MOLECULAR BASIS OF THALASSEMIA IN SLOVENIA

Transcription

1 MOLECULAR BASIS OF THALASSEMIA IN SLOVENIA Dijana Plaseska-Karanfilska, MD, PhD Research Centre for Genetic Engineering and Biotechnology Georgi D. Efremov, Macedonian Academy of Sciences and Arts, Skopje, R. Macedonia 5 th Congress of Slovenian Hematological Society, Lasko, Slovenia,

2 CONTENT Brief introduction Thalassemias in Slovenia Methodology Mutations Factors affecting fetal hemoglobin levels Slovenian db thalassemia patients

3 INTRODUCTION

4 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

5 Hb TYPES, GLOBIN GENE CLUSTERS AND DEVELOPMENTAL EXPRESSION OF GLOBIN GENES Higgs et al., The Lancet 2012, 379:373

6 Genotype Phenotype BETA THALASSEMIA GENOTYPE/PHENOTYPE HETEROGENEITY Heterozygote Homozygote Compound heterozygote Carriers Intermedia Major Silent Hematologic phenotype Mild Tfx independent Severe sporadic Tfx Tfx dependent

7 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) Severity of b thal phenotype a thalassemia mild/silent b alleles increased g chains Imbalance in globin chain synthesis

8 THALASSEMIAS IN SLOVENIA

9 THALASSEMIAS IN SLOVENIA MATERIAL STUDIED Date No of samples Material Feb blood June blood Nov blood Feb DNA Total 72 Missing hematological data 5 (1 Normal) 3 (2 Normal) 1 (1 Normal) 6 (1 Normal) 15 (5 Normal) Missing HbA 2 & HbF values / / / 10 (3 Normal) 10 (3 Normal)

10 METHODS 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 ) > α-globin triplication ααα anti3.7 ) Multiplex SNaPshot analysis:common Mediterannean mutations Sequencing analysis: HBB, HBA 2, HBA 1, HBD genes

11 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

12 THALASSEMIAS IN SLOVENIA Patients with thalassemia (n=53) 4% Unrelated patients with thlassemia (n=45) 2% 24% 72% deltabeta 29% beta alpha 69% deltabeta beta alpha

13 a-thalassemia Genotype No. of patients Ethnic origin Coinheritance - -(SEA)/aa 2 China (1) / a 3,7 /aa 1 Slovenia Sicilian db

14 Molecular characterization of a thalassemias MLPA analysis (a SEA deletion) Gap-PCR analysis

15 a THALASSEMIA DELETIONS Deletions of two a-genes (a 0 thalassaemia) Deletions of one a-gene (a + -thalassaemia) Hartveld & Higgs, Orphanet J Rare Dis 2010, 5:13.

16 DISTRIBUTION OF THE MOST COMMON a-thalassemia ALLELES Weatherall, DJ., Nature Reviews Genetics 2001;2(4):

17 b-thalassemia MUTATIONS Genotype No. of patients IVS-I-110G>A/N 5 Cd39C>T/N 5 IVS-II-745C>G/N 2 Ethnic origin Coinheritance Slovenia (4) Unknown (1) / Slovenia (4) Cuba (1) / Slovenia (1) Serbia (1) ααα anti3.7 (1) IVS-II-837T>G/N 1 Serbia (1) /

18 MOLECULAR CHARACTERIZATION OF b-thalassemia ALLELES HPLC analysis SNaPshot analysis Sequencing analysis

19 DISTRIBUTION OF THE MOST COMMON b-thalassemia ALLELES Weatherall, DJ., Nature Reviews Genetics 2001;2(4):

20 IVS-I-110 G>A HGVS name Effect on gene/protein function Type of thalassemia Ethnic background Heterozygotes Homozygotes HBB:c.93-21G>A Cryptic splice site (mrna processing) Beta + thalassemia Mediterranean Mild anemia Transfusion dependent thalassemia major

21 CD 39 C>T HGVS name Effect on gene/protein function Type of thalassemia Ethnic background Heterozygotes Homozygotes HBB:c.118C>T Nonsense codon (Translation) Beta 0 thalassemia Mediterranean Mild anemia Severe transfusion dependent thal major

22 IVS-II-745 C>G HGVS name Effect on gene/protein function Type of thalassemia Ethnic background Heterozygotes Homozygotes HBB:c C>G Cryptic splice site (mrna processing) Beta + thalassemia Mediterranean Mild anemia Severe transfusion dependent thal major

23 IVS-II-837 T>G HGVS name HBB:c T>G Effect on gene/ protein function Type of thalassemia Ethnic background Cryptic splice site (mrna processing) Beta + or Beta 0 thalassemia (unclear) Asian Indian (rare mutation)

24 db-thalassemia IN SLOVENIA Genotype No. of patients HbF (%) Ethnic origin Coinheritance Haplotype Sicilian db Thal/N 33 4,6-17,7 Slovenia (32) Unknown (1) ααα anti3.7 (2) α 3.7 (1) I (VII) Lepore BW/N 5 2,4 4,4 Slovenia (3) Serbia (1) B&H (1) / V

25 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, ,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

26 DELETIONS OF THE b-gobin GENE CLUSTER HPFH AND db THALASSEMIA Disorders of Hemoglobin, 2009

27 HPFH AND db THALASSEMIA db thalassemia HPFH Heterozygotes Hypochromia Microcytosis Normal A 2 (<3%) High HbF (5-15%) with heterocellular distribution Normal red cell indices Normal A 2 (<3%) Higher HbF (15-30%) with pancellular distribution Homozygotes Thalassemia intermedia Clinically normal with reduced MCV and MCH Compound heterozygotes with b thal Thalassemia major or intermedia Clinically very mild

28 HOW DO DELETIONS CAUSE RAISED HbF IN ADULTS Loss of regulatory regions Region between A g and d responsible for repressing g gene expression Competition between G g A g and db Globin gene expression involves interaction of LCR with gene promoters Promoter competition within the b-globin cluster Newly apposed enhancer sequences

29 MOLECULAR CHARACTERIZATION OF SICILIAN db THALASSEMIA HPLC analysis MLPA analysis Gap-PCR analysis

30 MOLECULAR CHARACTERIZATION OF LEPORE BOSTON WASHINGTON HPLC analysis Gap-PCR analysis

31 SICILIAN (db) 0 -THALASSEMIA HGVS name Mutation NG_ :g.64336_77738del13403 Deletion of nts from the delta to beta gene Type of thalassemia δβ-thalassaemia; GγAγ(δβ) 0 Ethnic origin Heterozygote Homozygote Haplotype Mediterannean Sicily, Italy, former Yugoslavia, Hungary, Greece, Turkey, Israel, Egypt Mild anemia Thalassemia intermedia (mild form) I (VII)

32 HB LEPORE BOSTON WASHINGTON HGVS name NG_ :g.63632_71046del Mutation Delta-beta hybrid (delta through 87; beta from 116) Type of thalassemia Ethnic background Heterozygotes Homozygotes Haplotype Hb variant and thalassemia (beta or deltabeta) 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 Mild anemia Variable (Thalassemia intermedia; thalassemia major) I, V

33 FACTORS AFFECTING HbF LEVELS

34 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

35 HB F VARIATION ASSOCIATED SNPs

36 SNPs ASSOCIATED WITH HIGH HbF - METHODS SNP XmnI (C>T) SNP BCL11A (T>C) RFLP analysis HBS1L-MYB (T>C) T/T T/C C/C HRM analysis

37 SNPs AFFECTING HbF IN SLOVENIAN SICILIAN db THALASSEMIA PATIENTS

38 SNPs AFFECTING HbF IN SLOVENIAN LEPORE BW PATIENTS

39 ITHANET WEBSITE

40 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