Download Thalassemia

Hemoglobinopathies
Dr Pupak Derakhshandeh, PhD
Ass Prof of Medical Science
of Tehran University
1
Hemoglobinopathies
Disorders of Hemoglobin
2
Disorders of Hemoglobin
5 % of world population:
carrier for genes, important
disorders of hemoglobin
3
Structure and function
of hemoglobin
 Oxygen carrier
 In vertebrate: red blood
cells
 Four subunits:
2α- and 2-chains
4
Hemoglobin
5
Each Subunits
 Globin: Polypeptide chain
 Heme : Prosthetic group
(Iron-Containing pigment)
Heme + Oxygene
Oxygene transporting
6
Normal adult hemoglobin
HbA:
 2 α globin chain (141 AA)
 2  globin chain (146 AA)
 α22
 Equal length
7
Normal adult hemoglobin
HbA2:
 2 α globin chain
 2 d globin chain
 a 2d 2
8
Normal adult hemoglobin
HbF:
 2 α globin chain
 2 γ globin chain
 α2γ2
9
Normal adult hemoglobin
10
Globin genes synthesis
Lessons from the thalasemia. Nature Reviews, Genetics, volume2, 2001
11
Thalassemia
 Onset: Childhood
 Hypo chromic / Microcytic anemia
 Low level of MCV / MCH
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
 -Thal: Elevated HbA2 (α2d2)
HbF (α2γ2)
 α-Thal: Normal HbA2, HbF
12
13
Thalassemia Minor
14
 Thalassemia major
15
 Thalassemia major
16
Thalassemia Minor
• Thalassemia minor is an inherited form
of hemolytic anemia that is less severe
than thalassemia major.
• This blood smear from an individual with
thalassemia shows small (microcytic),
pale (hypochromic), variously-shaped red
blood cells.
• These small red blood cells (RBCs) are
able to carry less oxygen than normal
RBCs .
17
Hematological values &
a-Thalassemia
a
a
a
a
s
Molecular diagnosis of hemoglobin disorders, Clin. Lab. Haem. 2004, 26, 159–176
18
Thalassemia Major
19
Thalassemia major
an inherited form of hemolytic anemia
red blood cell (hemoglobin)
abnormalities
the most severe form of anemia
the oxygen depletion in the body
becomes apparent within the first 6
months of life
20
If untreated, death usually results
within a few years
Note the small, pale (hypochromic),
abnormally-shaped red blood cells
associated with thalassemia major
The darker cells likely represent
normal RBCs from a blood
transfusion
21
Diesease
 Autosomal recessive
 Deficiency: Synthesis of α/globin
 Origin: Mediteranean, African,
Iranian, Indian, Southeast
Asian
 Resistant to malaria
22
Prevalence of -Thalassemia
 ~ 1.5 % in Africans and
African Americans
 ~ 30 % in Sardinia
23
Pathogenesis of
-Thalassemia
 In adequate Hb production
 Reduced MCV/MCH
 Unbalanced accumulation of a globin
subunits
 Ineffective Erythrocyt
 200 different mutations
 In Iran over 70 mutations
!
24
Prenatal diagnosis
 I. ARMS-PCR (22 common mut.)
 II. PCR-RFLP (9 inf. RFLPs)
 III. RDB (60 mut.)
 IV. Sequencing
25
ARMS-PCR
26
PCR-RFLP
1
2
3
M
4
5
6
7
27
 globin mutations
1. Transcriptional mutations (+)
 In promotor regulatory elements




-101(silent)
-92 (silent)
-88
-30
28
 globin mutations
2. RNA-Processing (º)
 Splice junction




IVSI-1 Cd30
IVSI-2
IVSI-3’ end del 25bp
IvsI-130
 Consensus splice sites (º/ +)
 IVSI-5
 IVSI-6
 IVSII-844
29
 globin mutations
 Cryptic splice sites in Introns (+)
 IVSI-110
 IVSII-745
 Cryptic splice sites in exons
 Cd 26 (HbE)
 Cd 121 (HbD panjab/O Arab)
30
-Thalassemia major








Onset: 6 months
Severe hemolytic anemia
Hb level< 7 g/dl
Skin: pale
Growth retardation
don’t eat or sleep well
Hepatosplenomegaly
Bone marrow expansion:
 Make more red cells





Expantion in face and skull
Spleen: destroy of young red cell
80% of untreated patients: † by 5 y.
Treatment: Cardiac/Hepatic: † by 30 y.
Transfusion +Chelation > 30y.
31
a-Thalassemia
32
Peripheral Blood Smear (1)
Normochrome Normocyte
MCV a
MCH a
33
Peripheral Blood Smear (2)
Hypochrome Microcyte
MCV
MCH
34
Defected globin chains
35
Prevalence of α-Thalassemia
 0.01 % in non malarial areas
ig. UK, Japan
 ~ 49 % in Southwest Pacific
Islands
36
37
α globin mutations
 Deletions: 80-85 % of αThalassemia
Del: 3.7 kb (most frequent)
Del: 4.2 kb



α2 InsI-5bp deletion (αHph1α)
α2 InCd T>C (αNco1α)
αº Variant:
--MED
--CAL
--SEA
38
a-Thalassemia
Trait -a/aa
– Hemoglobin is with in the reference
range.
– Reticulocyte count is within the
reference range.
– Mean corpuscular volume (MCV) is
75-85 fL.
– Mean corpuscular hemoglobin (MCH)
is 26 pg.
39
a-Thalassemia
Alpha1 thalassemia minor (--/aa)
– Hemoglobin is within the reference
range.
– Reticulocyte count is within the
reference range.
– MCV is 65-75 fL.
– MCH is 22 pg.
40
Hemoglobin H disease
Peripheral smear from a patient with hemoglobin H disease showing
target cells, microcytosis and hypochromia. Morphological
abnormalities are similar to those observed in beta thalassemia.
In alpha2 thalassemia (silent trait) only mild microcytosis is
41
observed.
HbH disease
• Hemoglobin H disease
–
–
–
–
–
Hemoglobin is 7-10 g/dL.
Reticulocyte count is 5-10%.
MCV is 55-65 fL.
MCH is 20 pg.
The peripheral blood smear shows small
misshapen red cells, hypochromia,
microcytosis, and targeting.
– Brilliant cresyl blue stain demonstrates
hemoglobin H inclusion bodies.
42
HbH disease








Functional α globin : 1
α: globin ratio : 0.3
Hb level: 7-9 g/dl
Genotype: --/-α
HbH Inclusion (Heinz body): Many
Moderate anemia
Hepatosplenomegaly
Galstones, infection, folic acid deficiency
43
Hydrops fetalis
– Hemoglobin is 4-10 g/dL.
– MCV is 110-120 fL.
– The peripheral blood smear
shows severe hypochromia, and
nucleated red blood cells.
44
Hydrops fetalis






Functional α globin : 0
α: globin ratio : 0.0
Genotype: --/-HbH Inclusion (Heinz body): Present
Severe anemia
Heart defect/fatal in utero/
shortly after birth
45
haemoglobinopathies
• Reduced synthesis of globin chains
(Thalassaemia)
• Synthesis of a structurally abnormal Hb
variant
Thalassaemia
β-thalassemia
( over 200 point mutations)
a-thalassemia
(over 80 deletions & point
mutations)
46
a-globin genes cluster
Chromosome16p13.3
Exon I
Intron I
Blood,Vol 91, No 7 (April 1), 1998: pp 2213-2222
Exon II
Intron II
Exon III
47
Alpha-Thalassemia
inheritance
Autosomal
recessive
48
a-Thalassemia mutations
1) alpha-globin gene deletions
approximately 90% of mutations
2) alpha-globin point mutations
approximately 10% of mutations
49
a-thalassemias phenotype

+
a
or a-thalassemia 2
- non-functional one a-globin gene (-a)

o
a or a-thalassemia
1
- non-functional both a-globin genes (--)
50
+
a -Thalassemia
Rightward
Leftward
Archives of Iranian Medicine, Vol 4, No 4, October 2001
51
a0-Thalassemia
~20 kb
~23 kb
~30 kb
> 300 kb
Southeast Asia
Philippines
Thailand
Chinese
Mediterranean
Greece, Turkey
Blood,Vol 91, No 7 (April 1), 1998: pp 2213-2222
(cd51a1)
52
Outcome of deletions a-globin genes
a-globin
genes
affected
production
a-globin
chains
Genotype
0
100%
aa/aa
Normal, healthy
1
75%
-a/aa
Silent carrier, healthy
2
50%
aa/- or
-a/-a
Carrier, a-thalassaemia trait,
Mild hypochromic microcytic
anemia
3
25%
--/-a
Hemoglobin H disease, mild
to severe hemolytic anemia
- -/- -
Hemoglobin Bart's, hydrops
fetalis
53
4
0%
Outcome
Haemoglobinopathies and clotting disorders. Vol. 36, No. 10, October 2007
HbH Disease
54
Method detection
a-Thalassemia mutations
Salting out DNA extraction
- human whole blood
PCR-Based Strategies
- Gap-PCR
- Multiplex Gap-PCR
DNA Sequencing
55
Gap-PCR
56
Molecular diagnosis of hemoglobin disorders, Clin. Lab. Haem. 2004, 26, 159–176
Primers Multiplex PCR (2)
Chong SS, et al. Single tube multiplex-PCR screen for common deletional
determinants Of a-thalassemia. Blood 2000;95:360–362.
57
Primers DNA Sequencing
Name
M13S
1
13
2
S6
3
S8
Sequence 5'-3'
5´ - CGA CGT TGT AAA ACG ACG GCC
AGT CGC CAG CCA ATG AGC GCC - 3´
5´ - TCC ATT GTT GGC ACA TTC CG - 3´
5´ - TGT CCA CGC CCA TGC TGG CAC - 3´
American Journal of Hematology 74:99–103 (2003)
58
Hba1 Sequence
59
Hba2 Sequence
60
Gap-PCR
61
MED Mutation
1. Positive control
2. Marker 200bp
3. Negative control
Cycle
Temperature
Time
1
94
5 minute
94
30 second
60
30 second
72
120 second
72
7 minute
35
1
62
Multiplex PCR
63
Multiplex PCR
1) Smart taq DNA Polymerase
2) AMS Buffer
3) DMSO
(5-10%)
4) Mgcl2 (50 mM)
Cycle
Temperature
Time
1
94
5 minute
94
60 second
60
60 second
72
120 second
72
7 minute
(2 mM)
5) dNTP (10mM)
(0.2 mM)
35
6) Primers (10pm)
(0.3 µl)
7) ddH20
8) Template
1
64
Result
65
Multiplex PCR (1)
66
Multiplex PCR (2)
67
Multiplex PCR (3)
68
Multiplex PCR (4)
69
Patient samples
Male
Female
RBC : 5.81
MCV :75.9
MCH :25.6
HbA2 :2.6
RBC : 5.41
MCV :75.8
MCH :25.0
HbA2 :2.7
70
Patient sample
HbH
-a3.7/-(a)20.5
71
Mutations in Khorasan
province
65 (57.5%)
41 (36.3%)
Deletion
Without Deletion
Point mutation
6 (6.2%)
72
Sequencing
950 bp PCR Product
a1 S8 Primer
a2 S6 Primer
Khorasan province
- a2: 7 sample
(4 point mutation)
- a1: 7 sample
(2 point mutation)
Khoozestan province
- a2: 4 sample
(4 point mutation)
- a1: 7 sample
( all normal)
73
Mutant Seq.
AAAAAA
Normal Seq.
AATAAA
74
a2 IVSII-55
Normal
CGC>CTC
75
a2 IVS II-24
Normal
TCT>TTT
76
Khorasan & Khoozestan
province
HbA
MCV MCH
2
RBC
Hb
Hct
aa/aa
5.53 +
0.51
13.52 +
1.45
41.73 +
3.61
73.96 +
6.09
24.48 +
2.23
2.96 +
1.21
--MED/aa
0
0
0
0
0
0
3.7
-a /aa
5.68 +
0.43
14.07 +
1.14
42.98 +
3.49
75.45 +
5.23
25.31 +
1.25
2.66 +
0.5
-a3.7/-a3.7
5.74 +
0.56
13.1 +
0.94
41.25 +
3.97
71.9 +
2.89
22.9 +
1.54
2.83 +
1.57
5.81 +
0.64
13.97 +
1.57
42.23 +
3.75
71.2 +
2.38
23.47 +
0.76
2.37 +
0.55
-a /aa
5.73 +
0.7
14.33 +
1.52
75.9 +
4.49
24.95 +
0.59
2.28 +
0.56
-a20.5/aa
0
0
0
0
077
3.7
4.2
-a /-a
4.2
43.45 +
2.78
0
Relationship of MCV to mutations
78
Relationship of MCH to mutations
79
Treatment
 Blood transfusion (3-4 weeks for
life)
Iron accumulation in body
 Remove the iron: Desferal:
Infused under the skin (8-12 h/6 times
a week)
 Bone marrow transplantation
A sib brother or sister
HLA matched
80
Treatment
• Avoid iron supplementation. It contributes to
iron overload
• Administer folate supplementation to provide
adequate amounts of the vitamin for increased
utilization resulting from the hemolytic process
and high bone marrow turnover rate.
• Provide prompt attention to infection, especially
in children who have had a splenectomy.
• Administer blood transfusions only if necessary.
• If chronic transfusion is needed (hemoglobin H
disease), iron chelation therapy should be
81
considered to avoid iron overloading.
Surgical Care
Hemoglobin H disease
– Perform a splenectomy if
transfusion requirements are
increasing.
– Surgical or orthodontic correction
may be necessary to correct
skeletal deformities of the skull
and maxilla due to erythroid
hyperplasia.
82
Sickle Cell disorder
83
Sickle Cell disorder
 Stuck the red cell in the vessels
 In children: Spleen, chest, wrists,ankles
 In adults: hips and shoulders
 Anemia (Hb 7-8 g/dl)
 Infections (take antibiotics)
 Painful crises (6-18 months)
 Swollen and inflamed (hand/food
syndrome)
84
What are the Complications?
•
•
•
•
•
•
•
•
•
pain episodes
increased infections
bone damage
yellow eyes or jaundice
early gallstones
lung blockage
kidney damage and loss of body water in urine
painful erections in men (priapism)
blood blockage in the spleen or liver
(sequestration)
• eye damage
• low red blood cell counts (anemia)
• delayed growth
85
The combination of
hemoglobinopathies
Doesn't cause any health problem:
 α+ Thalassemia / α+ Thalassemia (-a/-a)
HbH disease:
 αº Thalassemia / α+ Thalassemia (--/-a)
Hydrops fetalis:
 αº Thalassemia / αº Thalassemia (--/--)
86
Doesn't cause any
health problem






α+ / º Thalassemia/Thalassemia
α+ / º Thalassemia / HbC
α+ / º Thalassemia / HbD
α+ / º Thalassemia / HbE
α+ / º Thalassemia / HbO Arab
α+ / º Thalassemia / HbS
87
Thalassemia / Thalassemia
Caused severe
health problem!
88
Other combinations





HbC / Thalassemia (no problem)
HbD / Thalassemia (no problem)
HbE / Thalassemia (serious anemia)
Hbs / Thalassemia (intermediate-severe)
HPFH* / Thalassemia (no problem)
*Heriditary persistance of fetal hemoglobin
89
Doesn't cause any
health problem
 HbC / HbC
 HbC / D, E, O Arab, HPFH
 HbD / HbD
 HbD / C, E, O Arab, HPFH
 HbE / HbE
 HbE / C, D, O Arab, HPFH
90
Doesn't cause any health
problem
 HbO Arab / HbO Arab
 HbO Arab/ C, E, D, HPFH
 HPFH / HPFH
 HbH / Thalassemia !
 Thalassemia major/α+/º Thalassemia!
 Thalassemia major / HbC, D
91
serious anemia
 HbH / α+/º Thalassemia
 HbS /  Thalassemia
 HbS / HbC
 HbS / HbD
 HbS / HbE
 HbS / O Arab
92
Prenatal Diagnosis (PND)
93
ThankS for
Your
Attention
94