Genotypes/phenotypes Thalassemia in Asia  Suthat Fucharoen M.D.  Thalassemia R  Th l       i Research C t                 ...
                                                                                                                 ...
Pathophysiology of–Thalassemia/Hb E Disease
Interstitial deletional -thalassemias                                                                    --/-thalassem...
Types of Mutations Resulting in -Thalassemia  yp                        g > 200 mutations
Thalassemia in Thailand            ‐Thalassemia (‐thal1 and ‐thal2)                     20 ‐ 30%            Hb Constant...
Alpha Thalassemia in Thailand (2008)                             Central            South         Northeast          North...
-Beta Thalassemia in Thailand (2008)                                 Central          South         Northeast          No...
ANEMIA IN THALASSEMIAConcordance and Discordance Among Sib Pairs                        Fucharoen S et al. Am J Med Genet ...
Variable severity in -thalassemia disorder                                                 Intermediate              Norm...
Thalassemia:Genotype-phenotype InteractionG t       h t      I t    ti1. Better d t di1 B tt understanding of             ...
Factors affecting severity of -thalassemia diseaseHeterogeneity of Primary Mutations:H t       it f P i       M t ti    ...
Association Studies• Detect association between genetic variants and phenotype  across families       – Case-Control desig...
SCORING SYSTEM FOR CLASSIFYING THE DISEASE          SEVERITY IN  THALASSEMIA PATIENTS                      -THALASSEMIA ...
Distribution of 0-thalassemia/Hb E patients based on -globin gene mutations.                          50.0              ...
N -thalassemia                                         No th l      i                                                    ...
SNP map, Beta Globin Gene Cluster                                         map. ..                              Confirmed T...
XmnI–Gγ POLYMORPHISM AS A MODIFYING FACTOR IN β-THALASSEMIA                                                               ...
High correlation r2, value in β-globin cluster     correlation,                            46 kb           LCR     ε      ...
Genotype of 3 TagSNPs                                                 Genotypes                     95% CIbg2   bg11   bg2...
120                                                 Al cases (n=1081)                                                  l  ...
160 Candidate Modifier Genes/Regions      from Genomewide Association Study                                            APO...
2nd Genome‐wide Association Study (GWAS)           2nd Genome‐wide Association Study (GWAS)                               ...
Results of genome-wide associationNu uinoon et         t al. Hum                 m Geneti                        ics 125: ...
618 Thai β0 thalassemia/Hb E patients             618 Thai β0‐thalassemia/Hb E patients# Significant SNP from Sardinian po...
Galanello et al. Blood 114: 3935‐7, 2009
SardiniansGalanello et al. Blood 114: 3935‐7, 2009
All -globin gene mutations      618 0‐thalassemia/Hb E patients
Codon 41/42, -TCTT   297 0‐thalassemia/Hb E patients
The box plot shows the distribution of HbF (%) and absolute HbF (g/dl)
FETAL HEMOGLOBIN ANALYSIS                                                  Linear regression analysis     Number of       ...
Conclusion: 11. We have introduced a scoring system to classify  -thalassemia patients into mild, intermedia and severe  ...
Conclusion: 24. Genome wide scan for SNPs of the whole genome  revealed 150 SNP on 100 genes that showed strong        l d...
pVal = 0.0381                  pVal = 0.0158GeneGo Display of the largest        pVal = 0.0141network of genes with direct...
Systems Biology & ThalassemiaGlobin gene Red Cell             Anemia                                   e a             Iro...
AcknowledgementSiriraj Hospital, Mahidol University:            Noppadol SiritanaratanakulRamadhibodi Hospital, Mahidol Un...
Acknowledgement
Overview of Genotypes and Phenotypes of Thalassemia in Asia
Overview of Genotypes and Phenotypes of Thalassemia in Asia
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Overview of Genotypes and Phenotypes of Thalassemia in Asia

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Genotypes/phenotypes Thalassemia in Asia - by Suthat Fucharoen MD of the Thalassemia Research Center Institute of Molecular Biosciences, Mahidol University, Thailand.

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Overview of Genotypes and Phenotypes of Thalassemia in Asia

  1. 1. Genotypes/phenotypes Thalassemia in Asia Suthat Fucharoen M.D. Thalassemia R Th l i Research C t h CenterInstitute of Molecular Biosciences Mahidol University, Thailand
  2. 2.             Normal -Thalassemia Thalassemia -Thalassemia Thalassemia Hb A = 22 (97%) 4 = HbH Increased HbA2 HbA2 = 22 (2.5%) 4 = HbBart’s (4-6%) Hb F = 22 (0 5%) (0.5%)Normal Thalassemic RBC RBC
  3. 3. Pathophysiology of–Thalassemia/Hb E Disease
  4. 4. Interstitial deletional -thalassemias --/-thalassemia1 (--SEA, --THAI & --FIL)-thalassemia2 (-3.7 & -4.2)Hb Constant Spring -/Hb PakséRange 10-25% (modified from Higgs DR, Disorder of Hemoglobin, 2009)
  5. 5. Types of Mutations Resulting in -Thalassemia yp g > 200 mutations
  6. 6. Thalassemia in Thailand ‐Thalassemia (‐thal1 and ‐thal2) 20 ‐ 30% Hb Constant Spring ( -thal 2 like effect ) 1 ‐ 8% -Thalassemia Th l i 3 ‐ 3 9% Hemoglobin E 10 ‐ 53% Total number of thalassemic patients and the number of births per year (total births = 800,000/year) Diseases Couple at risk Birth Living (per year) (per year) patientsHomozygous -thalassemia thalassemia 828 207 2,070 2 070 -Thalassemia/Hb E 12,852 3,213 96,390 ,Hb Bart s hydrops fetalis 3332 833 0Hb H disease 22,400 5,600 336,000 Total , 39,412 9,853 , 434,460 , Fucharoen S. and Winichagoon P., 1988
  7. 7. Alpha Thalassemia in Thailand (2008) Central South Northeast North Mutations No. % No. % No % No. %Deletional Hb H  14 26.91. –SEA/‐α3.7 199 39.8 75 50 34 33.32. –SEA/‐α4.2 9 1.8 7 4.7 10 9.83 THAI/ α3.73. – /‐α 4 0.8 08 1 0.7 07 ‐ ‐4. etc 1 0.2 ‐ ‐ ‐ ‐Non‐Deletional Hb H1. –SEA/αCSα 247 49.4 55 36.9 35 67.3 54 532. –SEA/αPSα 28 5.6 4 2.7 3 5.8 3 33. –THAI/αCSα 2 0.4 2 1.3 1 14. etc 10 2 5 3.3 ‐ ‐Total l 500 00 100 00 149 9 100 00 52 2 100 00 102 02 100 00
  8. 8. -Beta Thalassemia in Thailand (2008) Central South Northeast North Mutations No. % No. % No % No. %1. codon 41/42 (‐TCTT) 50 36.3 109 15.1 19 31.6 108 49.52. codon 17 (A‐>T) 24 17.4 41 5.7 13 21.7 75 34.43. codon 35 (C‐>A) 2 1.4 4 0.6 0 0 ‐ ‐4. IVS1‐1 (G‐>T)4 IVS1 1 (G T) 3 2.2 22 35 4.8 48 1 1.7 17 15 6.9 695. codon 71/72 6 4.3 4 0.6 8 13.3 13 66. etc. 5 3.5 35 4.8 1 1.7 ‐ ‐7. IVS1‐5 (G‐>C) 7 5.1 98 13.6 0 0 ‐ ‐8. IVS2‐654 (C‐>T) 8 5.8 16 2.2 5 8.3 1 0.49. codon 19 (A‐>G) 3 2.2 57 7.9 0 0 ‐ ‐10. ‐28 (A‐>G) 20 14.5 20 2.8 1 1.7 3 1.4 ( )11. codon 26 (Hb E) ‐ ‐ 243 33.7 9 15 ‐ ‐12. etc. 10 7.3 9 1.2 ‐ ‐ 3 1.413.  δβ thalassemia ‐ ‐ 14 1.9 ‐ ‐ ‐ ‐14. Unknown ‐ ‐ 37 5.1 3 5 ‐ ‐Total 138 100 722 100 60 100 218 100
  9. 9. ANEMIA IN THALASSEMIAConcordance and Discordance Among Sib Pairs Fucharoen S et al. Am J Med Genet 1984; 19: 39-44
  10. 10. Variable severity in -thalassemia disorder Intermediate Normal Hemoglobin value 12 to 14 g/dl tientsNumb of Pat ber Severe Mild Fucharoen S et al Birth Defects 1988; 23(5A): 241-8
  11. 11. Thalassemia:Genotype-phenotype InteractionG t h t I t ti1. Better d t di1 B tt understanding of fgene-gene interaction,natural history, prognosis2. Decision of therapeutic pintervention eg. BM transplantation3.3 Prenatal diagnosis andinduced abortion4. New h4 N therapeutic i i intervention i
  12. 12. Factors affecting severity of -thalassemia diseaseHeterogeneity of Primary Mutations:H t it f P i M t ti  -Thalassemia mutations: -Thal, -Thal ,Genetic Modifiers: Coinheritance of -thalassemia Stimulation of Hb F production1. Concomitant inheritance of -thalassemia in -thalassemia/Hb E disease. Am J Hematol 1985; 20:217. ;2. Severity differences in -thalassemia/hemoglobin E syndromes; implication of genetic factor. Br J Haematol 1993; 83; 633.3. Genetic factors affecting clinical severity in beta-thalassemia syndromes. J Pediatrics Hematol/Oncol 2000; 22: 573.
  13. 13. Association Studies• Detect association between genetic variants and phenotype across families – Case-Control designs – Cohort designs Single Nucleotide Polymorphisms: SNPs • Biallelic polymorphism • Accounting for ~90% of human genetic variants • Occurring on coding and non-coding chromosome region SNP markers associated with modifier alleles GENE SNP modifier SNP SNP SNP marker allele marker marker marker
  14. 14. SCORING SYSTEM FOR CLASSIFYING THE DISEASE SEVERITY IN  THALASSEMIA PATIENTS -THALASSEMIA Clinical criteria Points scored 0 0.5 1 2Hb at steady state (g/dL) > 7.5 - 6-7.5 <6Age at first transfusion (yr) >10 - 5-10 ≤4Requirement for transfusion None/Rare - Occasional RegularSize of spleen (cm) <3 - 3-10 >10 or splenectomizedAge at presentation (y ) g p (yr) >10 3-10 ≤2Growth development >25th percentile 3rd-25th percentile <3rd percentile Severity grouping Total score range Mildly affected 0-3.5 Moderately affected 4-7 Severely affected 7.5-10 (Sripichai O et al, 2008) 15
  15. 15. Distribution of 0-thalassemia/Hb E patients based on -globin gene mutations. 50.0 00 45.0 40.0 entage of cases 35.0 30.0 f 25.0 20.0 15.0 50 Perce 10.0 5.0 0.0 Cod41/42 Cod17 IVS II-654 IVS I-1 IVS I-5 Cod35 Cod71/72 Rare Unchar (-TTCT) (A>T) (C>T) (G>T) (G>C) (C>A) (+A) All samples (n=517) 44.0 24.1 9.9 4.6 6.2 1.5 2.5 4.4 2.7 Pooled mild (n=197) 42.1 24.9 9.6 8.6 4.1 2.5 0.5 7.6 0.0 Pooled severe (n=198) 45.5 24.2 14.6 1.5 7.1 1.0 4.0 2.0 0.0
  16. 16. N -thalassemia No th l i With -thalassemia th l i 7.0 7.0 Mild with alpha- 6.0 60 Mild 6.0 60 thalassemia oglobin F (g/dl) oglobin F (g/dl) Severe 5.0 5.0 Mild without alpha- thalassemia 4.0 4.0 3.0 3.0Hemo Hemo 2.0 2.0 1.0 1.0 0.0 0.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 Hemoglobin ( /dl) H l bi (g/dl) Hemoglobin (g/dl)
  17. 17. SNP map, Beta Globin Gene Cluster map. .. Confirmed Thai polymorphisms (n = 503): . .. .. 10 C Common RFLPs RFLP . 54 Reference SNPs . . 3 Novel SNPs . . . Median Intermarker = 514 bp . . . ... . . . .. . . . . . .... . .. . . ... . . . . . .. . .. . .. .. .. . . ... LCR
  18. 18. XmnI–Gγ POLYMORPHISM AS A MODIFYING FACTOR IN β-THALASSEMIA β Allele distribution of XmnI in Mild & Association between Xmn I genotype and Severe β0-thalassemia/Hb E patients. Hb F level stratified by severity group (n=517). 100% 90% All subjects Mild MILD SEVERE Severe 80% 74% 70% 64% 60%% patients 53% 50% 40% 34% 34% 30% 20% 16% 10% 10% 2% 0% / / / XmnI +/+ XmnI +/ X X I X I +/- XmnI -/- I / XmnI + X I -/- +/- +/+ -/- +/- +/+ Xmn I-Gγ site Xmn I-Gγ site β-Thalassemia/Hb E patients with XmnI -/- are 35 times more likely to have severe disease than patients with XmnI +/+. β-Thalassemia/Hb E patients with XmnI +/+ and +/- h h l i / b i ih / d / have hi h Hb F level than higher b l l h patients with XmnI -/- (p-value < 0.001).
  19. 19. High correlation r2, value in β-globin cluster correlation, 46 kb LCR ε Gγ Aγ δ β 3 TagSNPs cover 88% of the genotypes in large LD block
  20. 20. Genotype of 3 TagSNPs Genotypes 95% CIbg2 bg11 bg200 Mild Severe Total odd ratio frequency for odd ratioAA AA CA 1 1 0.21AA AA CC 14 64 78 16.02 3.55 1.93 - 6.55AA AG CA 4 4 0.82AA AG CC 2 2 0.41AC AA CA 1 1 0.21AC AA CC 2 19 21 4.31 6.59 1.51 - 28.65AC AG CA 119 152 271 55.65 55 65 0.66 0 66 0.46 0.96 0 46 - 0 96AC AG CC 4 10 14 2.87 1.66 0.51 - 5.38AC GG AA 6 1 7 1.44 0.10 0.01 - 0.89CC AA CC 3 3 0.62 0 62CC AG CA 6 13 19 3.90 1.44 0.53 - 3.86CC AG CC 4 4 0.82CC GG AA 27 3 30 6.16 0.06 0.01 - 0.21CC GG CA 11 20 31 6.37 1.20 0.56 - 2.58CC GG CC 1 1 0.21
  21. 21. 120 Al cases (n=1081) l 100 Pooled cases (n=395) 80 - Unrelated patients l t d ti t N me o c s s u b r f aeRegionally-Matched 60 - Extremely SeverityDNA Pools 40 20 - Excluded knownConstruction 0 modifying factors 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 Disease Severity Score DNA1, DNA2 … DNAnMild DNA1, DNA2 … DNAnSevere Mild Severe DNA pool DNA pool (n=197) (n=198) 110,000 SNPs genotype using MassARRAY system , g yp g y SNPs p-value <0.05 <0 05 854 SNPs were genotype individually in 198 mild, 305 severe patients GENOMEWIDE SEARCH FOR DISEASE MODIFIER GENES IN β0-THALASSEMIA/HBE
  22. 22. 160 Candidate Modifier Genes/Regions from Genomewide Association Study APOPTOSIS & CELL CYCLE REGULATION Cell cycle regulation, Associated PROTEASE & UBIQUITINATION Fas apoptotic inhibitory molecule 3 Apoptosis Function REGULATORY FACTORS NIMA (never in mitosis gene a)- related kinase 11 calpain 1 (mu/I) large subunit 1,Proteolysis SIGNAL2TRANSDUCTION 1 sestrin high-mobility group box NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, T-box 2 INTERESTING BIOLOGICAL FUNCTION NACHT, leucine rich repeat and PYD containing 14 16kDa beta 4 binding protein mitogen-activated protein kinase kinase kinase 9 integrin leukemia sarcoma(TEL1 oncogene homolog 1 (avian) v-ros UR2 inhibitory virus receptor HtrA serine gene 6 factor ets variant assembly factor 1, subunit A (p150) chromatin peptidase oncogene) p rich interactive domain 5B ( protein-coupled receptor 22 (MRF1-like) AT p p ) oncoprotein induced transcript 3 2 phosphoinositide-3-kinase, thrombospondin type 1 motif, 6 phosphoinositide 3 kinase class 2, alpha polypeptide ADAM metallopeptidase withsuppressor of hairless adenylate kinase 5 protein recombining binding 2 (juvenile) hemochromatosis type FERM, RhoGEF and III (DNA directed) polypeptide D, 44kDa polymerase (RNA) pleckstrin domain protein (Drosophila) frataxin protein tyrosine kinaseinitiation factor 2C, 4 eukaryotic translation 2 beta gastric inhibitory polypeptide DnaJ (Hsp40) related, subfamily B, member 13 neurolysin (metallopeptidase M3 family) TAO kinaseconverting enzyme 1 endothelin 3 beta, acid 3 (cytosolic) glucosidase, receptor determining region Y)-box 5receptor axin(sex tyrosine inhibitor,M5 orphan kinase-like SRY p p S-transferase , clade B, member 11 g p glutathione serpin peptidase , Non-Associated i t d membrane associated guanylatefactor 3 WW and PDZ pre-B-cell l k lipase ll leukemia t B i transcription f kinase, g an ti Wilms tumor associated protein i late kinase t Regulatory carboxypeptidase E domain containing activity-1 transcription factor glucose transporter Function protein kinase, cGMP-dependent, type I interleukin protein 152 transposase fusion gene ring finger 24 transporting SET domain and mariner ATPase, Ca++ Factor src family associatedhomolog 1 (Drosophila) sex comb on midleg phosphoprotein 1 ATPase, H+ transporting F-box and WD-40 domain protein 11 2C, 3 Rho guanine nucleotide exchange factor (GEF) eukaryoticvoltage-gated channel potassium translation initiation factor 12 HSA275986 transcriptiond alpha-linkedd idi ddipeptidase-like 1 calcium h factor activity acidic di t N-acetylated l l activity-2 lt k d 2 N l i t lchannel,hvoltage-dependent tid t li lik transcription factor659 zinc finger protein SMIF Unknown Function chloride intracellular channel 5 vascular cell adhesion molecule 1 zinc adhesion cell finger protein 214 regulator of G-protein signalling like 2 zinc finger protein 609 Golgi function tumor necrosis factor receptor superfamily, member 11, mitochondrial protein activator of NFKB Hypothetical Protein
  23. 23. 2nd Genome‐wide Association Study (GWAS) 2nd Genome‐wide Association Study (GWAS) β0‐Thalassemia/Hb E patients 383 SEVERE CASES 235 MILD CASES GWAS analysis • QC data Quality Controls : MAF > 0.05, call rate > 0.99 Q • Significant levelsStatistical Analysis using chi‐square test for general model and Fisher exact test for dominant recessive and allelic model 
  24. 24. Results of genome-wide associationNu uinoon et t al. Hum m Geneti ics 125: 3 303‐14, 2010
  25. 25. 618 Thai β0 thalassemia/Hb E patients 618 Thai β0‐thalassemia/Hb E patients# Significant SNP from Sardinian population (Galanello et al, Blood 114: 3935‐7, 2009)  g p p ( , , )
  26. 26. Galanello et al. Blood 114: 3935‐7, 2009
  27. 27. SardiniansGalanello et al. Blood 114: 3935‐7, 2009
  28. 28. All -globin gene mutations 618 0‐thalassemia/Hb E patients
  29. 29. Codon 41/42, -TCTT 297 0‐thalassemia/Hb E patients
  30. 30. The box plot shows the distribution of HbF (%) and absolute HbF (g/dl)
  31. 31. FETAL HEMOGLOBIN ANALYSIS  Linear regression analysis Number of (Additive model, P value) high-HbF allele Locus rs ID SNP allele High-HbF HbF (%) Abs HbF (g/dL) 0 1 2 alleleBCL11A rs766432 A/C C 2.33E-07 1.20E-09 AA AC CCHBS1L-MYB rs9399137 A/G G 2.76E-18 5.93E-19 AA AG GGHBG2(XmnI) rs7482144 C/T T 2.52E-19 1.90E-23 CC CT TT 32
  32. 32. Conclusion: 11. We have introduced a scoring system to classify -thalassemia patients into mild, intermedia and severe cases. Mild patients have hi h Hb F levels. ti t h higher l l2. β-Thalassemia/Hb E patients with XmnI -/- are 35 β p times more likely to have severe disease than patients with XmnI +/+.3. Three “novel SNPs” on the -globin gene cluster show novel SNPs  globin strong association with disease severity and Hb F levels.
  33. 33. Conclusion: 24. Genome wide scan for SNPs of the whole genome revealed 150 SNP on 100 genes that showed strong l d SNPs h h d association with disease severity5. 2nd GWS revealed SNPs in 3 major regions that showed strong association with disease severity, namely -globin gene cluster, intergenic region between Myb cluster and HBS1L and BCL11A. The function of these regions in the modifying of disease severity need further studies. studies
  34. 34. pVal = 0.0381 pVal = 0.0158GeneGo Display of the largest pVal = 0.0141network of genes with directinteractions (6) pVal = 0.0013 pVal = 0.0834 (5) pVal = 0.0042 pVal = 0.00162 pVal = 0.0737 (2) pVal = 0.0123
  35. 35. Systems Biology & ThalassemiaGlobin gene Red Cell Anemia e a Iron Clinical mutation ManifestationGenotype Phenotype Drug Organ Erythropoiesis Metabolism Pathology Apoptosis Iron Absorption Ferrokinetic Oxidative Stress Antioxidants Heart GDF 15 Liver Endocrine glands g UGT Cyt P450 CVS High HbF Genes Epo
  36. 36. AcknowledgementSiriraj Hospital, Mahidol University: Noppadol SiritanaratanakulRamadhibodi Hospital, Mahidol University: Suporn Chancharunee, Tanyachai Sura, T h iS Ampaiwan ChuansumritChulalongkorn University: Issarang NuchprayoonChiangrai P hChi i Pachanukraw H k Hospital: it l Saranya S S Suwansings iMaharaj Nakornrajchasima Hospital: Somchai Intarasiripong, Nittaya VisanuyothinLampang Hospital:L H it l Ladda S ib ib L dd SriboriboonsiniUttaradit Hospital: Chuchart Koosirirat, Anong BenjakoraneeChonburi HCh b i Hospital: it l Kesada Ch K d ChansawangSaraburi Hospital: Soraya Thammarak, Bang-orn UbolMaharaj Nakornsrithammarat:M h jN k ith t Wanchai Kachonwattanakul W h iK h tt k lNakhonpathom Hospital: Piatip Boonmongkol, Navarat Chantrakul
  37. 37. Acknowledgement

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