THALASSEMIAS AND

HAEMOGLOBINOPATHIES

dr. Nyoman Suci W, M.Kes, SpPK

2012
WHAT IS THALASSEMIA?

Genetic blood disorder resulting in a

mutation or deletion of the genes that
control globin production.
Mutations in a given globin gene can
cause a decrease in production of that
globin, resulting in deficiency
Syndromes arising form decreased
rate or absence of globin chain
synthesis.
The resulting imbalance-globin chain
synthesis takes place, giving rise to
the excess amount of the normally
synthesized globin chain.
HAEMOGLOBINOPATHIES
The syndrome arising from the
synthesis of abnormal hemoglobin
or hemoglobin variants.
Point mutation in the globin gene
Amino acid substitution in globin chain
Rate of globin chain synthesis are
theoritically normal.
HAEMOGLOBIN
 4 polypeptide chains (globin)
2  (like)
2 non  (β)

 Each globin chain complexed with haem

Haem: Iron + Protoporphyrin ring
HAEMOGLOBIN MOLE CULE (Hb A)
 HAEMOGLOBIN
 Alpha globin genes coded on Chrom 16
 Each Chrom 16 has 2 alpha gene loci
 Four total per cell
 Non alpha globin genes on Chrom 11
 Arranged from embryonic expression to
adult expression (epsilon, gamma,
delta, beta)
 Adult chromosome has one copy of
beta gene, two per cell
Phenotypic expression of Hemoglobin
results from variations in “non-alpha”
globin genes
 Epsilon  during embryogenesis
 Gamma  2 gamma + 2 alpha = Fetal Hgb
 Delta  2 delta + 2 alpha = Hgb A2
 Beta  2 beta + 2 alpha = HgbA
Copyright ©1997 BMJ Publishing Group Ltd.
NORMAL HEMOGLOBIN
ADULT HAEMOGLOBINS

Hb A (22) ~ 97%

Hb A2 (22) 2 – 3%

Hb F (22) <1.0%
HOW TO NAME THALASSEMIA?
 Named after globin chain that is abnormally
synthesised
 Reduced or absent -globin chain  -thalassemia
 Reduced or absent -globin chain  -thalassemia

 Reduced or absent -globin chain   -thalassemia

 Reduced or absent -globin chain   -thalassemia

 Reduced or absent -globin chains

  -thalassemia
GLOBAL DISTRIBUTION OF THE THALASSEMIAS AND
HAEMOGLOBINOPATHIES
COMMON TYPES OF THALASSEMIA

-thalassemia
-thalassemia
  THAL  THAL
 Chrom 16- globin  Chrom 11-non- globin
1. All four work 1. Both beta proteins work
2. Only 3 work (trait) 2. One works (trait/minor)
3. Two work (minor) 3. Both affected, but not
4. 1 works (Hgb H) absent (intermedia)
5. None work (Hgb Barts) 4. Absent (major)
ALPHA THALAS S E MIA
ALPHA THALASSAEMIA

 Deletion of one or more of the four

 globin genes
 Molecular background :
Gene deletions
Point mutations
ALPHA THAL :COMMON DELETIONS
Bain, Barbara J., Haemoglobinopathy Diagnosis,
Blackwell Science, Oxford, 2001

Chromosome 16
 PATHOPHYSIOLOGY
of ALPHA THALASSAEMIA
  

2 2 2 2
(Hb F) (Hb A)

4 4
Hb Bart’s Hb H
FOETUS ADULT
ALPHA THALASSEMIA

 Mutation of 1 or more of the 4 alpha

globin genes on chromosome 16
 in classical α -thalassemia the defect is
caused by the deletion from
chromosome 16 of entire a-genes.
 Severity of disease depends on number
of genes affected
 Results in an excess of beta globins
ALPHA THALASSEMIA
CHROMOSOME 16:
ALPHA GENOTYPES

  Normal

 - + (Blacks, Asians, most

Mediteranneans)

 -- 0 (Asians, some Mediteranneans)

CLASSIFICATION & TERMINOLOGY
ALPHA THALASSEMIA

- : Indicates a gene deletion:

• Normal /
• Silent carrier - /
• Minor -/-
--/
• Hb H disease --/-
• Barts hydrops fetalis --/--
SILENT CARRIERS (HETEROZYGOTES +/-)
3functional  globin genes
No symptoms, but thalassemia could
potentially appear in offspring
ALPHA THALASSEMIA TRAIT
2 functional globin genes
 -thalassemia-1 or o-
thalassemia (--) --/

 -thalassemia-2or +-
thalassemia (-a) -/-

No serious symptoms
ALPHA0 THALASSAEMIA TRAIT
ALPHA0 THALASSAEMIA TRAIT

28 year old female SEA

Hb: 11,4 g/dL RCC: 5.3 x 1012/L

MCV: 69 fL Hb A2: 2.1%

MCH: 22 pg Hb F: <1%
Hb H stain: Occasional Hb H cells
HPLC, alk gel: NORMAL
Genotype: - - / 
HEMOGLOBIN H DISEASE
1 functional globin gene
 Results in very lightly coloured
red blood cells and possible
severe anemia
 Hemoglobin H is susceptible to
oxidation, therefore oxidant
drugs and foods are avoided
Hb H disease ( --/-)
 Haemoglobin H Disease
Genotype:  - / --

37 year old female

Hb A2: 1.2%
Hb: 9,5 g/dL
Hb F: <1.0%
MCV: 59 fL
Hb H: 10.2%
MCH: 19 pg
Hb H stain: Many Hb H
RCC: 4.6 x 1012/L cells
HAEMOGLOBIN H DISEASE
 HB H INCLUSION BODY TEST

Principle
 Hb H (4) is an unstable hemoglobin commonly
seen in -thalassemia.
 On incubation with some oxidative chemicals
such as brilliant cresyl blue (BCB), HbH is
oxidised, denatured and precipitated in the
erythrocytes and seen as small, evenly-
distributed, intra-erythrocytic blue dots which
termed HbH inclusion bodies.
 HB H INCLUSION BODY TEST

 Homozygous β-thalassemia IB : Negative

 HbE/b-thalassemia IB : Negative
 -thalassemia 1 heterozygote IB : 1/30,000 RBC
 -thalassemia 2 heterozygote IB : Rare
 Hb H disease IB : 50-100% of
total RBC
 Hb H DISEASE
ALKALINE ELECTROPHORESIS

Hb H levels up to 25%
Reduced Hb A2 level
Hb H
Disease

Bart’s

H
HB BART’S HYDROPS FOETALIS

No functional globin genes

Death before birth (embryonic
lethality)
Blood Film :
Hb Bart’s Hydrops Foetalis
 ALPHA THALASSAEMIAS
HAEMATOLOGY

Deletions Hb MCV/MCH Film Hb H Cells Hb EPG HPLC

 (- Normal ~Normal Normal Negative Normal Normal

2 (-- Normal Reduced Mild Difficult Normal Normal

(-- Normal Reduced Mild Occasional Normal Normal

 (--- Reduced Very reduced Bizarre Many Hb H band Hb H peak

NB: A DIAGNOSIS OF ‘NORMAL’ DOES NOT EXCLUDE THE

POSSIBILTY OF ALPHA THALASSAEMIA SILENT CARRIER
BETA THALASSEMIA
BETA THALASSEMIA
The syndrome arising form
decreased or absent rate of -
globin chain synthesis.
Mutations on chromosome 11
Hundreds of mutations possible in
the beta globin gene, therefore beta
thalassemia is more diverse
Results in excess of alpha globins
 BETA THALASSAEMIA
Point mutation in/near  gene

Reduced(+) or absent(0) production of  globin

Increased  (and sometimes ) output

+
Excess  globin

Hb A2 (22) +/- Hb F (22)

(>=4.0 %) * (up to 5 %)
BETA THALASSEMIA

HbF

HbA2
 MOLE CULAR BAS IS OF BE TA
THALAS S AE MIA

Types of mutations which produce beta thalassaemia :

Single base changes, small deletions and insertions. Frame shift (FS),
Nonsense (NS), Splicing (SPL),promotor, CAP, Poly A,Initiation.
Reference: “Essential Haematology”, Hoffbrand & Petit, 1993, page 102.
 Weatherall, DJ,
Phenotype-
Genotype
Relationships in
Monogeneic
Disease: Lessons
From the
Thalassaemias,
Nat. Rev. Gen.
2:245, 2001
 CLASSIFICATION & TERMINOLOGY
BETA THALASSEMIA
+: Indicates diminished, but some production of
globin chain by gene:
0 :Indicates no production of globin chain by gene:

• Normal /
• Minor /0
/+
• Intermedia 0/+
• Major 0/0
+/+
BETA THALASSEMIA TRAIT
Slight lack of beta globin
Smaller red blood cells that are
lighter in colour due to lack of
hemoglobin
MCV < 80 fl and or
MCH < 27
No major symptoms
 The severity of disease expression may only be
seen as mild anemia and a microcytic state.
 It thus may be difficult to distinguish from iron
deficiency.
 Differentiating features include the following:
Thalassemias are more apt to demonstrate
reticulocytosis and basophilic stippling in the
peripheral blood.
Heterozygous  thalassaemia

47 year old male

Hb: 13,8 g/dL RCC: 6.6 x 1012/L

MCV: 68 fL Hb A2: 5.3%

MCH: 21 pg Hb F: 4.2%

Genotype:  /  +
HETEROZYGOUS BETA THALASSAEMIA
ALKALINE ELECTROPHORESIS

CATHODE (-) ANODE (+)

NORMAL

HET  THAL

A2 FA
Normal
HETEROZYGOUS
BETA
THALASSAEMIA
BETA THALASSEMIA INTERMEDIA
 Lack of beta globin is more significant
 Bony deformities due to bone marrow
trying to make more blood cells to replace
defective ones
 Causes late development, exercise
intolerance, and high levels of iron in blood
due to reabsorption in the GI tract
 If unable to maintain hemoglobin levels
between 6 gm/dl – 7 gm/dl, transfusion or
splenectomy is recommended
BETA THALASSEMIA MAJOR
Complete absence of beta globin
Enlarged spleen, lightly coloured
blood cells
Severe anemia
Chronic transfusions required, in
conjunction with chelation therapy to
reduce iron (desferoxamine)
 BETA THALASSAEMIA MAJOR:
PATHOPHYSIOLOGICAL FEATURES
  
REF: “Sickle Cell

2  2
Disease &
Denaturation Thalasaemia”
Balliere’s Clinical
Degradation Haematology
11:1,129, 1998

Hb F Haemolysis Destruction of
Selective survival of red cell
HbF containing precursors
precursors

Increased levels of Splenomegaly Ineffective

Hb F in red cells (pooling, plasma erythropoiesis
volume expansion)
BETA THALASSAEMIA MAJOR:
PATHOPHYSIOLOGICAL FEATURES (CONT)
REF: “Sickle Cell
Increased levels of Splenomegaly Ineffective Disease &
Thalasaemia”
Hb F in red cells (pooling, plasma erythropoiesis Balliere’s
volume expansion) Clinical
Haematology
11:1,129, 1998

High oxygen affinity Anaemia

of red cells
Reduced O2 delivery Tissue hypoxia
Erythropoietin Transfusion

Marrow Expansion
Increased iron absorption
Iron Loading

Skeletal deformity
Increased metabolic rate
Wasting, Gout, Folate deficiency Endocrine deficiencies
Cirrhosis, Cardiac
failure
 Classical Presentation
 Frontal bossing/severe thalassemia facies (see
photo below)
 Hepatosplenomegaly
 Hypersplenism
 Pallor
 Cachexia
 Fatigue
 Poor appetite
THALASSEMIC FACE
THALASSEMIA FACE
HEPATOSPLENOMEGALY
Hair on End Appearance
DARK SKIN DUE TO IRON OVERLOAD
BETA THALASSAEMIA MAJOR

Bain, Barbara J.,

Haemoglobinopathy
Diagnosis,
Blackwell Science,
Oxford, 2001
Definisi Geno- HPLC Fenotip
tip
Normal β/β Normal Hb, MCV dan MCH
normal. Asimptomatik
Trait talasemia -/β HbA2 > 3,5 % * Anemia ringan, MCV dan
β MCH . Asimptomatik
Talasemia β -/β0 Hb F  bervariasi Anemia. MCV dan MCH
intermedia atau , splenomegali,
β+/β+ perubahan tulang
** bervariasi,
ketergantungan transfusi
bervariasi
Talasemia β -/- Hb F > 90% (tidak Anemia hemolitik berat,
mayor *** ditransfusi) MCV dan MCH ,
hepatosplenomegali,
transfussion dependent.
 Silent/Normal Hb A2: Globin production not
obviously affected (heterozygote)
*5’ flanking (CAP)
*RNA processing (promoter, transcription,
splicing reduced)
* thal
 Dominant: Heterozygous condition produces
disease
*Nonsense/ frameshift Exon 3
*/
‘SILENT’ BETA THAL MUTATIONS
ETHNIC POSITION MUT Hb MCV MCH Hb A2 Hb F
GROUP ATION
Medit -101 C->T 110-170 70-92 23-33 3.0-4.4 0.1-3.4

Medit -92 C->T 120-160 79-86 25-30 3.4-4.0 <1

Indian CAP+1 A->C 120-140 76-81 25-27 3.4-3.6 <1

(Asian)
Chinese CAP+8 C->T 98 34 3.0 1.8

Greek CAP+10 (-T) 140-160 94-102 30-33 2.5-2.7 <1

Greek CAP+33 C->G 120-140 85-86 28-29 2.8-3.0 1.6-2.0

Greek Terminal C->G 120-170 79-95 20-31 1.9-3.4 0.3-3.0

codon +6
Italian IVSII-844 C->G 130-160 73-90 26-31 3.0-3.8 <1

Bain, Barbara J., Haemoglobinopathy Diagnosis,

Blackwell Science, Oxford, 2001
 NORMAL HBA2 BETA THAL
Microcytic hypochromic
Frequently results from double heterozygosity for beta
and delta thalassaemia.
Delta thal most common in Mediterranean –Greek and
Italian
Hb A2 2.2 – 3.5%
Other mutations:
ETHNIC POSITION MUTA MCV MCH Hb A2
GROUP TION
Medit IVS1-6 T->C 71 23 3.5
Malta
Saudi IVS1-28 T->G 70 25 3.5

Medit Cd 27 G->T 71 25 2.1

Middle Ala->Ser Hb
East Knossos
THALASSEMIA /
HAEMOGLOBINOPATHIES &
IRON DEFICIENCY
Iron deficiency may
1. Mask thalassaemias
 : Hb H production reduced
 : Hb A2 production reduced
2. Reduce Hb variant levels
NEONATAL AND ADULT
HAEMOGLOBINS

% NEONATE ADULT

Hb A UP TO 30 ~97

Hb F 70-100 <1

Hb A2 ~NIL 2-3
ALPHA/BETA THALASSAEMIA INTERACTIONS

Alpha thalassaemia genes can ameliorate the

course of beta thalassaemia major due to
reduction in amount of free  globin
ie, globin chain imbalance is reduced.

NB: A DIAGNOSIS OF BETA THALASSAEMIA

TRAIT DOES NOT EXCLUDE THE POSSIBILTY OF
CO-EXISTENT ALPHA THALASSAEMIA.
HAEMOGLOBINOPATHIES
(HAEMOGLOBIN VARIANTS)
HAEMOGLOBINOPATHIES
Point mutation in  or  gene

Amino acid substitution in  or 

globin

Abnormal Hb
(2*2 or *22 + *22)
-STRUCTURAL VARIANTS
(469 VAR SUBMITTED, JULY 2002)

 Hb Anantharaj  Hb Suan Dok

cd11(Lys-Glu) cd109(Leu-Arg)
 Hb Mahidol  Hb Constant spring
cd74(Asp-His) cd142(stop-Gln)
 Hb Siam

cd15(Gly-Arg)
-STRUCTURAL VARIANTS
(649 VAR SUBMITTED, JULY 2002)

 Hb D-Punjab  Hb G-Siriraj
cd121(Glu-Gln) cd7(Glu-Lys)
 Hb J-Bangkok  Hb Tak
cd56(Gly-Asp) cd147(+AC)
 Hb S  Hb E
cd6(Glu-Val) cd26 (Glu-Lys)
GEOGRAPHIC DISTRIBUTION OF
HAEMOGLOBINOPATHIES
THALASSAEMIA & HB VARIANTS

  thal gene +  variant -> variant

  thal gene +  variant -> variant
  thal + Hb E -> thal major !!!!
  thal gene +  variant -> variant
  thal gene +  variant -> variant
HAEMOGLOBIN E
(26 GLU -> LYS)
GEOGRAPHIC DISTRIBUTION OF
Hb E
HB E HETEROZYGOTES PROPERTIES
EPG MOBILITY: Alkaline as Hb A2
Acid as Hb A
HPLC: Co-elutes with Hb A2
LEVEL: (Hb E + Hb A2) : 27 – 30% (cryptic splice site)

HAEMATOLOGY: MCV/MCH may be reduced

FILM : Mild. Occasional targets.
May have slight microcytosis
Heterozygous Hb E

29 year old female

Hb: 11,9 g/dL HbE (+ A2): 31.3%

MCV: 74 fL Hb F: 0.5%

MCH: 24 pg

RCC: 5.0 x 1012/L

Genotype:  /  E
HETEROZYGOUS HB E : BLOOD FILM
 HETEROZYGOUS Hb E
ALKALINE ELECTROPHORESIS

CATHODE(-) ANODE (+)

Hb E
 Homozygous Hb E
35 year old female

Hb: 10,2 g/dL Hb E(+Hb A2): 96.6%

MCV: 62 fL Hb F: 3.4%

MCH: 21 pg

RCC: 4.9 x 1012/L

Genotype:  E /  E
HOMOZYGOUS HB E
 HOMOZYGOUS Hb E
ALKALINE ELECTROPHORESIS
Homozygous Hb E

NB: Co-existent
alpha thalassaemia
not excluded
 DCIP PRECIPITATION TEST
Principle
HbE (2E2) has loose contact between 1
and 1-globin chains.
When it is incubated with dichlorophenol
indophenol (DCIP); oxidizing agent, it will
be denatured and precipitated.
The reaction is stopped by adding ascorbic
acid and the denatured HbE precipitates.
DCIP TEST

After adding 5% ascorbic acid

Pos Pos
Before Neg 5% ascorbic acid
adding

Neg Pos Pos

DCIP TEST

Normal Negative
Homo E 3+- 4+
HbE trait 1+- 2+
β-thal/HbE disease 1+- 2+
HbH disease 1+ - 2+

Report as positive or negative

HBE/B-THAL  THALASSEMIA
MAJOR

400x
THALASSEMIA DIAGNOSIS

History retrieve
Physical examination
Laboratory investigation
LABORATORY INVESTIGATIONS

Redcell studies
Hemoglobin studies
DNA analysis