia syndromes and
some rare b-thalassemias are diagnosed using Southern blot
hybridization of particular restriction enzyme digests to labeled
complementary gene probes.
• PCR techniques using allele-specific probes after globin gene
amplification, allele-specific primers, or deletion dependent
amplification with flanking primers are used
• in definition of known globin chain mutations/deletions, including
those for Hb S, E, D, and O, and several b-thalassemias . For
unknown mutations, several PCR-based methods,
• including denaturing gradient gel electrophoresis and single-strand
conformation polymorphism analysis, as well as sequencing of the
amplified globin gene DNA may be used.

16. THALASSEMIAS AND RELATED
DISORDERS
Quantitative Disorders of
Hemoglobin Synthesis

17. Introduction
• Heritable, hypochromic anemias-varying degrees of
severity
• Genetic defects result in decreased or absent production
of mRNA and globin chain synthesis
• At least 100 distinct mutations
• High incidence in Asia, Africa, Mideast, and
Mediterrenean countries

18. Overview
• According to the chain whose synthesis is impaired, the
thalassemias are called α-, β, γ-, δ-, δβ-, or ∊γδβ-
thalassemias.
• These subgroups have in common an imbalanced globin
synthesis,
• Consequently the globin produced in excess is
responsible for ineffective erythropoiesis (intramedullary
destruction of erythroid precursors) and hemolysis
(peripheral destruction of red cells).

19. Many phenotypes
• The application of recombinant DNA technology has led to
the understanding of the basic aspects of gene structure
and function and the characterization of the molecular
basis for deficient globin synthesis.
• The thalassemias result from the effect of a large number
of different molecular defects,
• These may interact, leading to a variety of clinical and
hematologic phenotypes.

20. History
• In 1925, Cooley and Lee1 first described a form of severe
anemia that occurred early in life and was associated with
splenomegaly and bone changes.
• In 1932, George H. Whipple and William L. Bradford published
a comprehensive account of the pathologic findings in this
disease.
• Whipple coined the phrase thalassic anemia and condensed it
to thalassemia, from ("the sea"), because early patients were
all of Mediterranean background.
• The true genetic character of the disorder became fully
appreciated after 1940.

21. Geographic Distribution, and the Role of Malaria
• That malaria had an influence in maintaining the high
prevalence of hemoglobinopathies in the world was first
suggested in 1948 by Haldane,
• The small red cells of the carriers of thalassemia could be
more resistant to the malaria parasites.
• Neel and Valentine had calculated that, in the absence of
selective pressure, the mutation rate for thalassemia had
to be in the order of 1 in 2,500.
• Selective pressure of malaria has amplified the β-
thalassemia genes to high frequency

22. World distribution of β-thalassemia

23. Terms describing severity of phenotype
• The disease described by Cooley and Lee is the homozygous
state of an autosomal gene.
• The heterozygous state is associated with much milder
hematologic changes.
• The severe homozygous condition became known as
thalassemia major.
• The heterozygous states were designated according to their
severity as thalassemia minor or minima.
• The term thalassemia intermedia was used to describe
disorders that were milder than the major form but more severe
than the traits.

25. Clinical syndromes in β-THALASSEMIAS
β-Thalassemia major Severe; requires blood
transfusions
β-Thalassemia
intermedia
Severe but does not
require regular blood
transfusions
β-Thalassemia minor Asymptomatic with mild
or absent anemia; red
cell abnormalities seen

26. β-Thalassemias Molecular Pathogenesis.
• The β-thalassemias are caused by mutations that
diminish the synthesis of β-globin chains.
• The causative mutations fall into two categories:
(1) β0 mutations, associated with absent β-globin
synthesis, and
(2) β+ mutations, characterized by reduced (but detectable)
β-globin synthesis.

27. Types of mutations in β- Thalassemia
• Splicing mutations.
These are the most common cause of β+-thalassemia.
Most of these mutations lie within introns, while a few are located
within exons.
• Promoter region mutations.
These mutations reduce transcription and some normal β-globin
is synthesized; thus, these mutations are associated with β+-
thalassemia.
• Chain terminator mutations and frame shift mutations
These are the most common cause of β0-thalassemia.
Common type creates a new stop codon within an exon;
Both block translation and prevent the synthesis of any
functional β-globin.

28. Distribution of β-globin gene mutations associated with β-
thalassemia.Arrows denote sites where point mutations giving
rise to β0 or β+ thalassemia have been identified.

30. Causes of anemia in β- Thalassemia
• Impaired β-globin synthesis results in anemia
by two mechanisms
1. The deficit in HbA synthesis produces
“underhemoglobinized” hypochromic, microcytic
red cells with subnormal oxygen transport
capacity.
2. Even more important is the diminished
survival of red cells and their precursors, which
results from the imbalance in α- and β-globin
synthesis.

31. Mechanism of hemolysis in β- Thalassemia
• Unpaired α chains precipitate within red cell precursors,
forming insoluble inclusions.
• These inclusions cause membrane damage and is the cause of
most red cell pathology.
• Many red cell precursors succumb to membrane damage and
undergo apoptosis.
• 70% to 85% of red cell precursors suffer this fate, which leads
to ineffective erythropoiesis.
• Those red cells that are released from the marrow also bear
inclusions and membrane damage and are prone to splenic
sequestration and extravascular hemolysis.

32. Consequences of ineffective erythropoiesis
• Severe uncompensated anemia leads to massive erythroid
hyperplasia in the marrow and extensive extramedullary
hematopoiesis.
• The expanding mass of red cell precursors erodes the bony
cortex, impairs bone growth, and produces skeletal
abnormalities.
• Extramedullary hematopoiesis involves the liver, spleen, and
lymph nodes, and in extreme cases produces extraosseous
masses in the thorax, abdomen, and pelvis.
• The metabolically active erythroid progenitors steal nutrients
from other tissues that are already oxygen-starved, causing
severe cachexia in untreated patients.

33. Secondary hemochromatosis
• Another serious complication of ineffective erythropoiesis
is the excessive absorption of dietary iron.
• Ineffective erythropoiesis suppresses the circulating levels
of hepcidin, a critical negative regulator of iron absorption.
• Low levels of hepcidin and the iron load of repeated blood
transfusions inevitably lead to severe iron overload unless
preventive steps are taken.
• Secondary injury to parenchymal organs, particularly the
iron-laden liver, often follows and sometimes induces
secondary hemochromatosis.

34. Pathophysiology of β-thalassemia

37. Morphology – peripheral smear
• Marked variation in size (anisocytosis) and shape (poikilocytosis),
microcytosis, and hypochromia.
• Target cells (so called because hemoglobin collects in the center of
the cell), basophilic stippling, and fragmented red cells are also
common.
• Inclusions of aggregated α chains are efficiently removed by the
spleen and not easily seen.
• The reticulocyte count is elevated, but it is lower than expected for the
severity of anemia because of the ineffective erythropoiesis.
• Variable numbers of poorly hemoglobinized nucleated red cell
precursors (normoblasts) are seen in the peripheral blood as a result
of “stress” erythropoiesis and abnormal release from sites of
extramedullary hematopoiesis.

38. . A. β-Thalassemia minor. Anisocytosis.
poikilocytosis. hypochromia. Occasional
spherocytes and stomatocytes. B.
Scanning electron micrograph of cells in
(A) showing more detail of the
poikilocytes. Note the knizocyte (pinch-
bottle cell) at the lower right. C. β-
Thalassemia major. Marked anisocytosis
with many microcytes. Marked
poikilocytosis. Anisochromia. Nucleated
red cell on the right. Small lymphocyte on
the left.

39. Thalassemia trait
blood film
Peripheral blood films
in β-thalassemia trait
may
demonstrate
microcytosis and
possibly
hypochromasia.
Multiple morphologic
changes including
target cells, teardrop
cells, and rare
fragments may occur.
These features can
appear identical to
the morphologic
picture of iron
deficiency.

40. Basophilic
stippling in
thalassemia
Peripheral blood film
demonstrating
microcytic
hypochromic RBCs
and basophilic
stippling (arrows).
Basophilic stippling
occurs in
thalassemia as well
as in other
hematologic
disorders

41. Basophilic stippling
• Thalassemia trait and major
● Hemolytic anemia
● Myelodysplastic syndrome/sideroblastic anemia
● Megaloblastic anemia
● Pyrimidine 5′ nucleotidase deficiency
● Heavy metal poisoning (coarse basophilic stippling)
Lead, zinc, arsenic, silver, mercury

42. Bone marrow and spleen
• In the bones of the face and skull the burgeoning marrow erodes
existing cortical bone and induces new bone formation, giving rise to
a “crew-cut” appearance on x-ray.
• Both phagocyte hyperplasia and extramedullary hematopoiesis
contribute to enlargement of the spleen, which can weigh as much as
1500 gm.
• The liver and the lymph nodes can also be enlarged by
extramedullary hematopoiesis.
• Hemosiderosis and secondary hemochromatosis, occur in almost all
patients.
• The deposited iron often damages organs, most notably the heart,
liver, and pancreas.

43. Bone marrow
in thalassemia.
Top and bottom
panels show bone
marrow aspirate and
biopsy, respectively,
from a case of
thalassemia trait. The
bone marrow has
increased numbers of
erythroid precursors
(a low myeloid to
erythroid ratio)
related to the
increased peripheral
RBC
destruction in this
disease.

44. Beta Thalassemia Major
• Reduced or non-existent production of β-globin.
• Poor oxygen-carrying capacity of RBCs
• Failure to thrive, poor brain development.
• The clinical course of β-thalassemia major is brief unless
blood transfusions are given.

45. Pathogenesis in β-thalassemia major
• Increased alpha globin production and precipitation and RBC
precursors are destroyed within the marrow –
• Hyperplastic Bone Marrow and Ineffective erythropoiesis
leading to poor bone growth, frontal bossing, bone pain.
• Increased splenic destruction of dysfunctional RBCs resulting
in Anemia, jaundice, splenomegaly
• Extramedullary erythropoiesis
• Iron overload—due to increased absorption and transfusions
• Endocrine disorders, Cardiomyopathy, Liver failure due to
secondary hemochromatosis.

46. Pathogenesis
of β-
thalassemia
major
Note that the
aggregates of
unpaired α-globin
chains, a hallmark of
the disease, are not
visible in routinely
stained blood smears.
Blood transfusions are
a double-edged
sword, diminishing the
anemia and its
attendant
complications, but also
adding to the systemic
iron overload.

47. Clinical course in β-Thalassemia major
• Untreated children suffer from growth retardation and die
at an early age from the effects of anemia.
• In those who survive long enough, the cheekbones and
other bony prominences are enlarged and distorted.
• Hepatosplenomegaly due to extramedullary
hematopoiesis is usually present.

48. β-Thalassemia facial bone abnormalities.
These changes include bossing of the
skull; hypertrophy of the maxilla,
exposing the upper teeth; depression of
nasal bridge; and
periorbital puffiness.
β-Thalassemia major. Note the pallor,
short stature, massive
hepatosplenomegaly,
and wasted limbs in this undertransfused
case of β-thalassemia major.

49. Thalassemia
x-ray film of the
skull showing
new bone
formation on the
outer table,
producing
perpendicular
radiations
resembling a
crew cut.

50. β -Thalassemia Major—Lab findings
• Hypochromic, microcytic anemia
• Target Cells, nucleated RBCs, anisocytosis and
reticulocytosis
• Hemoglobin electrophoresis shows
Increased Hb A2—delta globin production
Increased Hb F—gamma globin production
• Hyperbilirubinemia
• LFT abnormalities (late finding)
• TFT abnormalities, hyperglycemia (late endocrine
findings)

51. Chronic Transfusion Therapy
• Maximizes growth and development
• Suppresses the patient’s own ineffective erythropoiesis and excessive
dietary iron absorption
• PRBC transfusions often monthly to maintain Hgb 10-12
Chelation Therapy
• Binds free iron and reduces hemosiderin deposits
• 8-hour subcutaneous infusion of deferoxamine, 5 nights/week
• Start after 1year of chronic transfusions or ferritin>1000 ng/dl
Splenectomy--indications
• Trasfusion requirements increase 50% in 6mo
• PRBCs per year >250cc/kg
• Severe leukopenia or thrombocytopenia

52. Prognosis in β-Thalassemia major
• Blood transfusions improve the anemia and suppress
complications related to excessive erythropoiesis, but lead to
complications of their own.
• Cardiac disease resulting from progressive iron overload and
secondary hemochromatosis is an important cause of death,
• With transfusions and iron chelation, survival into the third
decade is possible, but the overall outlook remains guarded.
• Bone marrow transplantation is the only therapy offering a cure
and is being used increasingly.
• Prenatal diagnosis is possible by molecular analysis of DNA.

53. Complications and Emergencies
• Sepsis—Encapsulated organisms like Strep Pneumoniae
• Cardiomyopathy—presentation in CHF - Use diuretics,
digoxin, and deferoxamine
• Endocrinopathies—presentation in DKA
• Take care during hydration so as not to precipitate CHF
from fluid overload

54. β-Thalassemia Minor
• β-Thalassemia minor is much more common than β-thalassemia major
and affects the same ethnic groups.
• Most patients are heterozygous carriers of a β+ or β0 allele. These
patients are usually asymptomatic.
• Anemia, if present, is mild.
• The peripheral blood smear shows hypochromia, microcytosis, basophilic
stippling, and target cells.
• Mild erythroid hyperplasia is seen in the bone marrow.
• Hemoglobin electrophoresis usually reveals an increase in HbA2 (α2δ2)
to 4% to 8% of the total hemoglobin (normal, 2.5% ± 0.3%), which is a
reflection of an elevated ratio of δ-chain to β-chain synthesis.
• HbF levels are generally normal or occasionally slightly increased.

55. Importance identifying β-thalassemia trait
(1) differentiation from the hypochromic microcytic anemia
of iron deficiency
(2) genetic counselling.
Iron deficiency can usually be excluded through
measurement of serum iron, total iron-binding capacity,
and serum ferritin
The increase in HbA2 is diagnostically useful, particularly
in individuals (such as women of childbearing age) who
are at risk for both β-thalassemia trait and iron deficiency.

56. High-performance
liquid
chromatography
(HPLC)
sample demonstrating
increased hemoglobin A2
(arrow) in a case of β-
thalassemia trait. HPLC is
an automated way of
separating and identifying
variant hemoglobins and is
more accurate at
quantifying hemoglobin
A2 than is Hb
electrophoresis. It can
separate HbA2 from certain
hemoglobins, which is not
possible using hemoglobin
electrophoresis alone.

57. α-Thalassemias

58. α-Thalassemias
• The α-thalassemias are caused by inherited deletions that
result in reduced or absent synthesis of α-globin chains.
• The severity of α-thalassemia depends on how many α-
globin genes are affected.
• Anemia stems both from a lack of adequate hemoglobin
and the effects of excess unpaired non-α chains (β, γ, and
δ), which vary in type at different ages.

59. World distribution of +α (hatched areas)
and 0α-thalassemia (shaded areas).

60. Most common
deletional
α-thalassemia
defects. HS,
hypersensitive
site.

62. α-Thalassemias - pathophysiology
• In new borns with α-thalassemia, excess unpaired γ-globin
chains form γ4 tetramers known as hemoglobin Barts
• In older children and adults excess β-globin chains form β4
tetramers known as HbH.
• Free β and γ chains are more soluble than free α chains and
form fairly stable homotetramers, hemolysis and ineffective
erythropoiesis are less severe than in β-thalassemias.
• A variety of molecular lesions give rise to α-thalassemia, but
gene deletion is the most common cause of reduced α-chain
synthesis.

63. α-THALASSEMIAS
Silent carrier -/α α/α Asymptomatic; no
red cell abnormality
Mainly gene
deletions
α-Thalassemia trait -/- α/α (Asian) Asymptomatic, like
β-thalassemia
minor-/α -/α (black
African, Asian)
HbH disease -/- -/α Severe; resembles
β-thalassemia
intermedia
Hydrops fetalis -/- -/- Lethal in utero
without transfusions

64. Silent carrier
oThis is associated with the deletion of a single α-globin
gene,
ocauses a barely detectable reduction in α-globin chain
synthesis.
oThese individuals are completely asymptomatic, but they
have slight microcytosis.

65. α-Thalassemia Trait
• This is caused by the deletion of two α-globin genes from a single
chromosome (α/α α/α), or the deletion of one α-globin gene from each
of the two chromosomes (α/—α α/—α).
• The former genotype is more common in Asian populations, the latter
in regions of Africa.
• children of affected individuals, who are at risk of clinically significant
α-thalassemia (HbH disease or hydrops fetalis) only when at least
one parent has the α/—α haplotype.
• As a result, symptomatic α-thalassemia is relatively common in Asian
populations and rare in black African populations.
• The clinical picture in α-thalassemia trait is identical to that described
for β-thalassemia minor.

66. Hemoglobin H Disease
• Caused by deletion of three α-globin genes.
• HbH disease is most common in Asian populations.
• With only one normal α-globin gene, the synthesis of α chains is markedly
reduced, and tetramers of β-globin, called HbH, form.
• HbH has an extremely high affinity for oxygen and therefore is not useful for
oxygen delivery,
• Leading to tissue hypoxia disproportionate to the level of hemoglobin.
• HbH is prone to oxidation, which causes it to precipitate out and form intracellular
inclusions that promote red cell sequestration and phagocytosis in the spleen.
• The result is a moderately severe anemia resembling β-thalassemia intermedia.

67. Hemoglobin H
disease
This blood film
demonstrates
microcytosis,
hypochromasia, and
numerous
morphologic
abnormalities,
including target
cells,
microspherocytes,
and fragments.
Basophilic stippling
may occur.
Polychromasia is
present

68. Hydrops Fetalis
• This most severe form of α-thalassemia is caused by deletion of all
four α-globin genes.
• In the fetus, excess γ-globin chains form tetramers (hemoglobin
Barts) that have such a high affinity for oxygen that they deliver little
to tissues.
• 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 usually become evident by the third trimester of
pregnancy.
• The fetus shows severe pallor, generalized edema, and massive
hepatosplenomegaly similar to that seen in hemolytic disease of the
newborn.
• In the past, severe tissue anoxia led to death in utero or shortly after
birth; with intrauterine transfusion many such infants are now saved.
• There is a lifelong dependence on blood transfusions for survival, with
the associated risk of iron overload.
• Bone marrow transplantation can be curative.

69. Hydrops fetalis
at autopsy in
hemoglobin Bart
disease.
Hepatosplenomegaly in a
newborn with hemoglobin
Bart disease. The loss of
all four α-globin genes
results in severe
anemia, high-output heart
failure, splenomegaly,
edema, and intrauterine
or immediately
postpartum death for the
affected fetus. Dystocia,
eclampsia, and
hemorrhage can occur in
the
mother carrying the
affected fetus.

70. Management in severe thalassemias
• Oral Chelation Agents
• Pharmacologically upregulating gamma globin synthesis,
increasing Hgb F
• Carries O2 better than Hgb A2, Will help bind α-globin and
decreases precipitate
• Bone Marrow transplant
• Gene Therapy - Inserting healthy b genes into stem cells
and transplanting

71. Follow up and anticipatory guidance
• Immunizations—Hepatitis B, Pneumovax
• Follow for signs of diabetes, hypothyroid, gonadotropin
deficiency
• Follow for signs of cardiomyopathy or CHF
• Follow for signs of hepatic dysfunction
• Osteoporosis prevention
• Diet, exercise
• Hormone supplementation
• Osteoclast-inhibiting medications
• Follow ferritin levels

72. Important to remember
• All anemias, whether they are symptomatic or not should
be evaluated fully.
• Importance of early diagnosis and appropriate treatment
in every case, as any delay might lead to increased
mortality, skeletal defects and significant growth
retardation.
• Parents should be provided with adequate counseling
regarding the risks to the progeny.
• In carriers and symptomatic pregnant women, prenatal
diagnosis should be advised.