Dijana Plaseska-Karanfilska, MD, PhD Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov” Macedonian Academy of Sciences and Arts
STROKOVNI SESTANEK ZDRUŽENJA HEMATOLOGOV SLOVENIJE IN ZDRUŽENJA ZA TRANSFUZIJSKO MEDICINO SLOVENSKEGA ZDRAVNIŠKEGA DRUŠTVA
Term e Olim ia, Slovenia, April 12, 2013
The commonest monogenic diseases (3-4% of human population carry a gene for Hb disorder)
Two main groups Thalassemias – reduced rate or complete lack of production of one or more globin chains Abnormal hemoglobins - abnormal structure of globin chains Both groups are caused by mutations in the globin genes
Chromosome 16
HS-40
ζ
ψζ ψα2 ψα1
α2
α1
θ
5’
3’
EMBRYO ζ2ε2 α2ε2 ζ2γ2
Chromosome 11 5’
β−LCR
ε
FETUS α2γ2 (HbF)
Gγ Aγ
ADULT α2β2 (HbA) α2δ2 (HbA2)
ψβ
δ
β 3’
Clinical β thalassemia Minor β thalassemia Intermedia β thalassemia Major
Genetic β / β0 β / β+ β / δβ β / Lepore
β+ / β + β+ / Lepore Lepore / Lepore β+/ δβ β0 / β 0 β0 / β+ β0 / Lepore
Genotype
Homozygote Compound heterozygote
Phenotype
Heterozygote
Carrier
Silent
Hematologic phenotype
Intermedia
Mild Tfx independent
Severe sporadic Tfx
Major
Tfx dependent
mild/silent β alleles α thalassemia
increased γ chains
Imbalance in globin chain synthesis
β0 thalassemia mutation
No
β+ thalassemia mutation
No
Splice junction
22
Transcriptional mutants
26
Nonsense
15
Consensus splice site
12
Frameshift
62
IVS changes
6
Coding region
5
Initiation codon
7
Hyperunstable globin
31
Polyadenylation signal
6
Deletion
14
CAP site
1
3’ UTR
6
TOTAL
62
TOTAL
151
Clinical
Genetic
Normal
αα/αα
α+ thalassemia (silent)
−α/αα
α0 thalassemia (minor) HbH disease
−α/−α −−/αα −−/−α
Hydrops fetalis
−−/−− -
deletion
αT
non functioning gene
Population surveys in Macedonia and Former Yugoslavia
Structural characterization of Hb variants
Molecular characterization of different thalassemias
Factors affecting fetal Hb levels
Total number of screened subjects 35.425
1.611 (2.0/2.7%)
22.136 (2.6/3.8%)
Total number of screened subjects 22.136
SERBIA Kriva Palanka
91 (1.1%)
Kumanovo Tetovo
β thalassemia
2.6%
α thalassemia
1.5%
δβ thalassemia
0.2%
HPFH
0.3%
Abnormal Hbs
0.4%
572 (1.7%) 4437 (2.2%) Skopje
BULGARIA
Kocani
143 (0.7%) Gostivar 816 (2.9%) Radovis 400 (1.7%) Veles Debar 896 (5.5%) 8471 (2.5%) 147 (0.0%) Kicevo Strumica Kavadarci 1500 (6%) 384 (1.0%) 170 (5.9%) 505 (2.8%)
Stip
Prilep
69 (5.7%)
Struga
188 (1.1%)
Resen
199 (6.5%) Bitola 136 (4.4%) 1236 (3.6%) Ohrid
ALBANIA
Valandovo
302 (12.3%) Gevgelija
1474 (8.6%)
GREECE
*Le **SB *Z *L *S *S
*L
*L *K
*Sr *Sr
*L
*Sr *Sr **MS H
*B
*K*L*B *BI *L *OA *Sa
*K *L
*B
*B
*Sr
*L *B
*L *Bu *Y
*MHP
*S *Ha*B *S *JP *S *JP *C *L *B *OA *B *Sr*I * DP *JO *DP *S
B L Sr K Z JP Ha DP JO I Le BI H MS Bu Y S OA S MHP
Beograd Lepore Strumica Koln Zagreb J Paris Hamilton D Punjab J Oxford Icaria Leiden Beth Israel Hoshida M Saskatoon Bushwick Yokohama Sabine O Arab Savaria M Hyde Park
Abnormal Hb
Codon
Substitution
Hb Richmond
β 102; AAC → AAA
Asn → Lys
Efremov et al. 1969
Hb Savannah
β 24; GGT → GTT
Gly → Val
Huisman et al. 1971
Hb Istanbul
β 92; CAC → CAA
His → Gla
Askoy et al. 1972
Hb Beograd
β 121; GAA → GTA
Glu → Val
Efremov et al. 1973
Hb Novi Sad
β 63; CAT → TAT
His → Tyr
Efremov et al. 1974
Hb Strumica
α 112; CAC → CGC
His → Arg
Niazi et al. 1975
HbA2 Zagreb
δ 125; CAA → GAA
Gln → Glu
Juricic et al. 1983
His → Gln
Plaseska et al. 1994
Lys →Asn
Plaseska et al. 1994
β77; CAC → CAG
His → Gln
Hopmeier et al. 1998
B137-139 del
Val-Ala-Asn->Asp
B104-107; del-ins
4aa del->9aa ins
HbF Macedonia I
Aγ2;
HbF Macedonia I
Gγ104;
Hb Vienna Hb Stara Zagora Hb Jambol
CAT → CAG AAG → AAC
Reference
Petkov et al. 2006 Efremov et al. 2007
Mutation
Type of β-thal
References
-101 (C → T)
β+
Gonzales et al. 1989
-30 (T → A)
β+
Fei, Y. J. et al. 1988
Initiation codon ATG → ACG
β0
Jankovic, L. et al. 1990
Codon 6 (-A)
β0
Petkov, G. et al. 1990
Codon 82/83 (-G)
β0
Jankovic, L. et al. 1992
IVS-II-850 (G → C)
β0
Jankovic, L. et al. 1991
Poly A (AATAAA → AATGAA)
β+
Jankovic, L. et al. 1990
Poly A (AATAAA → AATAGA)
β+
Jankovic, L. et al. 1990
G → A at +22 3’ to the cap site
β+
Oner, R. et al. 1991
Thal. variants
Type
Ref.
Macedonian (δβ)0 - Thal
δβ0
Efremov, G. D. et al. 1986
Croatian (εγδβ) 0 - Thal
εγδβ0
Diez-Chico et al. 1988
- - (Med-II) α -Thal
α -Thal-1 (- - /)
Croatian β0 – Thal 1.605 bp deletion
β0
Dimovski, A. et al. 1993
Indonesian-Malay β-thal del. – 45 kb deletion
β0
Dimovski, A. et al. 1996
Kutlar, F. et al. 1989
Mutation
Population (No. of chromosomes and %) MK (459)
SR (46)
CR (53)
AL (55)
TU (36)
BG (801)
1 (2.8)
2 (0.2)
-101 (C → T)**
1 (0.2)
3 (5.5)
-30 (T → A)
2 (0.4)
10 (18.2)
CD 5 (-CT)
11 (2.4)
CD 6 (-A)**
17 (3.7)
3 (6.5)
CD 8 (-AA)
10 (2.2)
1 (2.2)
4 (7.5)
2 (3.6)
CD 8/9 (+G)
2 (0.2) 1 (2.8)
67 (8.4)
2 (5.6)
39 (4.9)
6 (16.7)
31 (3.9)
9 (25.0)
19 (2.4)
CD 39 (C → T)
58 (12.6)
5 (10.9)
6 (11.3)
21 (38.2)
5 (13.9)
233 (29.1)
IVS I-1 (G → A)
74 (16.1)
7 (15.2)
7 (13.2)
3 (5.5)
1 (2.8)
114 (14.2)
IVS I-6 (T → C)
83 (18.1)
8 (17.4)
6 (11.3)
8 (14.5)
4 (11.1)
54 (6.7)
173 (37.7)
16 (34.8)
15 (28.3)
7 (12.7)
5 (13.9)
184 (23.0)
2 (0.4)
1 (2.2)
4 (7.5)
1 (2.8)
7 (0.9)
18 (3.9)
5 (10.9)
3 (5.7)
1 (1.8)
1 (2.8)
30 (3.7)
4/16
5/8
7/14
6/8
6/11
6/18
IVS I-110 (G → A) IVS II-1 (G → A) IVS II 745 (C → G)
No. mutations > 85 % (100%)
Level
Methods
Hematological
Hb, MCV, MCH, blood smears
Protein (biochemical)
HPLC (Hbs, globin chains), Hb electrophoresis, in vitro chain synthesis
Molecular (genetic)
Point mutations PCR based: ASO, ASP, reverse line blot, SNaPshot, DNA Sequencing Deletions Southern blot, gap PCR, MLPA
Common β thal mutations by SNaPshot
Blood test Family history Routine Hb analysis
Sequencing of β globin gene
Deletions in βglobin cluster by MLPA
Variant Hb specific PCR
Sequencing of α or β globin gene
Common α thal deletions by gap PCR
Deletions in αglobin cluster by MLPA
Sequencing of α globin genes
β thalassemia high Hb A2
Normal
δβ thalassemia high Hb F
Hb Lepore
HbA HbF
HbA1c (3.1%)
HbF
(0.5%)
(15.1%)
HbA2
(2.6%)
HbA2
(4.8%)
HbLepore (9.2%)
Multiplex SNaPshot analysis
DNA sequencing analysis
MLPA analysis Gap PCR analysis
No of Cooley anemia patients
25 20 15 10 5 0
1961-1970
1971-1980
1981-1990
1991-2000
Period (years)
2001-2010
Ackgnowledgements Prof. Georgi D. Efremov and collaborators
