The molecular spectrum of hemoglobinopathy; Thalassemia and Hb variants

Cornelis L. Harteveld

Dpt. Of Clinical Genetics, Hemoglobinopathy Reference Center

LEIDEN UNIVERSITY MEDICAL CENTER

I have no personal or financial interests to declare:

I have no financial support from an industry source at the

current presentation.

대한혈액학회 Korean Society of Hematology

COI disclosureName of author ： 홍 길 동 or Gildong Hong

Epidemiology hemoglobinopathies

7% world population is carrier of hemoglobinopathy

~350,000 baby’s born each year with Sickle Cell Anemia (SCA) or Thalassemia Major (TM)

Wednesday, March 20, 2019C.L.Harteveld, LUMC 4

From Hemoglobin to Hemoglobinopathy

Hb molecule:

16p

11p

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11p

16p ζ α2 α1

ε Gγ Aγ δ β

Alpha-globin gene cluster

Beta-globin gene cluster

Globin genes

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Hemoglobine-switch

11p

16p ζ α2 α1

ε Gγ Aγ δ β

HbF HbA

Embryonisch HbHbA2

Hb switch: embryo (Hb Gower I,II, Hb Portland) > foetus (HbF) > adult (HbA)

C.L.Harteveld, LUMC

Sickle Cell Disease

Structural defect: sickle cell anemia (SCA)

C.L.Harteveld, LUMC

β-Thalassemia

Expression defect: Thalassemia

α-thalassemiaβ-thalassemia

Pathology determined by unbalance between α- and β-chains

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Patient β-thal intermedia/major: αα/αα vs β+β+ , β+β0 or β0β0

Patient α-thal to HbH disease: -α/αα, --/αα , --/-α, --/-- vs β/β

βα

Prevention and management of Hemoglobinopathy

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Implementation of basic health interventions1. Management of Sickle Cell Anemia (SCA)

-early diagnosis-penicillin prophylaxis-vaccination

To reduce excess mortality amongst children under 5 years

3. Prevention of SCA and TM-carrier detection -information and counseling-prenatal diagnosis

To reduce the number of affected births

2. Management of Thalassemia Major (TM)-early diagnosis-transfusion /chelation therapy

To prevent unnecessary complications of treatment

Carrier detection

through laboratory diagnosis

Diagnostics of hemoglobinopathy

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HemoCytometry

Hbseparation

DNA ClinicalDiag-nosis

C.L.Harteveld, LUMC

Flow Chart for Diagnostics of HbP

Hematology HPLC and Hb electrophoresis

Gap-PCR for7 most commonα-thal deletions

normal

Sequencing α-genes

MicrocyticHypochromicNormal iron

MLPA

Hb variant

Sequencing β-gene

HbA2 HbF

RBC (x 1012 /l)

1716151413121110987

7

6

5

4

Hb(mmol/land g%)

normal βT/β βT/ βT

Hemocytometry thalassemia genotypes

normal βT/β βT/ βT

MCV (fl)100

90

80

70

60

50

MCH (fmoland pg)

323028262422201816

normal βT/β βT/ βT

normal βT/β βT/ βT

βT/β trait

normal

βT/ βT

Intermediamajor

10.5109.58.78.17.56.96.25.654.5

2.001.871.741.621.491.371.251.121.00

normal

α-thaltrait

HbH disease

C.L.Harteveld, LUMC

Flow Chart for Diagnostics of HbP

Hematology HPLC and Hb electrophoresis

Gap-PCR for7 most commonα-thal deletions

normal

Sequencing α-genes

MicrocyticHypochromicNormal iron

MLPA

Hb variant

Sequencing β-gene

HbA2 HbF

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Capillarys 2 FlexPiercing (Sebia)

Hb electrophoresis

Hb separation equipment: HPLC and CE

Premier Resolution (PR)Trinity Biotech(Menarini)

High Performance Liquid Chromatography (HPLC)

Together they make a strong combi….

C.L.Harteveld, Hemoglobinopathies Lab

HPLC and CE : Hb variants and quantitation

Normal HbS carrier

HbSHbAHbA97.5%

HbA22.5% HbA2

5.6%

β-thal carrier

C.L.Harteveld, LUMC

Flow Chart for Diagnostics of HbP

Hematology HPLC and Hb electrophoresis

Gap-PCR for7 most commonα-thal deletions

normal

Sequencing α-genes

MicrocyticHypochromicNormal iron

MLPA

Hb variant

Sequencing β-gene

HbA2 HbF

Fully Automated sample preparation and Sanger sequencing

• Approx. 10% of all alpha-thal defects• All Hb variants of the alpha-globin chain

S13F S6R S13F S8RF S18R

S3F R

2A

2BF S18R

S3F R

1A

1B

α2 α1

Direct sequencing for point mutation analysis

β

HBLBF1 HBLBR1

HBLBF2 HBLBR2

HBLBF3 HBLBR3

HBLBF4 HBLBR4

• Detection rate approx. 90% of all beta-thal defects• All Hb variants of the beta-globin chain

~10% deletions

~90% deletions

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ε Gγ Aγ δ βNorm HetLW Het SEA Het MedI Het20.5 Het FILNorm HetRW HomRW α-tripl.

3000

20002500

150012001000

500

Multiplex gap-PCR for the most common thalassemia deletions

Other deletion types of α-thalassemia: MLPA

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C.L.Harteveld, Hemoglobinopathies Lab

…. Sometimes patterns are more complex…

Can’t do without

DNA analysis

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fundamental genetics

α2 α1

δ β

HbA22.6%

HbA97.4%

Simplified genetics:

Case 1: complex CE pattern

Hb Bart’s (γ4)

HbA (α2β2)

HbF (α2γ2)+ HbAQ (αQ2β2)

HbF Q-Thailand (αQ2γ2)

HbE (α2βE2)

HbE Q-Thailand (αQ2βE2)

Fraction % Hb type

Proband18 days

Child detected during neonatal screening:

Molecular analysis revealed 3 defects:

- Hb Q-Thailand (α1cd74 Asp>His)- -α4.2 deletion- HbE (βcd26Glu>Lys)

HbE HbA HbA2

α2 α1

γγ δ β

HbF

HbQE HbQ HbQA2 HbQF

HbBart’s

Fraction % Hb type

HbA (α2β2)HbF (α2γ2)+HbA Q-ThailandHbF Q-Thailand (αQ2γ2)HbE (α2βE2)HbA2 (α2δ2)HbE Q-Thailand (αQ2βE2)HbA2 Q-Thailand (αQ2δ2)

Proband 18 daysProband87 days

HbE HBA HbA2

α2 α1

γγ δ β

HbF

HbQE HBQ HbQA2 HbQF

HbBart’s

Case 1: complex CE pattern

Child detected during neonatal screening:

Molecular analysis revealed 3 defects:

- Hb Q-Thailand (α1cd74 Asp>His)- -α4.2 deletion- HbE (βcd26Glu>Lys)

HbBart’s

HbF+HbQ

HbA HbQFHbEHbQE

Hom HbE Het -α4.2HbQ-Thailand

Het -α4.2HbQ-Thailand /het HbE

2.5 wks

3 months2 yrs

Case 1: complex CE pattern

HbE particularly common in South-East Asia

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Kaart Azië met verdeling hbE en beta-thal

HbE/beta-thal

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Type of mutation determines phenotypic severity ….HbE is an Hb variant with a beta-thalassemic effect:

Cd 26 GAG>AAG β

mRNA1 βE

mRNA2 thalassemia

HbE carrier: ~25-30% HbE

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This was reported asHbE carrier (!)

HbE coincides with HbA2 peak….but usually 30%, NOT 37.9%

Case 2: Pitfalls ….to (β)E or not to (β)E.

…but this is how a real HbE carrier looks like!

Hb D-IranSilent alpha-variantNormalhematology

Hb EMicrocytichypochromic

CE

CE

Unbalanced α / β synthesis

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Hemolytic anemia in beta-thalassemia intermedia/major

Due to unbalanced α- / β-chain synthesis

- alpha-thalassemia; excess beta-chains- beta-thalassemia; excess alpha-chains

Proteolytic processes degrade excess,

…..but if unbalance becomes too excessive this may result in

β-thal-intermedia

Everything depends on the balance in life

Case 3 Child is beta-thal carrier, but unexpected severely anemic

αααanti 3.7-α3.7 Cd 39 CAG>TAG

(HBB:c.118C>T)

1 2 3

Hb 15.5 11.6 7.2 g%

RBC 5.31 5.39 3.67 1012/l

Ht 0.46 0.37 0.25 l/l

MCV 86 69 67 fl

MCH 29.1 21.6 19.9 pg

ZPP 62 83 270 μmol/mol heme

Hp - - <24.3 Mg/100ml serum

HPLC A2 2.9 5.1 4.8 %

CE A2 2.8 5.2 4.4 %

1 2

3

Example : genetic consequences

1 2

-α3.7

Dupαα

DuplicationDeletion -α3.7

Child: MLPA Alpha-gene cluster (P140B2, MRC-Holland)

Example : genetic consequences

αααanti 3.7-α3.7

1 2

3

ααTel MRE normal

Case 3 Child is beta-thal carrier, but unexpected severely anemic

Cd 39 CAG>TAG(HBB:c.118C>T)

αααα

ααTel MRE ααMRE-α3.7Tel MREβ0

β5 x α1 x β

Example : genetic consequences

Counseling:Triplication+ quadruplication+ beta-thal =Severe beta-thalintermedia

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A beta-thal carrier becoming transfusion dependent later in life: “late-onset TM”

Case 4: other case of ‘late-onset TM’ from Greece:

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Daughter (norm)

Hb (g/l) 75 140

RBC (x10 12/l)

- 4.8

MCV (fl) 79.7 87

MCH (pg) 27.4 29

HbF (%) 6.7 0

HbA2 3.2 2.5

treatment Transfusion dependent

HBB:c.315+1G>A (IVS2-1g>a)

5 Mb deletion

Two different mechanisms:Mosaicism due to ‘second’ (somatic) mutation!

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1. Uni Parental Iso Disomy (UPID) 11p

Greek patient2. Deletion 5Mb on 11p

Italian patient

*

* *

*

5Mb deletion takes away HBB and H19 + IGF2 :

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CNV’s patient

CNV’s mother

SNPs patient

SNPs mother

Affymetrix CytoScan HD Array with Chromosome Analysis Suite (ChAS, version 3.0) (Thermo Fisher Scientific, CA, USA).

Chromosoom 11

IGF2 H19

IGF2 H19

M

Pmethylated

This might explain the late onset: growth advantage for cells deprived of beta-synthesis

IGF2 H19

IGF2 H19

P

Pmethylated

methylated

Conclusion

• More laboratory tools become available in time (Next Generation Sequencing)

• Clinical genetic lab is changing

• Whole Exome/Genome Sequencing is still expensive, but becomes cheaper

• More emphasis on functionality of Variants of Unknown Significance (VUS) found by WES/WGS

• Therefore RNA and protein technologies will become more important

• Hemoglobinopathy diagnosis relies on phenotype-genotype correlation:Hematology, Hb separation and DNA analysis

Acknowledgements

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LUMC/LDGA:Sandra ArkesteijnGreet BakkerJeanette ter HuurneSharda BisoenRianne SchaapMaaike VerschurenLinda VijfhuizenClaudia RuivenkampCathy Bosch

Frank BaasChristi van Asperen

LUMC/LGTC:Rolf VossenEmile de MeijerQuint HottentotStefan WhiteHenk Buermans

Human and Clinical Genetics:Johan den Dunnen

National and KapodistrianUniversity of Athens:

Joan Traeger-Synodinos