hemoglobin & hemoglobinopathies palm springs -...

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Hemoglobin & Hemoglobinopathies

beyond the peripheral slide Jane Merschen MT (ASCP), MPH"

An Emerging Global Health Burden"

Breakdown of the annual number of births with the different hemoglobin disorders

Major hemoglobin disorder No. of annual births

β-thalassemia major 22 989 Hb E thalassemia 19 128 Hb H disease 9 568 Hb Bart hydrops 5 183 Hb SS disease 217 331 Hb Sβ-thalassemia 11 074 Hb SC disease 54 736

Weatherall DJ, Blood, 2010;115:4331-4336.

Distribution of sickle cell trait Historical distribution of malaria

Modern distribution of malaria

What is hemoglobin ?"

Hemoglobin is a protein in red blood cells that carries oxygen

Hemoglobin structure"Tetrameric structure made by 2 dissimilar pairs of globin

chains each linked to a single HEME molecule"

•  Alpha globin chains: (α) "

•  Non-alpha globin chains:" Beta (β), gamma (γ), delta (δ) "

Normal adult Hbs consists of 2 (α) chains + another pair "

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Hemoglobin structure"Highly conserved protein""Proteins are made of amino acids""Gas transporting molecule that makes up 95% of the RBC""Carries Oxygen"

Adult Hemoglobins" HbA (α2 β2) "§  Major adult hemoglobin "§  95% of total"

HbF (α2 γ2)"§  ‘about’ 1%"

HbA2 (α2 δ2) "§  ~ 2.5% of adult Hb "§  δ chain differs from β at ~ 10% of residues"§  function unclear"

Embryonic : Gower I (ζ2 ε2), Gower II (α2 ε2) Portland (ζ2γ2)"

Fetal Hemoglobins"

Fetal : HbF (α2 γ2)"

β, γ, δ, ε chains can all form tetramers: Hb Bart’s (γ4) HbH (β4)" α does not form tetramers"

Ø  When synthesis is un-balanced:"

Globin Synthesis and Hemoglobin Assembling"

Hemoglobin HbA" HbF" HbA2"

Chains" α2 β2 α2 γ2 α2 δ2

Newborn" ~10-30%" ~70-80%" ~ 0%"

Adult" ~94-96%" ~ 1%" ~2,5%"

At birth Adult

Transition of the hemoglobin chains"

Rat

io (％

)

Antenatal Birth Post-natal

0 3 6 3 6

10

20

30

40

50

0

β

γ α

ε

δ

Newborn chromatogram on HPLC"

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ζ α2 α1

Embryonic Fetal Adult Hb Gower1 (ζ2ε2) Hb F (α2γ2) Hb A (α2β2)

Hb Gower2 (α2ε2) Hb A2 (α2δ2)

Hb Portland (ζ2γ2)

ε Aγ δ β Gγ

HEMOGLOBIN SWITCHING" Healthy adult chromatogram on HPLC"

Genetics"

Genes encoding for globin chains"

ε Gγ Αγ ψβ δ β 5`

3`

Chromosome 11

ζ ψζ ψα1 α2 α1 5` 3`

ψα2 θ

Chromosome 16

Hemoglobin gene clusters"

From Gene to Hemoglobin" We have 4 α genes and only 2 β genes "

Genotype!

Phenotype!

Presumptive or Definitive Identification ?"

ELECTROPHORESIS

CE-HPLC

IEF

MICROCOLUMNS

CE TO SCREEN HEMOGLOBINS

HPLC TO DIFFERENTIATE GLOBINS

MS TO CONFIRM AA SEQUENCE

DNA ANALYSIS TO IDENTIFY GENE MUTATIONS

ANALYTES METHODS OF ANALYSIS RESULTS

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Hemoglobinopathies"

Hemoglobinopathies"

Structural variants: § Mutation changes an amino acid in the globin chain sequence"§ Change may alter the normal hemoglobin function"§ Change may also cause thalassaemia"

The thalassemias:

§ Mutation results in either the absence or a reduction in the synthesis of one or more globin chains"§ Causes an imbalance in the ratio of the amounts of the two globin chains that make up a Hb (eg Hb A: a/b chains)"

Hemoglobinopathies"

§ More than 80% result from a substitution mutation"§ Many populations affected"§ Multiple abnormalities can be inherited"§ Less than 30% of variants result in clinical outcomes"§ Some hemoglobinopathies are ‘screen negative’"

" " " " " " " ""www.globin.cse.psu.edu!

ü  Hemoglobin variants: 1015 currently described ü  Thalassemia mutations: 393 mutations currently described

Heterogeneous group of inherited conditions comprising of either:

Hb Variants - Types"β-chain variants 362 α-chain variants 217 γ-chain variants 70 δ-chain variants 32 They can be: •  Variants with one amino acid replacement 612 •  Variants with two amino acid replacements 19 •  Variants with hybrid chains 10 •  Variants with elongated chains 13 •  Variants with deletions (17), insertions (6), •  and deletions + insertion (4) 27

Common Hb Variants "

HbS is the commonest variant Exceeds 20% in parts of Africa. Also found in Turkey, Iran, Saudi Arabia, & India

HbE is the second commonest variant Exceeds 50% in the “Hb triangle” – ie parts of Thailand, Laos and Cambodia

HbC is found mainly in West Africa Found at a frequency of up to 50% on the Ivory Coast High frequencies are due to heterozygotes having increased resistance to infection by malaria (P. falciparum)

Inheritance of Hemoglobin Disorders"

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Carrier!Carrier!

25% Affected

50% Carrier

25% Unaffected

What is autosomal recessive inheritance?"

Limitations of Hemoglobin Electrophoresis"

Identification dependent on technical performance § Hemoglobin concentration"§ Amperage"§ Running temperature"§ Length of electrophoresis run""

Identical or similar migrations § Discrete differences "§ Lack of control specimens"

METHODOLOGIES"QUALITATIVE

§ Cellulose Acetate Electrophoresis (pH 8.6)"§ Citrate Agar Electrophoresis (pH 6.2)"§  Isoelectric Focusing (pH gradient)"§ Globin Chain Electrophoresis (Alkaline and Acid)""

QUANTITATIVE § Column Chromatography (Hb A2)"§  Alkali Denaturation (Hb F)"§ Radial Immunodiffusion (Hb F)"§ Densitometry (Hb A2,Hb F, and hemoglobin fractions)"§  Kleihauer-Betke Stain (Hb F)""

AUTOMATED (Qualitative and Quantitative) § Capillary Zone Electrophoresis (CZE)"§ High Performance Liquid Chromatography (HPLC)"

HELENA CELLULOSE ACETATE, pH 8.6

HELENA CITRATE AGAR, pH 6.2

A

F

S

C

A

F

S

C

D-‐Punjab, G-‐Phil, Lepore

A2, E, O-‐Arab

D, G, Lepore, A2, E, O-‐Arab

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The thalassemias"

Did you Know…"Ø  Thalassemia is sometimes called Mediterranean anemia or Cooley’s anemia

Ø  The name “ thalassemia “ comes from the greek thalassa which means “ sea” and haema , which means “blood” .

Ø  Thalassemia is prevalent in people from the Mediterranean region and middle east

Ø  India, Central Asia, and Southeast Asia also have a high prevalence of thalassemia

Ø The highest prevalence of thalassemia is geographically located in the world where there is or was malaria.

The thalassemias"

α - thalassemia types: α+ and α0 β - thalassemia types: β+ and β0 δ - thalassemia types: δ+ and δ0 δβ - thalassemia types: (δβ)0, (Aγδβ)0

γ - thalassemia type: GAγ hybrid εγδβ - thalassemia types: (εγδβ)0

Thalassemia: Quantitative defects:

Reduced synthesis of a normal globin chain § α + or β + thalassemia"

Absent synthesis of a normal globin chain § α 0 thalassaemia or β 0 thalassaemia"

Unequal α and β globin chain production

What are α- and β- thalassemias ?"

Range of Clinical Symptoms §  β-thalassemia minor"

-Asymptomatic to mild microcytic anemia

§  β-thalassemia intermedia"Range of symptoms

§  β-thalassemia major"Fatal if untreated Transfusion Dependency Hepatosplenomegaly Severe Anemia Skeletal Deformities

§  α-thalassemia trait (-α/αα) (-α/-α) (--/αα)"Asymtomatic

§  Hemoglobin H Disease (--/-α)"Chronic hemolytic anemia

§  Hydrops Fetalis"Stillbirth

Clinical Information: thalassemias " Transfusion therapy"

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β-thalassemia"

: α-Thalassemia : β-Thalassemia

: A group of inherited disorders in which globin chain synthesis is impaired.

Thalassemia

α β

δγ

Normal

~97% Hb A=α2β2 ~2.5% HbA2=α2δ2 ~0.5% Hb F=α2γ2

α β

δγ

α-Thalassemia

β4=HbH γ4=HbBart’s

α β

δγ

β-Thalassemia

Increased HbA2 (4-6%) Increased HbF (10-70%)

PATHOPHYSIOLOGY OF THALASSEMIA "

Thalassemia genes!Defective α- or β-chain synthesis, Excess α- or β-chains !

Inclusion bodies!Membrane damage!

Increased RBC destruction!Iron excess!Increased bilirubin!production

Hyperurcemia

RE hyperplasia!

Hepato-splenomegaly!

Anemia!

Massive erythropoiesis!

Hypersplenism!Extramedullary hematopoiesis!Jaundice!

Gallstones!Bone changes!Inadequate nutrition!

Defective development!

Iron overload! Increased infection!

Cardiac failure, Cirrhosis!

Blood transfusion!

Beta thalassemia "

§ Inherited mutation defect on beta gene § Mutation results in an either the absence or a reduction in the synthesis of b-globin chains:

β+ type: reduced expression of b-globin β0 type: no expression of b-globin

Causes an imbalance in the ratio of the amounts

of the two globin chains that make up a Hb"

β-thalassemia Genotypes"

Ø β-thalassemia TRAIT (β0 or β+ type) both types can be mild or severe

Ø  β-thalassemia MAJOR (β0/β0 or β+/β+)

β+ is a severe type

Ø β-thalassemia Intermedia (β0/β+ or β+/β+)

β+ is a mild type (β+ +)

Phenotypes of β-thalassemia"

β+-thalassemia (trait)

Silent (normal MCH & normal HbA2) normal HbA2 (reduced MCH & normal HbA2) Mild (reduced MCH & raised HbA2) Severe (reduced MCH & raised HbA2)

β0-thalassemia (trait)

Mild (reduced MCH & raised HbA2) Severe (reduced MCH & raised HbA2) Dominant (reduced MCH & raised HbA2 , plus anemia and splenomegaly)

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There is great variety in β Thal alleles- and approx 20 diff alleles comprise 80% of the mutations worldwide.

Frequencies of common β-thalassemia mutations"

UK Romania Spain Greece Egypt Tunisia

Arab Penins

ula India China African

IVS110 (G→A) 5.5 34.7 10.2 42.5 28.5 20.0 CD 39 (C→T) 3.4 13.7 35.5 19.6 2.1 45.4 IVSI-6 (T→C) 2.2 17.0 9.4 8.4 19.7 1.7 IVSII-745 (C→G) 1.7 12.1 1.5 4.8 3.7 3.1 IVS1-1 (G→ A) 1.9 7.2 25.0 13.4 15.1 3.9 CD 8/9 (+G) 9.8 14.1 12.0 IVSI-5 (G→C) 22.5 32.4 619 bp deletion 1.7 13.1 IVSI-1 (G→T) 1.6 10.4 1.3 CD41/42 (-TCTT) 9.0 6.3 42.3 -28 (A→G) 0.7 12.4 CD17 (A→T) 1.0 16.6 CD 71/72 (+T) 0.1 5.8 IVSII-654 (C→T) 1.6 12.1 -29 (A→G) 2.3 2.3 68.4 -88 (C→T) 4.8 24.3 CD 24 (T→G) 0.2 2.7

α-thalassemia"

Alpha thalassemia"§  Inherited deletion or mutation defect on alpha genes "§ Deletion or mutation results in an either the absence or a reduction in the synthesis of α-globin chains:""

α + type: reduced expression of α-globin genes α 0 type: no expression of α-globin genes

§ Causes an imbalance in the ratio of the amounts of the two globin chains that make up a Hb (eg Hb A: α/β chains):"§ Excess of γ chains in the fetal stage can generate γ4 tetramers: Hb Bart’s"§ Excess of β chains in the adult stage can generate β4 tetramers: HbH""

Genetics of α-thalassemia "

Caused by the loss of both the α1 and the α2 genes on the same chromosome

5 common mutations: --SEA, --FIL, --THAI, & --MED, -α20.5

Two rare deletions have been described in Asian-Indian population

αo-thalassemia "

Genetics of α-thalassemia "

Ø  Single gene deletion: 3.7kb (- α3.7) Mediterranean, Middle east, Africa, India 4.2kb (- α4.2) India, Southeast Asia, Pacific populations

Ø  Non-deletion mutation on either α1 or α2 gene 60 mutations – some result in hyper-unstable globins

α+-thalassemia "

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Hb Constant Spring"

Ø  non-deletional form of α-thalassemia"Ø  α2 globin allele contains a point mutation "Ø  the globin chains αCS is elongated by 31 amino acids"Ø  Hb Constant Spring is highly unstable"Ø  HbCS in heterozygotes constitutes only 1-2% of the " total hemoglobin"Ø  HbCS interacting with deletional αo-thalassemia can " cause a severe HbH disease ( --/ αCS α)"

Alpha Thalassemia genetics "

•  4 alleles = Normal α

α α

α

1 gene deletion = silent carrier

α

α α

- •  asymptomatic •  may see small amount of Hb Bart’s (γ4) at birth •  normal red cell indices – normal looking morphology •  detected only by DNA methods


2 gene deletion = α-thalassemia trait

•  mild anemia •  small red blood cells (low MCV) •  Hb Bart’s at birth •  difference between cis and trans becomes important when considering inheritance

α

α -

-

α

-

α

-

CIS

TRANS


3 gene deletion = hemoglobin H disease

•  moderate to marked anemia •  very small red blood cells •  Hb Bart’s (γ4) present at birth, replaced by HbH (β4)

4 gene deletion = hydrops fetalis

α

- -

-

-

-

-

-

•  extreme anemia produces congestive heart failure, edema in utero •  stillbirth or early neonatal death

Risk for HbH"

A pregnancy is at risk for HbH disease (3 gene deletion) if:

a) One parent has 1 α gene deletion and the other parent has 2 α gene deletion (CIS or α°-thalassemia heterozygote)

α

α -

-

α

α α

- +"

or"b) One parent has 1 α gene deletion and the other parent has HbH disease

+"α

- -

-

α

α α

-

α

α -

-

Risk for Hydrops Fetalis"

A pregnancy is at risk for Hydrops Fetalis if:

a) Both parents have a 2 α gene deletion (CIS or α°-thalassemia heterozygote)

α

α -

- +"

or"

b) One parent has 2 α gene deletion (CIS)and the other parent has HbH disease

+"α

- -

-

α

α -

-

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Alpha thalassemia hematology "

Red Blood Cell Index

Normal Affected Carrier

Male Female Hb Bart’s Hydrops Fetalis

HbH disease α-thalassemia trait (-α/-α)1

α-thalassemia silent carrier

Mean Corpuscolar volume (MCV,fL ) 89.1+/-5.01 87.6+/-5.5 136+/-5.1

56+/-5 (children) 61+/-4 (adults) 71.6+/-4.1 81.2+/-6.9

Mean Corpuscolar hemoglobin (MCH, pg)

30.9+/-1.9 30.2+/-2.1 31.9+/-9 18.4+/-1.2 22.9+/-1.3 26.2+/-2.3

Hemoglobin (Hb,dL) 15.9+/-1.0 14.0+/-0.9 3-8

M10.9+/-1.0 F9.5+/-0.8

M13.9+/-1.7 F12.0+/-1.0

M14.3+/-1.4 F12.6+/-1.2

§ Galanello R. ; Cao A. ; Last update : Nov 2005 ; Alpha-Thalassemia In: GeneReviews at GeneTests: Medical Genetics Information Resource (database online).

Copyright, University of Washington, Seattle. 1997-2008. Available at http://www.genetests.org. Accessed [5-26-08].

Red Blood Cell Indices in Adults with Alpha-Thalassemia§

(1) α-thalassemia carriers with the ( - -/ α α ) genotype have slightly lower RBC indices.

Alpha thalassemia hemoglobin types "

Hemoglobin Type Normal

Affected Carrier

Hb Bart’s Hydrops Fetalis

HbH disease α-thalassemia trait or α°-thalassemia

α-thalassemia silent carrier or α+-thalassemia

HbA 96-98% 0 60-90%) 96-98% 96-98%

HbF < 1% 0 <1% <1% <1%

Hb Bart’s 0 85-90% 2-5% 0 0

HbH 0 0 0.8-40% 0 0

HbA2 2-3% 0 <2.0% 1.5-3% 2-3%

§ Galanello R. ; Cao A. ; Last update : Nov 2005 ; Alpha-Thalassemia In: GeneReviews at GeneTests: Medical Genetics Information Resource (database online). Copyright, University of Washington, Seattle. 1997-2008. Available at http://www.genetests.org. Accessed [5-26-08].

Hemoglobin Patterns in Alpha-Thalassemia (After 12 Months of Age) §

Newborns chromatograms on HPLC"

Affected"Healthy"

Hb Bart’s"

HbH" δβ-thalassemia & HPFH phenotypes"

δβ – thalassemia" Reduced MCH Hb F 4-20% Heterocellular

Deletion HPFH" Normal MCH Hb F 20-35% Pancellular

Non - deletion HPFH" Normal MCH Hb F 3-35% Heterocellular

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HPFH - Hereditary Persistance of Fetal hemoglobin § Deletional and non-deletional "

"(deletional can have a Hb F of 25-35%)"§ Cellular distribution (pancellular or homocellular)"§ D-10 (A1c) can report A1c in the presense of Fetal hemoglobin up to 10%. Using the Dual Mode, the c/o for Fetal is 17%."

HPFH"Location and size of deletion mutants of "

hereditary persistence of fetal hemoglobin (HPFH)

5’ 3’

HPFH-1 HPFH-2 HPFH-3 HPFH-4 HPFH-5 HPFH-6

-20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

ε Gγ Aγ ψβ δ β

Prenatal Diagnosis"Significant hemoglobinopathies that require prenatal diagnosis by DNA analysis:

Sickle cell diseases: Hb SS, SD-Punjab, SC, SO-Arab β-thalassemia major β-thalassemia intermedia (severe genotypes) HbE/ β-thalassemia HbS/ β-thalassemia α-thalassemia

–  Hb Bart’s hydrops fetalis syndrome –  Hb H hydrops fetalis syndrome


4α genes Normal

α

α α

α - α / - α

2α genes deleted α+-thal homozygote

- α/αα 1α genes deleted

α+-thal heterozygote

- - / - α 3α genes deleted Hb H disease

- - / α α 2α genes deleted

αo-thal heterozygote

- - / - - 4 genes deleted Hydrops fetalis

Alpha thalassemia main deletions:

α -3.7 Kb

α -4.2 Kb α+ thalassemias αο thalassemias del SEA

del MED

α

α α

-

α

-

α

-

α

α -

-

α

- -

-

αα/αα

- α / α α

- α / - α

-- / α α

- α / --

-- / --

4

3

2*

2*

1

0

Normal

Normal

MCV, MCH

MCV, MCH

HbH disease

Hb Barts hydrops fetalis

α-thalassemia"

Genotype No. Genes Functional

Phenotype

Normal RBCs Alpha-thal RBCs Beta-thal RBCs

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Normal Thalassemia

RED BLOOD CELLS" THALASSEMIA "RED BLOOD CELLS"

Normal RBCs

Beta-thal minor RBCs

Beta-thal major RBCs

• Separates Hbs based on charge differences • Positively charged Hbs are separated by their absorption on a negatively charged stationary phase in a column • The cations (positive charge) in the mobile phase (buffers with increasing ionic strength) compete with the absorbed Hbs eluting them off • The fractions are detected optically by a spectrophotometer that measures the concentration of Hgb in each fraction which is quantified by calculating the area under the peaks

High Performance Liquid Chromatography (HPLC)"

High Performance Liquid Chromatography (HPLC)"

Polyaspartic acid column bound to a silica gel support, producing a weak cation exchange resin Two mobile phases (buffers) produce gradient of increasing ionic strength Blending of the phases, the flow rate and the physical characteristics of the hemoglobin determine the retention time on the column Elution from column measured at 415 nm. The resulting chromatograph is integrated and the % hemoglobins calculated

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HPLC controls

A S F A2

Manufacturer-Assigned Windows"

P1-Window 0.63 - 0.85 minutes F-Window 0.98 - 1.20 minutes P2-Window 1.24 - 1.40 minutes P3-Window 1.40 - 1.90 minutes A0-Window 1.90 - 3.10 minutes A2-Window 3.30 - 3.90 minutes D-Window 3.90 - 4.30 minutes S-Window 4.30 - 4.70 minutes C-Window 4.90 - 5.30 minutes

Healthy Adult

αAγA αAδA αAβA

Dx: Hb S trait

67 y.o. M RBC 4.67 MCV 87.2

HB 13.6 MCH 29.2

HCT 40.7 RDW 14.8

αAγA αAδA

αAβA αAβS

2 d.o. F RBC 4.89 MCV 112.2

HB 18.0 MCH 36.9

HCT 54.9 RDW 17.8

6 m.o. F RBC 4.38 MCV 96.7

HB 13.6 MCH 31.0

HCT 42.4 RDW 16.1

Dx: Healthy Newborn Dx: Newborn with Hb SS disease

αAγA

αAβS αAβA

αAγA

Dx: Hb E trait

18 y.o. F RBC 5.0 MCV 74.0

HB 12.4 MCH 24.8

HCT 37.0 RDW 12.8

αAγA

αAδA

αAβA αAβE

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26 M RBC 3.77 MCV 97.4

HB 12.8 MCH 29.4

HCT 36.8

Infant Father Mother

Dx: Hb E trait

αAγA αAβE

αAγA

αAβA

αAβE αAγA αAβA

αAβE

Dx: Hb D-‐Punjab Trait

26 y.o. F RBC 3.81 MCV 83.8

HB 11.2 MCH 29.3

HCT 32.0 RDW 13.0

αAγA αAδA

αAβA αAβD

Dx: Hb D-‐Punjab trait + α-‐thal

1 y.o. M RBC 4.95 MCV 60.6

HB 10.5 MCH 19.7

HCT 32.0 RDW 12.8

αAγA αAδA

αAβA

αAβD

α+ + β+ slow α+ + β-‐ normal α+ + β2-‐ fast

Bunn and Forget, Hemoglobin: Molecular, GeneYc and Clinical Aspects, 1986

THANK YOU [email protected]

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