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Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
Departmental seminar Genetic determinants of b-thalassaemia phenotype
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Departmental seminar Genetic determinants of b-thalassaemia phenotype

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  • 1. Molecular Haematology I Globin Disorders Dr Edmond S K Ma Division of Haematology Department of Pathology The University of Hong Kong
  • 2. Thalassaemia • First described by Thomas B. Cooley in 1925 • The term thalassaemia was first coined in 1932 based on the Greek word θαλασσα (thalassa) meaning the sea
  • 3. Prevalence of thalassaemia in Hong Kong Chinese α-thalassaemia 5% β-thalassaemia 3.1%
  • 4. Prevalence of thalassaemia in Hong Kong Chinese α-thalassaemia (--SEA ) α-thalassaemia deletion 90% β-thalassaemia codons 41-42 (-CTTT) β0 45% IVSII-654 (C→T) β0 20% nt-28 (A→G) β+ 16% codon 17 (A→T) β0 8%
  • 5. Carrier detection • Antenatal screening – Obstetrical Units of the Hospital Authority – Maternal and Child Health Centres – Private sector • Pre-marital and pre-pregnancy testing – Family Planning Association • Community based thalassaemia screening – Children’s Thalassaemia Foundation
  • 6. Detection of thalassaemia • Red cell indices (MCV, MCH) • Determine iron status • HPLC analysis • Hb and globin chain electrophoresis • Detection of HbH inclusion bodies
  • 7. Laboratory diagnosis of thalassaemia by HPLC
  • 8. Haemoglobin electrophoresis
  • 9. Detection of HbH inclusion bodies
  • 10. New approaches in diagnosis of SEA deletion: gap-PCR for SEA deletion
  • 11. New approaches in diagnosis of SEA deletion: detection of ζ-globin chains in adults
  • 12. α-globin gene mutations • Deletional (common) --SEA -α3.7 -α4.2 • Non-deletional (rare) Hb CS Hb QS codon 30 deletion Hb Q-Thailand Hb Westmead α2 codon 31 α2 codon 59 Others
  • 13. Prevalence of thalassaemia in Hong Kong Chinese (MCV < 80 fL) α-thalassaemia (--SEA ) α-thalassaemia deletion 90%
  • 14. Single α-globin gene deletion and triplicated α-globin gene • Prevalence – 6% for –α3.7 and –α4.2 • Hb 13.6 ± 0.12 g/dL (11.8 – 15.6) • MCV 83.0 ± 0.33 fL (77.9 – 88.1) • MCH 27.2 ± 0.16 pg (24.1 – 29.7) – 1.5% for αααanti-3.7 and αααanti-4.2 • Hb 13.5 g/dL, MCV 85.5 fL, MCH 28.7 pg
  • 15. Single α-globin gene deletion (-α) and triplicated α-globin gene (ααα) configuration
  • 16. Molecular diagnosis of α-thalassaemia Clark & Thein, Clin Lab Haematol 26: 159-76; 2004 • Deletions – Gap PCR – Southern blotting • Non-deletional mutation: on specifically amplified α2 or α1 genes – Restriction digest – ARMS-PCR – ASO – Direct sequence analysis
  • 17. Multiplex PCR for 3 commonest α-thalassaemia deletion LIS1 control α-2 gene SEA deletion 3.7 kb deletion 4.2 kb deletion
  • 18. LIS internal control (2350 bp) -α3.7 (2022/2029 bp) α2 (1800 bp) -α4.2 (1628 bp) --SEA (1349 bp) water αα/--SEA ladder -α3.7 /--SEA -α4.2 /--SEA αα/αα blank Multiplex PCR for 3 commonest α-thalassaemia deletion
  • 19. Restriction fragment length polymorphism (RFLP) The principle of RFLP as shown is used to diagnose the different types of α-globin genotypes relevant to α-thalassaemia. Gel Smaller fragment Larger fragment Key restriction enzyme sites probe region
  • 20. A typical RFLP result of different α-thal genotypes: Genotypes αα/αα αα/- α3.7 - α3.7 /-α3.7 αα/ - α4.2 - α4.2 / - α4.2 Bam HI probes with α-globin 14.5 kb 14.5 kb; 10.5 kb 10.5 kb 14.5 kb; 10.5 10.5 Bgl II probed with α-globin 12.6 kb; 7.0 16.0 kb 12.6; 7.0 16.0 12.6; 7.0 7.0
  • 21. 16kb 10.5 kb 14.5kb 12.6 kb 7.0 kb
  • 22. Multiplex ARMS for the 3 commonest non-deletional α2-globin gene mutations Internal control (930 bp) cd30(ΔGAG) (772 bp) HbQS (234 bp) HbCS (184 bp)
  • 23. Reverse dot blot Chan V et al, BJH 104: 513-5, 1999
  • 24. Multiplex mini-sequencing screen Wang W et al, Clin Chem 49: 800 – 803, 2003
  • 25. Molecular screening of non-deletional α-globin gene mutations by denaturing HPLC Guida V et al, Clin Chem 50: 1242 – 1245, 2004
  • 26. Thalassaemia array Chan K et al, BJH 124: 232 – 239, 2004
  • 27. Thalassaemia array
  • 28. β-thalassaemia phenotypes β-thalassaemia trait • Aymptomatic • Hypochromic microcytic red cells • High HbA2 • Variable ↑ HbF • Genotype: simple heterozygotes for β-thalassaemia alleles
  • 29. β-thalassaemia phenotypes β-thalassaemia major • Onset < 1 year • Transfusion dependent • Many complications • Markedly HcMc RBC • Nucleated reds • Majority HbF • Genotypes: homozygous or compound heterozygous for β-thalassaemia alleles
  • 30. β-thalassaemia syndromes
  • 31. Defining disease severity • Age at diagnosis • Steady state or lowest haemoglobin level • Age at first transfusion • Frequency of transfusion • Splenomegaly or age at splenectomy • Height and weight in percentile
  • 32. Why study genotype phenotype relationship? • Genetic counselling • Management decisions
  • 33. Genetic factors affecting disease severity • Nature and severity of β-globin mutation • Co-inheritance of α-thalassaemia or triplicated α-globin genes • Genetic determinant(s) for enhanced γ-globin chain production
  • 34. Mutation detection by dot blot hybridization
  • 35. Detection of five β-thalassaemia mutations by ARMS 1 2 3 4 5 6 7 8 Panel 1: 1-6 1: -28 Heterozygote 2: -28/71-72 Compound Heterozygote 3: Codon 17 Heterozygote 4: Codon 43 Heterozygote 5: 100 bp DNA Ladder 6: Reagent Blank Control Panel 2: 7-8 7: IVS 2-654 Heterozygote 8: Reagent Blank Control Internal control -28 17 43 71-72 654 Internal control
  • 36. Southern blot hybridization with α-probe
  • 37. PCR-based mutation detection α-multiplex PCR δβ-thalassaemia PCR
  • 38. The spectrum of β-thalassaemia alleles in Chinese
  • 39. Genotype phenotype correlation in β0 /β0 thalassaemia
  • 40. Genotype phenotype correlation in β0 /β+ thalassaemia
  • 41. Homozygous β0 /β0 and compound heterozygous β0 /β+ thalassaemia
  • 42. Clinical phenotype of β+ /β+ thalassaemia
  • 43. Clinical phenotype of HbE / β-thalassemia
  • 44. Molecular pathology of β-thalassaemia
  • 45. Thalassaemia intermedia: family study 1
  • 46. Thalassaemia intermedia: family study 2
  • 47. Thalassaemia screening using MCV and MCH cutoff
  • 48. Co-inheritance of α-thalassaemia determinants significantly ameliorates the phenotype of severe β-thalassaemia Yes β0 /β0 homozygotes + two α-globin gene deletion or non-deletional α2-globin gene mutation β+ -thalassaemia homozygotes or compound heterozygotes + single α-globin gene deletion No β0 /β0 homozygotes + single α-globin gene deletion
  • 49. Co-inheritance of α-thalassaemia determinants significantly ameliorates the phenotype of severe β-thalassaemia Points to note: • Molecular heterogeneity of α-thalassaemia and β-thalassaemia alleles results in wide range of clinical outcomes • Small numbers of patients in each category • Variations among different populations (e.g. in Thai patients α-thalassaemia ameliorates severe β-thalassaemia only in the presence of at least one β+ -thalassaemia allele)
  • 50. Co-inheritance of α-thalassaemia in severe β-thalassaemia
  • 51. Co-inheritance of α-thalassaemia in severe β-thalassaemia
  • 52. Co-inheritance of α-thalassaemia in severe β-thalassaemia Conclusion The co-inheritance of (--SEA ) α-thalassaemia (SEA) deletion ameliorates the clinical phenotype of β0 /β+ but not necessarily β0 /β0 -thalassaemia in Chinese patients
  • 53. Co-inheritance of α-thalassaemia in severe β-thalassaemia Implications 1. Detection of SEA deletion in couples at risk of offspring affected by β0 /β+ -thalassaemia (~ 8 / year) 2. At prenatal diagnosis, a genotype of β0 /β+ -thalassaemia + SEA deletion is predictive of thalassaemia intermedia, but the same cannot be said for β0 /β+ -thalassaemia alone or β0 /β0 -thalassaemia + SEA deletion
  • 54. Triplicated α-globin gene in β-thalassaemia heterozygotes • Observed in 15% of thalassaemia intermedia, not seen in thalassaemia major • Presentation in adulthood • May also be associated with a phenotype of thalassaemia trait
  • 55. Triplicated α-globin gene in β-thalassaemia heterozygotes
  • 56. Triplicated α-globin gene in β-thalassaemia heterozygotes • Distinction from simple β-thalassaemia heterozygotes – Presence of red cell abnormalities – Circulating normoblasts – More anaemic – Higher HbF levels • Explain the inheritance of families in which only one parent is thalassaemic
  • 57. Triplicated α-globin gene in β-thalassaemia heterozygotes
  • 58. Genetic basis for phenotypic variation in the Chinese • Severity of β-thalassaemia mutation β0 /β0 severe β0 /β+ 2/3 severe; 1/3 intermedia β0 /β+++ intermedia β+ /β+ intermedia (mild) • Concurrent α-thalassaemia SEA deletion ameliorate β0 /β+ only but not necessarily β0 /β0 • Triplicated α-globin gene in β-thalassaemia heterozygotes Often associated with thalassaemia intermedia phenotype
  • 59. Genetic basis for phenotypic variation in the Chinese • Determinants of HbF production – XMnI G γ-promoter polymorphism: inconsistent effect – Familial determinants of high HbF remains to be defined
  • 60. Effect of XMnI G γ-promoter polymorphism
  • 61. Genotype phenotype correlation in β0 /β0 thalassaemia
  • 62. Genetic determinants of high HbF
  • 63. Genetic determinants of high HbF A γβ+ -HPFH: nt -196 C→T Subject Sex/Age Hb (g/dL) MCV (fL) MCH (pg) HbA2 (%) HbF (%) HbH bodies α-genotype β-genotype Index F/42 8.2 61.3 21.8 4.5 34.9 Negative ζζζαα/ζζαα β41/42(-CTTT) /βA Elder brother 1 M/52 11.8 59.9 20.3 5.8 0.8 Negative ζζζαα/ζζαα β41/42(-CTTT) /βA Elder brother 2 M/46 11.4 58.3 19.2 4.8 45.3 Negative ζζζαα/ζζαα β41/42(-CTTT) /βA Elder Sister F/48 12.6 91.5 29.9 2.2 13.3 Negative ζζαα/ζζαα βA / βA Daugther F/13 10.5 60.2 19.6 5.7 0.8 Negative ζζαα/ζζαα β41/42(-CTTT) /βA Son of elder brother 2 M/12 12.3 62.3 18.3 5.6 1.5 Negative ζζαα/ζζαα β41/42(-CTTT) /βA Note: All subjects are negative for XmnI G γ-polymorphism
  • 64. Genetic modifiers of single gene disorders Primary modifiers Secondary modifiers Tertiary modifiers
  • 65. Hyperbilirubinaemia Jaundice Gall stones
  • 66. UGT1A1 mutations and hyperbilirubinaemia • Uridine-diphosphoglucuronate glucuronosyltransferase – UGT1 gene : 12 isoforms with alternative first exons – UGT1A1 contributes most significantly to bilirubin glucuronidation – Mutations in coding region and promoter
  • 67. UGT1A1 alleles in Chinese Hsieh S-Y et al, Am J Gastroenterol 96: 1188 - 1193, 2001
  • 68. Detection of UGT1A1 polymorphisms • UGT1A1 promoter genotype – direct sequencing of PCR product • Gly71Arg mutation at exon 1 – PCR restriction analysis of MspI cleavage site
  • 69. M A B C 143b p119b p 24bp 143bp 119bp 24bp M W h W W W W H h h W W H Homozgyous (TA)6 Homozgyous (TA)7 Heterozgyous (TA)6/(TA)7
  • 70. Prevalence of UGT1A1 polymorphisms (TA)7 = 25 cases (19.6%); G71R = 34 cases (26.8%) Major Intermedia (TA)7 homozygous 0 2 (TA)7 heterozygous 14 (2) 9 (1) G71R homozygous 4 2 G71R heterozygous 24 (2) 4 (1)
  • 71. Predictors of bilirubin level
  • 72. Predictors of gall stones
  • 73. Genetic haemochromatosis and iron overload in β-thalassaemia • Homozygosity for HFE alleles C282Y and H63D – predisposes to iron overload in β-thalassaemia • Prevalence in Chinese patient cohort Allele Frequency C282Y 0% H63D 1.3% S65C 0%
  • 74. Transferrin receptor-2 (TFR2) mutations and iron overload • Homologue of transferrin receptor with 48% identity and 66% similarity • Common affinity for diferric transferrin • Lack of affinity for HFE protein
  • 75. Transferrin receptor-2 (TFR2) polymorphisms • Allelic frequency Polymorphism Patients Control p-value exon 5 I238M 7.1% 4.7% 0.24 IVS16+251 -CA 24.5% 22.2% 0.54
  • 76. TFR2 polymorphism and iron overload in transfusion independent β-thalassaemia intermedia
  • 77. Genetics of osteoporosis in thalassaemia • Heterozygous (Ss) or homozygous (ss) polymorphism of COLIA1 gene: ↓ BMD – Perrotta et al, Br J Haematol 111: 461, 2000 • VDR BB genotype: ↓ spine BMD than bb genotype – Dresner Pollak et al, Br J Haematol 111: 902, 2000 • VDR FF genotype: shorter stature and ↓ BMD – Ferrara et al, Br J Haematol 117: 436, 2002
  • 78. Conclusions • Disease severity explainable by nature of β-thalassaemia mutation and interacting α-thalassaemia • Problem of discordant phenotype in β0 /β+ • Genetic modifiers may play in role in modulating phenotype (especially complications)

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