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Vip Viprakasit, M.D., D.Phil.(Oxon) Department of Pediatrics and thalassaemia Center

Recent Advances in Molecular Understanding of NTDT: 2012. 124 yrs of experience in medical care in Thailand. Vip Viprakasit, M.D., D.Phil.(Oxon) Department of Pediatrics and thalassaemia Center Faculty of Medicine, Siriraj Hospital Mahidol University, THAILAND. Overview :.

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Vip Viprakasit, M.D., D.Phil.(Oxon) Department of Pediatrics and thalassaemia Center

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  1. Recent Advances in Molecular Understanding of NTDT: 2012 124 yrs of experience in medical care in Thailand Vip Viprakasit, M.D., D.Phil.(Oxon) Department of Pediatrics and thalassaemia Center Faculty of Medicine, Siriraj Hospital Mahidol University, THAILAND

  2. Overview: • Globin gene regulation and associated disorders • Molecular basis of NTDT in a and b thalassaemia-haemoglobinopathies • - Conventional causes • - Unconventional causes • Roles of genetic modifiers in NTDT

  3. HS-40 FETUS ADULT EMBRYO Haem z2e2 a2g2 a2b2 -LCR b HS 4 3 2 1 b Structure and Expression of the Globin Genes 20 0 -20 40 -40 a2 a1 z Chromosome 16 e g g b Chromosome 11 20 60 0 -20 40 -40

  4. Structure and Expression of the Globin Genes Newborn Adult Hb A a2b2 25% 96% Hb A2 a2d2 <1% 3% Hb F a2g2 75% 1%

  5. Deletions Deletions Point Mutations The Molecular Basis for a and b thalassaemia -20 0 20 40 -40 Chromosome 16 a2 a1 z HS-40 Chromosome 11 b-LCR e g g b 3 2 1 HS 4 0 20 40 60 -20

  6. Overview: • Globin gene regulation and associated disorders • Molecular basis of NTDT in a and b thalassaemia-haemoglobinopathies • - Conventional causes • - Unconventional causes • Roles of genetic modifiers in NTDT

  7. Molecular basis of a0 and a+ thalassaemias: deletions & non-deletions aTa/aa aaT/aa --/aa -a/aa (modified from Higgs, Disorder of Hemoglobin, 2001 with permission)

  8. Hb H disease is the most common form of NTDT --SEA/aCSa --SEA/-a3.7 • Patients with non-deletional type of Hb H disease (--/aTa) had more severe clinical symptom than those with deletional type of Hb H (--/-a) Review in Chui DH, Chan V. & Fucharoen S. Blood 2004

  9. Hb H HydropsFetalis • Hydrops fetalis due to an unusual form of Hb H disease.By Chan V et al, Blood. 1985, 66(1):224-8. • Hydropic fetus with 20-25% of Hb Bart’s • Molecular pathology: • - - TOT/aCD30a, del. GAG • - - SEA/aCD35a, TCCCCC • - - SEA/aCD59a, GGCGAC • - - SEA/aCD66a, CTGCCG • apolyAa/apolyAa* • --SEA/aQSa • aCSa/aCSa and --SEA/aCSa Viprakasit V. et al, Br J Haematol. 2002;117(3):759-62. Fucharoen S. & Viprakasit V. Hematology (ASH) 2009

  10. Common causes of NTD-b thalassaemia b-Thalassaemia intermedia b0 thal / b+ thal b+ thal / b+ thal Hb E / b thal. b0 thal / HPFH b+ thal / HPFH b0 thal / db thal b+ thal / db thal Courtesy of Prof. V. S.Tanphaichitr

  11. A wide spectrum of b globin mutations in Thailand Summary of known b-globin genes mutations identified in Thailand: 2010 • Each mutation can be classified as b0 or b+ thalassaemia based on the output of b globin transcripts Suchada Riolueang et al. 16th National Thalassaemia Conference, Bangkok 2010

  12. b thalassaemia intermedia (Hb E/b thalassaemia) b 0/ b + + bE Courtesy of Prof. V. S.Tanphaichitr

  13. Most common bglobin gene cluster deletions found in Asia-Pacific Nipon Chalaow, Suchada Riolueang, ChompunutKarnjanakorn, Chanin Limwongse & Vip Viprakasit; To be presented at APIA 2012, Taiwan

  14. Summary of HPFH & db thalassemia interactions with NTDT and TDT phenotype Vip Viprakasit: Unpublished data 2012

  15. Overview: • Globin gene regulation and associated disorders • Molecular basis of NTDT in a and b thalassaemia-haemoglobinopathies • - Conventional causes • - Unconventional causes/novel mechanism/trans acting mutations • Roles of genetic modifiers in NTDT

  16. Upstream deletional a0-thalassaemia Fucharoen S & Viprakasit V, Hematology (ASH) 2009

  17. Unusual deletions causing a0 thalassaemia and Clinical Hb H disease Viprakasit V. et al, British Journal of Haematology, 2003 120(5): 867-75

  18. Weatherall DJ, Higgs DR, Bunch C, et al, Hemoglobin H disease and mental retardation. A new syndrome or a remarkable coincidence? N Engl J Med305: 607-612. 1981

  19. Gibbons RJ, Higgs DR., Am J Med Genetics, 2000 X-linked thalassemia/mental retardation (ATR-X) syndrome ATRX-gene or XNP2 or XH2 Gibbons RJ, Picketts DJ, Villard L, Higgs DR. Mutations in a putative global transcriptional regulator cause X-linked mental retardation with a-thalassemia (ATR-X syndrome). Cell80: 837-845. Other syndromes with ATRX mutations: Carpenter syndrome, Juberg-Marsidi syndrome, Smith-Fineman-Myers syndrome, X-linked mental retardation with spastic paraplegia

  20. Interaction of b thal alleles with extra copies of a globin genes b0 thal /aaa/aa b0 thal /aaaa/aa b0 thal /aaaaa/aa b+ thal /aaa/aa b+ thal /aaaa/aa b+ thal /aaaaa/aa • 24-year old Thai woman with underlying anemia since 7 years old • She received less than 7 blood transfusions in her lifetime • Hb 7.5 g/dL Hct 26% RBC 4.8 x106/mL, MCV 62 fL, MCH 24 pg MCHC 33 pg/dL, RDW 27 % Retic. 7% • Hemoglobin typing: A, A2 (7%), F (10%) • DNA analysis: • aaa/aa and b/b41/42

  21. b39CT/ b39CT b39CT/--- • Patient A • 42 yrs-male with Hb 7-9 g/dL • Clinical diagnosis with bthal. Intermedia • S/P splenectomy • Hb typing: A 81% A2 4.8% F 14.2% • a/b+gglobin synthesis: 1.79 • Patient B • 23 yrs-female with Hb 7-9 g/dL • Clinical diagnosis with bthal. Intermedia • Marked splenomegaly (10 cm BLCM) • Hb typing: A 87% A2 4.3% F 8.6% • a/b+gglobin synthesis: 1.65 Baden C et al, Lancet 2002: 359; 131-2, Galanello R et al. BJH 2004: 127: 604-6

  22. A594P XPD mutations in Patients with Trichothiodystrophy Causes b thalassaemia phenotype Hb 11.5 g/dL Hct 34% MCV 65 fL MCH 24 pg Hb A A2 (A2 4.5%) Haematological phenotype mimic that of mild b thalassaemia Viprakasit, et al. Human Molecular Genetics 2001:2797-802 (2001)

  23. Null and missense TTD specific mutations Null and missense XP specificmutations Schematic Representation of Seven-Conserved Helicase Domains of XPD Gene and Mutations in TTD and XP-D Patients Viprakasit, et al. Human Molecular Genetics 2001:2797-802 (2001)

  24. Further Example of Trans Acting Mutations Causing Thalassaemia HbA2 levelwas at 3.8%-4.4% (normal range: 1.8%-3.4%) The a : b chainsynthesis ratios were 0.55 to 0.67 in affected individuals NicholsKEet al.Nature Genetics24, 266 - 270 (2000)

  25. Further Example of Trans Acting Mutations Causing Thalassaemia Familial dyserythropoieticanaemia and thrombocytopenia due to an inherited mutation in GATA1 NicholsKEet al.Nature Genetics24, 266 - 270 (2000)

  26. We identified three Families one from PNG and two from Vanuatu with HbH disease aThalassaemia in the South Pacific a thalassaemia is common in the South Pacific Some Individuals with a thalassaemia have an apparently normal genotype (aa/aa)

  27. aa/aa aa/aa aa/aa HbH Disease In a Family From Papua New Guinea

  28. Haematological Phenotypes 0.39-0.52 NR 1.06 ± 0.11 a/b Globin Synthesis Ratio a/b Globin mRNA Ratio ~0.5 based on qPCR NR 1 ± 0.2 Is this really HbH disease? RNA FISH analysis confirmed a down regulation at the transcriptional level De Gobbi M* & Viprakasit V* et al. Science 2006; 1215-7

  29. Differences in the region underlying the new peak of expression 7 After Removal of known SNPs 1 In linkage with the disease aa/aa 141 T/T aa/aa 31 T/C aa/aa 5 C/C Identifying a Candidate SNP 50 100 150 200 250 300    D

  30. Overview: • Globin gene regulation and associated disorders • Molecular basis of NTDT in a and b thalassaemia-haemoglobinopathies • - Conventional causes • - Unconventional causes • Roles of genetic modifiers in NTDT

  31. Roles of genetic modifiers on b-thal intermedia Thein SL, Brit J Haematol 2004, p 264-274)

  32. Roles of genetic modifiers on a-thal syndromes b genes a genes Hb Bart’s Excess b globin chains Thein SL, Brit J Haematol 2004, p 264-274)

  33. Summary: • Molecular basis of NTDT either a and bglobin genes related are heterogeneous with several novel molecular defects have been increasingly found • Identification of molecular pathology of NTDT patients will be useful for management plan (early treatment and intervention including transfusion programme), prevention & control measures • Basic globin gene analyses should be first performed to identify common molecular pathology, if not found, a further detail study is highly recommended and this can provide new insights to understand gene regulation and genome biology

  34. Acknowledgements Faculty of Medicine, Siriraj Hospital Mahidol University, THAILAND Vip’s Laboratory Nipon Chalaow Suchada Ruengleung Nuntawut Chat-Utai Rapeepun Shompoopuong Punyanuch Pornpanich Kalaya Tachavanich Warapron Glomglao Supachai Ekwattanakit Chompunut Kanjanakorn Dr. Chanin Limwongse Prof. Suthat Fucharoen Prof. Voravarn S. Tanphaichitr Thailand Research Fund BIOTEC, Thailand

  35. Disorders of Globin Chain Production 1. Quantitative defects: Thalassaemia syndrome causing imbalance of globin chain production 2. Qualitative defects: Haemoglobinopathies result from substitution, deletion or insertion of one or more amino acid 3. Hereditary of Persistence of Fetal hemoglobin

  36. Genetic Analysis of the a Globin Locus 50 100 150 200 250 300  D   Excluded deletions and rearrangements No point mutations No other abnormalities De Gobbi M* & Viprakasit V* et al. Science 2006; 1215-7

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