View
1
Download
0
Category
Preview:
Citation preview
MOLECULAR BASIS OF
THALASSEMIA IN SLOVENIA
Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov”,
Macedonian Academy of Sciences and Arts, Skopje, R. Macedonia
Dijana Plaseska-Karanfilska, MD, PhD
5th Congress of Slovenian Hematological Society, Lasko, Slovenia, 07-09.2016
CONTENT
Brief introduction
Thalassemias in Slovenia
Methodology
Mutations
Factors affecting fetal hemoglobin levels
Slovenian db thalassemia patients
INTRODUCTION
THALASSEMIAS
First recognized in 1925 in USA and Italy
Widespread occurrence - from Africa and the Mediterranean region, through the Middle Eastern and Indian subcontinent, to Southern China and Southeast Asia
Distribution parallels that of Plasmodium falciparum
Inherited in a Mendelian
recessive fashion
Two main forms: a and b
thalassemia
Hb TYPES, GLOBIN GENE CLUSTERS AND
DEVELOPMENTAL EXPRESSION OF GLOBIN GENES
Higgs et al., The Lancet 2012, 379:373
BETA THALASSEMIA GENOTYPE/PHENOTYPE HETEROGENEITY
Heterozygote
Gen
oty
pe
Ph
enoty
pe
Homozygote
Compound
heterozygote
Carriers Intermedia Major
Hematologic
phenotype
Mild Tfx
independent
Severe
sporadic Tfx
Tfx
dependent Silent
Imbalance in globin chain synthesis
a
thalassemia
mild/silent
b alleles
increased
g chains
Severity of b thal phenotype
Primary modifiers – broad diversity of b globin gene mutations
Secondary modifiers - involved in modifying the degree of globin chain imbalance (coinheritance of a-thal, a-triplication, increased g-chains)
Tertiary modifiers – effect on complications of the disease (genes involved in iron absorption, billirubin metabolism, bone metabolism, susceptibility to infection)
THALASSEMIAS IN SLOVENIA
Date No of
samples
Material
Missing hema-
tological data
Missing HbA2
& HbF values
Feb 2013
24
blood
5
(1 Normal) /
June 2013
15
blood
3
(2 Normal) /
Nov 2014
13
blood
1
(1 Normal) /
Feb 2016
20
DNA
6
(1 Normal)
10
(3 Normal)
Total
72
15
(5 Normal)
10
(3 Normal)
THALASSEMIAS IN SLOVENIA MATERIAL STUDIED
Protein analysis
High Performance Liquid Chromatography (HPLC)
Molecular characterization of thalassemia
Multiplex Ligation Probe Amplification analysis (MLPA)
Gap-PCR assays
> Sicilian db deletion
> Hb Lepore
> α-globin deletions (α-3.7, α-med, α-20.5)
> α-globin triplication αααanti3.7)
Multiplex SNaPshot analysis:common Mediterannean mutations
Sequencing analysis: HBB, HBA2, HBA1, HBD genes
METHODS
b-globin gene haplotypes
8 polymorphic sites in the b-globin gene cluster by PCR-RFLP
Factors affecting fetal hemoglobin levels
PCR-RFLP analysis :
Gg Xmn1 polymorphic site
Bcl11A polymorphic site
High Resolution Melting analysis: MYB polymorphic site
METHODS
THALASSEMIAS IN SLOVENIA
72%
24%
4%
Patients with thalassemia (n=53)
deltabeta
beta
alpha 69%
29%
2%
Unrelated patients with thlassemia (n=45)
deltabeta
beta
alpha
a-THALASSEMIA
Genotype No. of
patients Ethnic origin Coinheritance
- -(SEA)/aa 2 China (1) /
a3,7/aa 1 Slovenia Sicilian db
MLPA analysis (a SEA deletion)
Molecular characterization of a thalassemias
Gap-PCR analysis
a THALASSEMIA DELETIONS
Deletions of one a-gene
(a + -thalassaemia) Deletions of two a-genes
(a 0 –thalassaemia)
Hartveld & Higgs, Orphanet J Rare Dis 2010, 5:13.
DISTRIBUTION OF THE MOST COMMON a-THALASSEMIA ALLELES
Weatherall, DJ., Nature Reviews Genetics 2001;2(4):245-55.
b-THALASSEMIA MUTATIONS
Genotype No. of
patients Ethnic origin Coinheritance
IVS-I-110G>A/N 5 Slovenia (4)
Unknown (1) /
Cd39C>T/N 5 Slovenia (4)
Cuba (1) /
IVS-II-745C>G/N 2 Slovenia (1)
Serbia (1)
αααanti3.7 (1)
IVS-II-837T>G/N 1 Serbia (1) /
MOLECULAR CHARACTERIZATION OF b-THALASSEMIA ALLELES
HPLC analysis SNaPshot analysis
Sequencing analysis
DISTRIBUTION OF THE MOST COMMON b-THALASSEMIA ALLELES
Weatherall, DJ., Nature Reviews Genetics 2001;2(4):245-55.
IVS-I-110 G>A
HGVS name HBB:c.93-21G>A
Effect on gene/protein function Cryptic splice site (mRNA processing)
Type of thalassemia Beta+ thalassemia
Ethnic background Mediterranean
Heterozygotes Mild anemia
Homozygotes Transfusion dependent thalassemia major
http://www.ithanet.eu
CD 39 C>T
HGVS name HBB:c.118C>T
Effect on gene/protein function Nonsense codon (Translation)
Type of thalassemia Beta0 thalassemia
Ethnic background Mediterranean
Heterozygotes Mild anemia
Homozygotes Severe transfusion dependent thal major
http://www.ithanet.eu
IVS-II-745 C>G
HGVS name HBB:c.316-106C>G
Effect on gene/protein function Cryptic splice site (mRNA processing)
Type of thalassemia Beta+ thalassemia
Ethnic background Mediterranean
Heterozygotes Mild anemia
Homozygotes Severe transfusion dependent thal major
http://www.ithanet.eu
IVS-II-837 T>G
HGVS name HBB:c.316-14T>G
Effect on gene/
protein function
Cryptic splice site
(mRNA processing)
Type of thalassemia Beta+ or Beta0 thalassemia (unclear)
Ethnic background Asian Indian (rare mutation)
db-THALASSEMIA IN SLOVENIA
Genotype No. of
patients
HbF
(%)
Ethnic
origin Coinheritance
Haplo-
type
Sicilian db
Thal/N 33
4,6 -
17,7
Slovenia (32)
Unknown (1)
αααanti3.7 (2)
α3.7 (1) I (VII)
Lepore
BW/N 5
2,4 –
4,4
Slovenia (3)
Serbia (1)
B&H (1)
/ V
b AND db CARRIERS WITH a TRIPLICATION
b-thal
Mutation
a globin
genes
No.
pat. Hb HCT MCV MCH Hb A2 Hb F
IVS-II-745 aa/aa 1 12,8 38,0 63,6 20,9 4,5 0,9
IVS-II-745 aa/aaa 1 10,1 32,0 65 20,5 5,9 3,5
b-thal
Mutation
a globin
genes
No.
pat Hb HCT MCV MCH Hb A2 Hb F
Sicilian db aa/aa 25 12,1 37,3 67,3 21,5 2,4 8,4
Sicilian db aa/aaa 2 11,0 32,5 74,8 23,7 2,3 16,6
DELETIONS OF THE b-GOBIN GENE CLUSTER HPFH AND db THALASSEMIA
Disorders of Hemoglobin, 2009
HPFH AND db THALASSEMIA
db thalassemia HPFH
Heterozygotes Hypochromia
Microcytosis
Normal A2 (<3%)
High HbF (5-15%) with
heterocellular distribution
Normal red cell indices
Normal A2 (<3%)
Higher HbF (15-30%) with
pancellular distribution
Homozygotes Thalassemia intermedia Clinically normal with
reduced MCV and MCH
Compound hetero-
zygotes with b thal
Thalassemia major or
intermedia
Clinically very mild
HOW DO DELETIONS CAUSE RAISED HbF IN ADULTS
Loss of regulatory regions
Region between Ag and d responsible for repressing g gene expression
Competition between GgAg and db Globin gene expression involves interaction of
LCR with gene promoters
Promoter competition within the b-globin cluster
Newly apposed enhancer sequences
MOLECULAR CHARACTERIZATION OF SICILIAN db THALASSEMIA
MLPA analysis HPLC
analysis
Gap-PCR
analysis
MOLECULAR CHARACTERIZATION OF LEPORE BOSTON WASHINGTON
Gap-PCR analysis HPLC analysis
SICILIAN (db)0-THALASSEMIA
HGVS name NG_000007.3:g.64336_77738del13403
Mutation Deletion of 13378 nts from the delta to beta gene
Type of thalassemia δβ-thalassaemia; GγAγ(δβ)0
Ethnic origin Mediterannean
Sicily, Italy, former Yugoslavia, Hungary, Greece,
Turkey, Israel, Egypt
Heterozygote Mild anemia
Homozygote Thalassemia intermedia (mild form)
Haplotype I (VII)
HB LEPORE BOSTON WASHINGTON
HGVS name NG_000007.3:g.63632_71046del
Mutation Delta-beta hybrid (delta through 87; beta from 116)
Type of thalassemia Hb variant and thalassemia (beta or deltabeta)
Ethnic background Wordwide; the most common Hb Lepore type;
found mainly in Italian families; it has also been observed
in families from Romania, Yugoslavia, Turkey, Cyprus,
Jamaica, Cuba, Greece, England, Australia, Mexico
Heterozygotes Mild anemia
Homozygotes Variable (Thalassemia intermedia; thalassemia major)
Haplotype I, V
FACTORS AFFECTING HbF LEVELS
FETAL HEMOGLOBIN
Strong modifier in hemoglobinopathies’ severity
Variable and inducible quantitative trait in humans
High levels are associated with thalassemia intermedia
Mechanisms of gene expression and developmental gene regulation
Targeted approaches for ameliorating severity of beta hemoglobinopathies
HB F VARIATION ASSOCIATED SNPs
SNP XmnI (C>T) SNP BCL11A (T>C)
HBS1L-MYB (T>C)
SNPs ASSOCIATED WITH HIGH HbF - METHODS
T/T T/C C/C
RFLP
analysis
HRM
analysis
SNPs AFFECTING HbF IN SLOVENIAN
SICILIAN db THALASSEMIA PATIENTS
SNPs AFFECTING HbF IN SLOVENIAN
LEPORE BW PATIENTS
ITHANET WEBSITE
http://www.ithanet.eu
ACKNOWLEDGEMENTS
RCGEB “Georgi D. Efremov”, MASA
Marija Dimishkovska
Dr. Emilija Shukarova-Stefanovska
Department of Hematology, University Medical Centre Ljubljana
Prof. Peter Cernelc
Dr. Biljana Todorova
Clinic of Hematology, Medical Faculty, Skopje
Prof. Oliver Karanfilski
Recommended