Thalassemia 89 90

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HemoglobinopatHemoglobinopathieshies

Dr PupakDr Pupak Derakhshandeh, PhDDerakhshandeh, PhDAss Prof of Medical Science Ass Prof of Medical Science

of Tehran Universityof Tehran University

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HemoglobinopatHemoglobinopathieshies

Disorders of Disorders of HemoglobinHemoglobin

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Disorders of Disorders of HemoglobinHemoglobin

5 % of world population: 5 % of world population: carrier for genes, carrier for genes, important disorders of important disorders of hemoglobinhemoglobin

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Structure and Structure and function of function of hemoglobinhemoglobin

Oxygen carrierOxygen carrier In vertebrate: red In vertebrate: red blood blood cellscells

Four subunits: Four subunits: 22αα- and 2- and 2-chains-chains

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HemoglobinHemoglobin

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Each SubunitsEach Subunits

Globin: Polypeptide chainGlobin: Polypeptide chain Heme : Prosthetic groupHeme : Prosthetic group

(Iron-Containing (Iron-Containing pigment)pigment)

Heme + OxygeneHeme + OxygeneOxygene transportingOxygene transporting

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Normal adult Normal adult hemoglobinhemoglobin

HbAHbA::

22 αα globin chain (141 AA) globin chain (141 AA) 2 2 globin chain (146 AA) globin chain (146 AA) αα2222

Equal lengthEqual length

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HbA2HbA2::

22 αα globin chain globin chain 2 2 globin chain globin chain

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HbFHbF::

22 αα globin chain globin chain 2 2 γγ globin chain globin chain αα22γγ22

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Globin genes synthesisGlobin genes synthesis

Lessons from the thalasemia. Nature Reviews, Genetics, volume2, 2001Lessons from the thalasemia. Nature Reviews, Genetics, volume2, 2001

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ThalassemiThalassemiaa

Onset: ChildhoodOnset: Childhood Hypo chromic / Microcytic Hypo chromic / Microcytic anemiaanemia Low level of MCV / MCHLow level of MCV / MCH

Mean corpuscular volume (MCV)Mean corpuscular hemoglobin (MCH)

-Thal: Elevated HbA-Thal: Elevated HbA2 2 ((αα2222))

HbF (HbF (αα22γγ22)) αα-Thal: Normal HbA-Thal: Normal HbA22, HbF, HbF

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Thalassemia Thalassemia MinorMinor

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Thalassemia majorThalassemia major

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Thalassemia majorThalassemia major

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Thalassemia MinorThalassemia Minor

• Thalassemia minor is an inherited Thalassemia minor is an inherited form of hemolytic anemia that is form of hemolytic anemia that is less severe than thalassemia majorless severe than thalassemia major..

• This blood smear from an individual This blood smear from an individual with thalassemia shows small with thalassemia shows small ((microcyticmicrocytic)), pale , pale ((hypochromichypochromic)), , variouslyvariously--shaped red blood cellsshaped red blood cells..

• These small red blood cells These small red blood cells ((RBCsRBCs) ) are able to carry less oxygen than are able to carry less oxygen than normal RBCsnormal RBCs. .

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Hematological values &a-Thalassemia

Hematological values &a-Thalassemia

Molecular diagnosis of hemoglobin disorders, Clin. Lab. Haem. 2004, 26, 159–176

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Thalassemia Thalassemia MajorMajor

http://www.snn-rdr.ca/snn/old/may2000/may2000/images/thalassemia.jpg

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an inherited form of hemolytic an inherited form of hemolytic anemiaanemia

red blood cell red blood cell ((hemoglobinhemoglobin)) abnormalitiesabnormalities

the most severe form of anemiathe most severe form of anemia

the oxygen depletion in the bodythe oxygen depletion in the body becomes apparent within the first becomes apparent within the first 6 months of life6 months of life

Thalassemia major

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If untreated, death usually results If untreated, death usually results within a few yearswithin a few years

Note the small, pale Note the small, pale ((hypochromichypochromic)), abnormally, abnormally--shaped red blood cells associated shaped red blood cells associated with thalassemia majorwith thalassemia major

The darker cells likely represent The darker cells likely represent normal RBCs from a blood normal RBCs from a blood transfusiontransfusion

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DieseaseDiesease Autosomal recessive Autosomal recessive Deficiency: Synthesis of Deficiency: Synthesis of

αα//- - globinglobin Origin: Mediteranean, Origin: Mediteranean,

African, African, Iranian, Indian, Iranian, Indian, Southeast Southeast AsianAsian

Resistant to malariaResistant to malaria

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Prevalence of Prevalence of --ThalassemiaThalassemia

~ 1.5 % in Africans and ~ 1.5 % in Africans and African AmericansAfrican Americans

~ 30 % in Sardinia~ 30 % in Sardinia

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Pathogenesis Pathogenesis of of -Thalassemia-Thalassemia

In adequate Hb productionIn adequate Hb production Reduced MCV/MCHReduced MCV/MCH

Unbalanced accumulation of Unbalanced accumulation of globin subunitsglobin subunits Ineffective ErythrocytIneffective Erythrocyt

200 different mutations200 different mutations

In Iran over 70 mutations In Iran over 70 mutations !!

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Prenatal diagnosisPrenatal diagnosis

I. ARMS-PCR (22 common I. ARMS-PCR (22 common mut.)mut.)

II. PCR-RFLP (9 inf. RFLPs)II. PCR-RFLP (9 inf. RFLPs)

III. RDB (60 mut.)III. RDB (60 mut.)

IV. SequencingIV. Sequencing

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ARMS-PCR

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PCR-RFLPPCR-RFLP1 2 3 M 4 5 6 7

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globin mutationsglobin mutations

1.1. Transcriptional mutations Transcriptional mutations ((++))

In promotor regulatory In promotor regulatory elementselements -101(silent)-101(silent) -92 (silent)-92 (silent) -88-88 -30-30

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globin globin mutationsmutations

2.2. RNA-Processing ( RNA-Processing (ºº)) Splice junctionSplice junction

IVSI-1 Cd30IVSI-1 Cd30 IVSI-2 IVSI-2 IVSI-3’ end del 25bpIVSI-3’ end del 25bp IvsI-130IvsI-130

Consensus splice sites Consensus splice sites ((º/ º/ ++)) IVSI-5IVSI-5 IVSI-6IVSI-6 IVSII-844IVSII-844

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globin mutationsglobin mutations

Cryptic splice sites in Introns Cryptic splice sites in Introns ((++)) IVSI-110IVSI-110 IVSII-745IVSII-745

Cryptic splice sites in exonsCryptic splice sites in exons Cd 26 (HbE)Cd 26 (HbE) Cd 121 (HbD panjab/O Arab)Cd 121 (HbD panjab/O Arab)

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-Thalassemia -Thalassemia majormajor

Onset: 6 monthsOnset: 6 months Severe hemolytic anemiaSevere hemolytic anemia Hb level< 7 g/dlHb level< 7 g/dl Skin: paleSkin: pale Growth retardationGrowth retardation don’t eat or sleep welldon’t eat or sleep well HepatosplenomegalyHepatosplenomegaly Bone marrow expansion:Bone marrow expansion:

Make more red cellsMake more red cells Expantion in face and skullExpantion in face and skull Spleen: destroy of young red cellSpleen: destroy of young red cell 80% of untreated patients: † by 5 y.80% of untreated patients: † by 5 y. Treatment: Cardiac/Hepatic: † by 30 y.Treatment: Cardiac/Hepatic: † by 30 y. Transfusion +Chelation > 30yTransfusion +Chelation > 30y..

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--ThalassemiaThalassemia

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Peripheral Blood Smear Peripheral Blood Smear (1)(1)


Normochrome Normocyte Normochrome Normocyte Normochrome Normocyte Normochrome Normocyte MCV MCV MCH MCH MCV MCV MCH MCH

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Hypochrome MicrocyteHypochrome Microcyte MCV MCV

MCH MCH

MCV MCV

MCH MCH

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Defected globin Defected globin chainschains

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Prevalence of Prevalence of αα--ThalassemiaThalassemia

0.01 % in non malarial 0.01 % in non malarial areasareas

ig. UK, Japanig. UK, Japan ~ 49 % in Southwest ~ 49 % in Southwest

Pacific Pacific IslandsIslands

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αα globin globin mutationsmutations

Deletions: 80-85 % of Deletions: 80-85 % of ααThalassemiaThalassemiaDel: 3.7 kb (most frequent)Del: 3.7 kb (most frequent)Del: 4.2 kbDel: 4.2 kb

αα22 InsI-5bp deletion ( InsI-5bp deletion (ααHph1Hph1αα))

αα22 InCd T>C ( InCd T>C (ααNco1Nco1αα))

ααºº Variant:Variant:--MED--MED--CAL--CAL--SEA--SEA

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-Thalassemia-Thalassemia Trait -

– Hemoglobin is with in the reference Hemoglobin is with in the reference range.range.

– Reticulocyte count is within the reference Reticulocyte count is within the reference range.range.

– Mean corpuscular volume (MCV) is 75-85 Mean corpuscular volume (MCV) is 75-85 fL.fL.

– Mean corpuscular hemoglobin (MCH) is Mean corpuscular hemoglobin (MCH) is 26 pg.26 pg.

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a-Thalassemiaa-Thalassemia Alpha1 thalassemia minor (--/)

– Hemoglobin is within the reference Hemoglobin is within the reference range.range.

– Reticulocyte count is within the Reticulocyte count is within the reference range.reference range.

– MCV is 65-75 fL.MCV is 65-75 fL.– MCH is 22 pg.MCH is 22 pg.

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Hemoglobin H diseaseHemoglobin H disease

Peripheral smear from a patient with hemoglobin H Peripheral smear from a patient with hemoglobin H disease showing target cells, microcytosis and disease showing target cells, microcytosis and hypochromia. Morphological abnormalities are similar to hypochromia. Morphological abnormalities are similar to those observed in beta thalassemia. In alpha2 those observed in beta thalassemia. In alpha2 thalassemia (silent trait) only mild microcytosis is thalassemia (silent trait) only mild microcytosis is observed.observed.

http://www.emedicine.com/cgi-bin/foxweb.exe/makezoom@/em/makezoom?picture=%5Cwebsites%5Cemedicine%5Cmed%5Cimages%5CLarge%5C1345thalmin.jpg&template=izoom2

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HbH diseaseHbH disease•Hemoglobin H disease

– Hemoglobin is 7-10 g/dL.Hemoglobin is 7-10 g/dL.– Reticulocyte count is 5-10%.Reticulocyte count is 5-10%.– MCV is 55-65 fL.MCV is 55-65 fL.– MCH is 20 pg.MCH is 20 pg.– The peripheral blood smear shows The peripheral blood smear shows

small misshapen red cells, small misshapen red cells, hypochromia, microcytosis, and hypochromia, microcytosis, and targeting.targeting.

– Brilliant cresyl blue stain Brilliant cresyl blue stain demonstrates hemoglobin H demonstrates hemoglobin H inclusion bodies.inclusion bodies.

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HbH diseaseHbH disease

Functional Functional αα globinglobin : 1: 1 αα:: globin ratio : 0.3 globin ratio : 0.3 Hb level: 7-9 g/dlHb level: 7-9 g/dl Genotype: --/-Genotype: --/-αα HbH Inclusion (Heinz body): ManyHbH Inclusion (Heinz body): Many Moderate anemiaModerate anemia HepatosplenomegalyHepatosplenomegaly Galstones, infection, folic acid Galstones, infection, folic acid

deficiencydeficiency

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Hydrops fetalisHydrops fetalis

– Hemoglobin is 4-10 g/dL. Hemoglobin is 4-10 g/dL. – MCV is 110-120 fL. MCV is 110-120 fL. – The peripheral blood smear The peripheral blood smear shows severe hypochromia, shows severe hypochromia, and nucleated red blood and nucleated red blood cells.cells.

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Hydrops fetalisHydrops fetalis

Functional Functional αα globinglobin : 0: 0 αα:: globin ratio : 0.0 globin ratio : 0.0 Genotype: --/--Genotype: --/-- HbH Inclusion (Heinz body): HbH Inclusion (Heinz body):

PresentPresent Severe anemiaSevere anemia Heart defect/fatal in utero/Heart defect/fatal in utero/

shortly after birthshortly after birth

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haemoglobinopathies

haemoglobinopathies

• Reduced synthesis of globin chains Reduced synthesis of globin chains (Thalassaemia)(Thalassaemia)

• Synthesis of a structurally abnormal Synthesis of a structurally abnormal Hb Hb variantvariant

Thalassaemia

β-thalassemia

( over 200 point mutations)

a-thalassemia

(over 80 deletions & point mutations)

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a-globin genes cluster

a-globin genes cluster

Chromosome16p13.3

Blood,Vol 91, No 7 (April 1), 1998: pp 2213-2222

Exon I Exon II Exon III Intron I Intron II

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Alpha-Thalassemia inheritance

Alpha-Thalassemia inheritance

Autosomal Autosomal recessiverecessiveAutosomal Autosomal recessiverecessive

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1)1) alpha-globin alpha-globin gene deletionsgene deletions approximately approximately 90%90% of mutations of mutations

2)2) alpha-globin alpha-globin point mutationspoint mutations approximatelyapproximately 10%10% of mutations of mutations

-Thalassemia mutations-Thalassemia mutations

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a-thalassemias phenotype

a-thalassemias phenotype

++ or or --thalassemia 2thalassemia 2 - non-functional one - non-functional one -globin gene -globin gene (-(-))

o o oror --thalassemia 1 thalassemia 1 - non-functional both - non-functional both -globin genes-globin genes (--)

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a+-Thalassemiaa+-Thalassemia

Archives of Iranian Medicine, Vol 4, No 4, October 2001

Rightward

Leftward

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a0-Thalassemiaa0-Thalassemia

Blood,Vol 91, No 7 (April 1), 1998: pp 2213-2222

> 300 kb> 300 kb

Southeast AsiaSoutheast Asia

PhilippinesPhilippines

ThailandThailand

ChineseChinese

MediterraneanMediterranean

Greece, TurkeyGreece, Turkey

~~30 kb30 kb

~~20 kb20 kb

~~23 kb23 kb

(cd51(cd511)1)

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a-globingenes

affected

production a-globin chains

Genotype Outcome

0 100% Normal, healthy

1 75% Silent carrier, healthy

2 50%

or

Carrier, a-thalassaemia trait, Mild hypochromic microcytic anemia

3 25%--/- Hemoglobin H disease, mild

to severe hemolytic anemia

4 0%- -/- - Hemoglobin Bart's, hydrops

fetalisHaemoglobinopathies and clotting disorders. Vol. 36, No. 10, October 2007

Outcome of deletions a-globin genesOutcome of deletions a-globin genes

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HbH DiseaseHbH Disease

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Method detection a-Thalassemia

mutations

Method detection a-Thalassemia

mutations

Salting out DNA extractionSalting out DNA extraction -- human whole bloodhuman whole blood

PCR-Based Strategies PCR-Based Strategies - Gap-PCR- Gap-PCR - Multiplex Gap-PCR- Multiplex Gap-PCR

DNA Sequencing DNA Sequencing

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Gap-PCRGap-PCRGap-PCRGap-PCR

Molecular diagnosis of hemoglobin disorders, Clin. Lab. Haem. 2004, 26, 159–176

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Primers Multiplex PCR (2)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.

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Primers DNA SequencingPrimers DNA Sequencing

Name Sequence 5'-3'

1M13S

135´ - CGA CGT TGT AAA ACG ACG GCC AGT CGC CAG CCA ATG AGC GCC - 3´

2 S6 5´ - TCC ATT GTT GGC ACA TTC CG - 3´

3 S8 5´ - TGT CCA CGC CCA TGC TGG CAC - 3´

American Journal of Hematology 74:99–103 (2003)

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Hba1 SequenceHba1 Sequence

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Hba2 SequenceHba2 Sequence

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Gap-PCRGap-PCR

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MED MutationMED Mutation1.1. Positive controlPositive control

2.2. Marker 200bpMarker 200bp

3.3. Negative controlNegative control

Cycle Temperature Time

1 94 5 minute

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94 30 second

60 30 second

72 120 second

1 72 7 minute

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Multiplex PCR

Multiplex PCR

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Multiplex PCRMultiplex PCR

Cycle Temperature Time

1 94 5 minute

35

94 60 second

60 60 second

72 120 second

1 72 7 minute

1) Smart taq DNA Polymerase 2) AMS Buffer 3) DMSO (5-10%)

4) Mgcl2 (50 mM) (2 mM)

5) dNTP (10mM) (0.2 mM)

6) Primers (10pm) (0.3 µl)

7) ddH20 8) Template

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ResultResult

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Multiplex PCR (1)Multiplex PCR (1)

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Patient samplesPatient samplesMale RBC : 5.81MCV :75.9MCH :25.6HbA2 :2.6

Female RBC : 5.41MCV :75.8MCH :25.0HbA2 :2.7

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Patient samplePatient sample

HbHHbH

-a-a3.73.7/-(a)/-(a)20.520.5

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Mutations in Khorasan province

Mutations in Khorasan province

6 (6.2%)

41 (36.3%)65 (57.5%) Deletion

Without Deletion

Point mutation

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Sequencing Sequencing

Khorasan provinceKhorasan province - 2: 7 sample (4 point mutation)

- 1: 7 sample (2 point mutation)

Khoozestan provinceKhoozestan province - 2: 4 sample (4 point mutation)

- 1: 7 sample ( all normal)

950 bp PCR Product950 bp PCR Product

S8 PrimerS8 Primer

2 S6 Primer 2 S6 Primer

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Normal Seq.

AATAAA

Mutant Seq.

AAAAAA

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NormalNormal

2 IVSII-552 IVSII-55

CCGGC>CC>CTTCC

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NormalNormal

2 IVS II-242 IVS II-24

TTCCT>TT>TTTTT

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Khorasan & Khoozestan province

Khorasan & Khoozestan province

RBC Hb Hct MCVMCV MCHMCHHbA

2

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 0 0 0 0 0 0

5.68 + 0.43

14.07 + 1.14

42.98 + 3.49

75.45 + 5.23

25.31 + 1.25

2.66 + 0.5

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

5.73 + 0.7

14.33 + 1.52

43.45 + 2.78

75.9 + 4.49

24.95 + 0.59

2.28 + 0.56

0 0 0 0 0 0

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Relationship of MCV to mutationsRelationship of MCV to mutationsRelationship of MCV to mutationsRelationship of MCV to mutations

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Relationship of MCH to mutationsRelationship of MCH to mutations

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TreatmentTreatment Blood transfusion (3-4 weeks for Blood transfusion (3-4 weeks for

life)life)Iron accumulation in bodyIron accumulation in body

Remove the iron: Desferal:Remove the iron: Desferal:Infused under the skin (8-12 h/6 Infused under the skin (8-12 h/6

times a week)times a week) Bone marrow transplantationBone marrow transplantation

A sib brother or sisterA sib brother or sisterHLA matchedHLA matched

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TreatmentTreatment• 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 considered to avoid iron overloading.

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Surgical CareSurgical CareHemoglobin 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.

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Sickle Cell disorderSickle Cell disorder

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Sickle Cell disorderSickle Cell disorder

Stuck the red cell in the vesselsStuck the red cell in the vessels In children: Spleen, chest, In children: Spleen, chest,

wrists,ankleswrists,ankles In adults: hips and shouldersIn adults: hips and shoulders Anemia (Hb 7-8 g/dl)Anemia (Hb 7-8 g/dl) Infections (take antibiotics)Infections (take antibiotics) Painful crises (6-18 months)Painful crises (6-18 months) Swollen and inflamed (hand/food Swollen and inflamed (hand/food

syndrome)syndrome)

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What are the What are the Complications?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

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The combination of The combination of hemoglobinopathieshemoglobinopathies

Doesn't cause any health Doesn't cause any health problem:problem: αα+ + Thalassemia / Thalassemia / αα+ + Thalassemia (-a/-a)Thalassemia (-a/-a)

HbH disease:HbH disease: ααº º Thalassemia / Thalassemia / αα+ + Thalassemia (--/-a)Thalassemia (--/-a)

Hydrops fetalis:Hydrops fetalis: ααº º Thalassemia / Thalassemia / ααº º Thalassemia (--/--)Thalassemia (--/--)

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Doesn't cause any Doesn't cause any health problemhealth problem

αα+ / º + / º

Thalassemia/Thalassemia/ThalassemiaThalassemia αα+ / º + / º Thalassemia / HbCThalassemia / HbC αα+ / º + / º Thalassemia / HbDThalassemia / HbD αα+ / º + / º Thalassemia / HbEThalassemia / HbE αα+ / º + / º Thalassemia / HbO ArabThalassemia / HbO Arab αα+ / º + / º Thalassemia / HbSThalassemia / HbS

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Thalassemia / Thalassemia / ThalassemiaThalassemia

Caused severe Caused severe health health problem!problem!

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Other Other combinationscombinations

HbC / HbC / Thalassemia (no problem)Thalassemia (no problem) HbD / HbD / Thalassemia (no problem)Thalassemia (no problem) HbE / HbE / Thalassemia (serious anemia)Thalassemia (serious anemia) Hbs / Hbs / Thalassemia (intermediate-Thalassemia (intermediate-

severe)severe) HPFH* / HPFH* / Thalassemia (no problem)Thalassemia (no problem)

*Heriditary persistance of fetal hemoglobin*Heriditary persistance of fetal hemoglobin

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HbC / HbCHbC / HbC HbC / D, E, O Arab, HPFHHbC / D, E, O Arab, HPFH

HbD / HbDHbD / HbD HbD / C, E, O Arab, HPFHHbD / C, E, O Arab, HPFH

HbE / HbEHbE / HbE HbE / C, D, O Arab, HPFHHbE / C, D, O Arab, HPFH

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HbO Arab / HbO ArabHbO Arab / HbO Arab HbO Arab/ C, E, D, HPFHHbO Arab/ C, E, D, HPFH HPFH / HPFHHPFH / HPFH

HbH / HbH / Thalassemia !Thalassemia ! Thalassemia major/Thalassemia major/αα+/º +/º

Thalassemia! Thalassemia! Thalassemia major / HbC, DThalassemia major / HbC, D

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serious serious anemiaanemia

HbH / HbH / αα+/º +/º ThalassemiaThalassemia HbS / HbS / Thalassemia Thalassemia HbS / HbCHbS / HbC HbS / HbDHbS / HbD HbS / HbEHbS / HbE HbS / O ArabHbS / O Arab

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Prenatal Diagnosis Prenatal Diagnosis (PND)(PND)

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Thalassemia 89 90