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Haemoglobinopathies
Henrik Birgens, MD Department of Haematology L
Herlev Hospital University of Copenhagen
Haemoglobinopathies Due to resistance against malaria, carrier states of hereditary haemoglobinopathies are the most common monogeneic disorders in the world WHO has calculated, that about 7 per cent of the worlds population carry a haemoglobinopathy gene Each year 300.000 -400.000 children are born with a serious haemoglobin disorder Weatherall & Clegg. Bulletin of the WHO 2001;79:704
Center for Haemoglobin Disorders at Herlev Hospital
Field of activity: 1. Diagnostic 2. Prenatal diagnosis 3. Genetic Councelling 4. Advising and education of health care
persons 5. Treatment of patients with
haemoglobinopathies Videncenter for Hæmoglobinsygdomme
Hemoglobin
Hæmoglobinopathies
Kvalitative mutations: Mutations leding to a change in an amino acid (missense): Examples: Hb S, Hb C, Hb D Hb Volga, Hb Philadelphia + approx. 600 others
Kvantitative mutations: Mutations leading to reduced synthesis of globin chains (deletions, nonsense, frameshift, promotor mutations and mutations involving splicing. Examples: α-thalassæmia, β-thalassæmia, δβ-thalassæmia, Hb Lepore, Hb E
Embryo ζ2,ε2 Ηb Gower I α2,ε2 Ηb Gower II ζ2,γ2 Portland I ζ2,β2 Portland II
Fetus α2,γ2 Hb F
Adult α2,β2 Hb A
HS-40 ζ α2 α1
5’ 3’
LCR
ε Gγ Aγ δ β
5’ 3’
Chromosome 16
Chromosome 11
α2,δ2 Ηb Α2
Geografic Distribution of the Haemoglobinopathies
HB, 2007
Geografic Distribution of the Haemoglobinopathies
HB, 2007
Migration
Geografic origin of immigrants to the Nordic countries during the recent
decade
In the period 1995-1999, the net annual immigragion to Norway: 18.000 Denmark: 16.000 Sweden: 53.000 Finland: 10.000
β-Thalassemia
Screening for Haemoglobinopathies
HB,2007
Screening for Haemoglobinopathies
HB,2007
1998
Ny i 2008
Screening for Haemoglobinopathies
HB,2002
β-thalassaemia: Homozygous β-thalassaemia, β-thalassaemia/ haemoglobin E disease
α-thalassaemia: Homozygous α0-thalassaemia
Sickle cell disease: Hb SS disease, Hb S/β-.thalassaemia
Diagnosis
• Tillväxtretardation, muskelatrofi, fettreduktion
• Hyperplastisk expanderande märg – Facies thalassemica. – Psudotumörer i rörben o kotpelare. – Generell osteoporos - patolog. frakturer
• Ökad plasmavolym pga extramedullär hematopoes och hepatosplenomegali
• Hypersplenism (hemolys, leukopeni, trombocytopeni) • Gallsten • Ökad järnresorption i tarmen
b-Thalassemia major b-Thalassemia major Beta-Thalassemia major
β-thalassæmia major
HB,2002
β-thalassæmia major
α-Thalassemia
Normal
α+, silent carr.
α+, homozyg.
α0, trait HbH (β4) Hb Bart (χ4)
Synonym α-thal. 2 α-thal. 1 α-thal. 1
Fung. Gener
4 3 2 2 1 0
α/β-ratio 1.0 0.8 0.6 0.6 0.3 0 Genotyp αα/
αα -α/αα -α/-α --/αα --/-α --/--
MCV 90 +/-5
81 +/- 7 69 +/-4 69 +/-4 65 +/-7 110/120
Hb u.a. u.a. mild anemi
mild anemi
mod/svår hemolytisk anemi, splenomegali, akut hemolytisk kris
död in utero/neonat.
Sickle cell anemia
HB,2002
Screening for haemoglobinopathies
HB,2007
All from the Mediterranean countries, Iran and Iraq with MCV < 78 fl.
All from Africa, including afroamericans, the Middle East, the Indian subcontinent, Southeast Asia and Polynesia.
Screening of pregnant immigrants:
0
20
40
60
80
100
120
140
160
180
200
Antal
<55 55-59 60-64 65-69 70-74 75-78 >78
MCV (fl)
Beta-ThalassæmiAlfa0-Thalassæmi
Distribution of mean cellular volume in carriers for β-thalassaemia in Denmark
(n=485) and α0-thalassaemia (n=110)
13
15
17
19
21
23
25
27
29
31
50 55 60 65 70 75 80 85
MCV (fl)
MC
H (p
g)
Relationship between MCV and MCH in carriers of β-thalassaemia (+),
α+-thalassaemia (°) or α0-thalassaemia (•)
Screening for Haemoglobinopathies
HB,2007
1995: 2,8 % (14/205) 1996: 4,2 % (25/578) 1998: 4,2 % (16/381)
The frequency of serious beta-globin allels among pregnant immigrants in Copenhagen County
1 3 2
1 30 30(3) 104 105 146
C ATG GT AG GT AG TAA P
5’UTR 3’UTR 5’ 3’ 130 bp 850 bp
IVS I IVS II
Nonsense and frameshift mutations (β0
Mutations affecting splicing of RNA from introns (β0 and β+)
Mutation affecting initiation of translation (β0)
CACCC
CCAAT
TATA
Promoter
Promoter mutations (β+)
Mutations affecting RNA cleavage (β+)
Mutations affecting transcription (β+)
)
Mutations in ß-thalassaemia
-α3.7 -α4.2 --MED --SEA -(α)20.5
--FIL --THAI
ζ2 α2 α1 3’ 5’
Iran, Irak,Indien, Sydøst asien, Indonesien, Afrika }
Middelhavet Sydøstasien
Middelhavet Sydøstasien Sydøstasien
Geografi ψζ1 ψα2 ψα1 θ1
0 10 kb 20 kb 30kb
HB 2002
α-thalassaemia deletions
Haemoglobinopathy Diagnostic Laboratory Methods:
Screening: High-pressure liquid chromatography (HPLC) Isoelectric focusing Unknown mutations in the β-globin gene: PCR followed by sequencing Denaturing gradient gel electrophoresis (DGGE Mutations in the α-globin gene: Deletions: GAP-PCR (multiplex) Small mutations: PCR followed by sequencing HB/1999
Normal
HbA
HbA
2
Heterozygot β-thalassæmi
HbA
HbA
2
Heterozygot HbS
HbA
HbA
2
HbS
Hæmoglobin Lepore
HbF
HbA
HbL
epor
e H
bA2
Haemoglobinopathies HPLC
Haemoglobinopathies
HB, 2002
Isoelectric focusing
A new thalassaemic nonsense mutation in the b-globin gene (codon 37;tgg > tag) in an Afghanistani
family
Mother
Father
Child
Kornblit B, Taaning P, Birgens H. Hemoglobin 2005; 29: 209-13
ψζ1 ζ2 ψζ2 ψα1 α2 α1 θ1
THAI(F)
FIL(F) 20,5(F)
MED(F)
a/SEA(F) a(R) 20,5(R) MED(R)
FIL(R) THAI(R)
SEA(R)
a0-Thalassæmi PCR multiplex
-(α)20,5 -(α)20,5 / Lane 1 Lane 2 αα/αα Lane 3 -(α)20,5 / - -MED
Lane 4 --SEA /--SEA
Lane 5 αα/ -(α)20,5
Lane 6 αα / --MED
Lane 7 αα / --SEA
Lane 8 αα / --FIL
Lane 9 αα / --THAI
HB/2003
Inter-HVR
β-thalassæmia
HB,2002
(Indien)CD8-9 +G, beta012%
(Medit)IVSI 110 G>A, beta+11%
(Medit)IVSII 1G>A, beta011%
(Medit)CD39 C>T, beta09%
(Medit)IVSI 1G>A, beta07%
(Medit)CD5 -CT, beta05%
(Medit)IVS I 6T>C, beta+5%
(Asiea)CD41-42 -TCTT, beta04%
(Medit)CD8 -AA, beta03%
(Medit)CD15 G>A, beta03%
(Asia)CD17 A>T, beta02%
(Iran)CD36-37 -T, beta01%
Andre14%
(Medit)IVSI 5G>C, beta013%
Distribution of 245 mutations in the β-globin gene associated with β-thalassaemi in the Danish immigrant population
-α3.7 -α4.2 --MED --SEA -(α)20.5
--FIL --THAI
ζ2 α2 α1 3’ 5’
Iran, Irak,Indien, Sydøst asien, Indonesien, Afrika }
Middelhavet Sydøstasien
Middelhavet Sydøstasien Sydøstasien
Geografi ψζ1 ψα2 ψα1 θ1
0 10 kb 20 kb 30kb
HB 2002
a-thalassaemia deletions
α-Thalassemia
HB,2002
α-Thalassemia SEA Deletion
Prevalence of (--SEA) α-Thalassemia Deletion in Southeast Asia.
Region Proportions of Population who are Heterozygous Carriers (%)
Hong Kong 4,5 Northern Taiwan 3,5 Southern China 5,0-8,8 Northern Thailand 14,0 Central Thailand 3,7
SEA73%
MED20%
20,54%
FIL1%
THAI2%
Distribution of various α0-thalassaemia alleles in 109 patients with α0-thalassaemia and 32 patients with HbH
disease in the Danish immigrant population
Thalassemia in 165 immingrants with MCV < 80 fl
Total 165 samples: β-thalassemia: 30 (18.2 %)
β-thalassemia minor 29; β-thalassemia intermedia 1
α-thalassemia: 39 (23.6 %) αα/-α3.7 27; -α3.7/-α3.7 5; αα,-SEA 2; αα/-MED 2; αα/-(α)20.5 1; αα/αααanti 3.7 1; -α4.2/-(α)20.5 1
3 pts have α and β thalassemia simultaneously
Videncenter for Hæmoglobinsygdomme
Sickle cell anemia
The Procedure for Screening of Haemoglobinopathy in Pregnants of relevant etnic origin in Copenhagen
Primary Health Care, General Practitioner
The Laboratory of General Practitioners in Copenhagen The pregnant comes from a mediterran- ean country, Iran or Iraq
MCV < 78 MCV > 78
The pregnant comes from Africa, the Middle east, the Indian subcontinent, or Southeast Asia
Haemoglobin analysis (HPLC)
The sample is stopped
If the sample shows heterozygosity for beta-thalassaemia, alpha0-thalassaemia, Hemoglobin S or hemoglobin E
All samples
Blood sample
A direct contact from the laboratory to the GP con- cerning a blood sample from the fa- ther
Chorion villus sampling
Far. Cd5 -CT
Foster. Cd5 -CT 1 2 3 4 5 6
Lane 1 hetero controle Lane 2 mother Lane 3 fetus Lane 4 fetus Lane 5 mother Lane 6 normal controle
Prenatal diagnosis
619 bp deletion
HB,2002
Beta-thalassaemia Homozygous: 4 Heterozygous: 2 Normal: 1 Sickle cell disease Homozygous: 1 Normal: 1 Alpha-thalassaemia Homozygous SEA: 1 Heterozygous 3.7: 1 Normal: 1
Number and results of CVS performed at Herlev Hospital in the period 2002-2007
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