Departmental seminar Genetic determinants of b-thalassaemia phenotype

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Departmental seminar Genetic determinants of b-thalassaemia phenotype

  1. 1. Molecular Haematology I Globin Disorders Dr Edmond S K Ma Division of Haematology Department of Pathology The University of Hong Kong
  2. 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. 3. Prevalence of thalassaemia in Hong Kong Chinese α-thalassaemia 5% β-thalassaemia 3.1%
  4. 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. 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. 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. 7. Laboratory diagnosis of thalassaemia by HPLC
  8. 8. Haemoglobin electrophoresis
  9. 9. Detection of HbH inclusion bodies
  10. 10. New approaches in diagnosis of SEA deletion: gap-PCR for SEA deletion
  11. 11. New approaches in diagnosis of SEA deletion: detection of ζ-globin chains in adults
  12. 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. 13. Prevalence of thalassaemia in Hong Kong Chinese (MCV < 80 fL) α-thalassaemia (--SEA ) α-thalassaemia deletion 90%
  14. 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. 15. Single α-globin gene deletion (-α) and triplicated α-globin gene (ααα) configuration
  16. 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. 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. 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. 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. 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. 21. 16kb 10.5 kb 14.5kb 12.6 kb 7.0 kb
  22. 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. 23. Reverse dot blot Chan V et al, BJH 104: 513-5, 1999
  24. 24. Multiplex mini-sequencing screen Wang W et al, Clin Chem 49: 800 – 803, 2003
  25. 25. Molecular screening of non-deletional α-globin gene mutations by denaturing HPLC Guida V et al, Clin Chem 50: 1242 – 1245, 2004
  26. 26. Thalassaemia array Chan K et al, BJH 124: 232 – 239, 2004
  27. 27. Thalassaemia array
  28. 28. β-thalassaemia phenotypes β-thalassaemia trait • Aymptomatic • Hypochromic microcytic red cells • High HbA2 • Variable ↑ HbF • Genotype: simple heterozygotes for β-thalassaemia alleles
  29. 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. 30. β-thalassaemia syndromes
  31. 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. 32. Why study genotype phenotype relationship? • Genetic counselling • Management decisions
  33. 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. 34. Mutation detection by dot blot hybridization
  35. 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. 36. Southern blot hybridization with α-probe
  37. 37. PCR-based mutation detection α-multiplex PCR δβ-thalassaemia PCR
  38. 38. The spectrum of β-thalassaemia alleles in Chinese
  39. 39. Genotype phenotype correlation in β0 /β0 thalassaemia
  40. 40. Genotype phenotype correlation in β0 /β+ thalassaemia
  41. 41. Homozygous β0 /β0 and compound heterozygous β0 /β+ thalassaemia
  42. 42. Clinical phenotype of β+ /β+ thalassaemia
  43. 43. Clinical phenotype of HbE / β-thalassemia
  44. 44. Molecular pathology of β-thalassaemia
  45. 45. Thalassaemia intermedia: family study 1
  46. 46. Thalassaemia intermedia: family study 2
  47. 47. Thalassaemia screening using MCV and MCH cutoff
  48. 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. 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. 50. Co-inheritance of α-thalassaemia in severe β-thalassaemia
  51. 51. Co-inheritance of α-thalassaemia in severe β-thalassaemia
  52. 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. 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. 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. 55. Triplicated α-globin gene in β-thalassaemia heterozygotes
  56. 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. 57. Triplicated α-globin gene in β-thalassaemia heterozygotes
  58. 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. 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. 60. Effect of XMnI G γ-promoter polymorphism
  61. 61. Genotype phenotype correlation in β0 /β0 thalassaemia
  62. 62. Genetic determinants of high HbF
  63. 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. 64. Genetic modifiers of single gene disorders Primary modifiers Secondary modifiers Tertiary modifiers
  65. 65. Hyperbilirubinaemia Jaundice Gall stones
  66. 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. 67. UGT1A1 alleles in Chinese Hsieh S-Y et al, Am J Gastroenterol 96: 1188 - 1193, 2001
  68. 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. 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. 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. 71. Predictors of bilirubin level
  72. 72. Predictors of gall stones
  73. 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. 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. 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. 76. TFR2 polymorphism and iron overload in transfusion independent β-thalassaemia intermedia
  77. 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. 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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