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