Molecular Haematology I Globin Disorders

1. Molecular Haematology IGlobin 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 qalassa (thalassa) meaning the sea

3. Prevalence of thalassaemia in Hong Kong Chinese a-thalassaemia 5% b-thalassaemia 3.1%

4. Prevalence of thalassaemia in Hong Kong Chinese a-thalassaemia (--SEA) a-thalassaemia deletion 90% b-thalassaemia codons 41-42 (-CTTT) b0 45% IVSII-654 (CT) b0 20% nt-28 (AG) b+ 16% codon 17 (AT) b0 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 z-globin chains in adults

12. Deletional (common) --SEA -a3.7 -a4.2 Non-deletional (rare) Hb CS Hb QS codon 30 deletion Hb Q-Thailand Hb Westmead a2 codon 31 a2 codon 59 Others a-globin gene mutations

13. Prevalence of thalassaemia in Hong Kong Chinese (MCV < 80 fL) a-thalassaemia (--SEA) a-thalassaemia deletion 90%

14. Single a-globin gene deletion and triplicated a-globin gene • Prevalence • 6% for –a3.7 and –a4.2 • 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 aaaanti-3.7 and aaaanti-4.2 • Hb 13.5 g/dL, MCV 85.5 fL, MCH 28.7 pg

15. Single a-globin gene deletion (-a) and triplicated a-globin gene (aaa) configuration

16. Molecular diagnosis of a-thalassaemiaClark & Thein, Clin Lab Haematol 26: 159-76; 2004 • Deletions • Gap PCR • Southern blotting • Non-deletional mutation: on specifically amplified a2 or a1 genes • Restriction digest • ARMS-PCR • ASO • Direct sequence analysis

17. Multiplex PCR for 3 commonest a-thalassaemia deletion LIS1 control 3.7 kb deletion a-2 gene 4.2 kb deletion SEA deletion

18. Multiplex PCR for 3 commonest a-thalassaemia deletion water αα/--SEA ladder -α3.7/--SEA -α4.2/--SEA αα/αα blank LIS internal control (2350 bp) -α3.7 (2022/2029 bp) α2 (1800 bp) -α4.2 (1628 bp) --SEA (1349 bp)

19. Larger fragment Gel Smaller fragment Key restriction enzyme sites probe region Restriction fragment length polymorphism (RFLP) The principle of RFLP as shown is used to diagnose the different types of -globin genotypes relevant to -thalassaemia.

22. 16kb 14.5kb 12.6 kb 10.5 kb 7.0 kb

23. Multiplex ARMS for the 3 commonest non-deletional a2-globin gene mutations Internal control (930 bp) cd30(ΔGAG) (772 bp) HbQS (234 bp) HbCS (184 bp)

26. Reverse dot blot Chan V et al, BJH 104: 513-5, 1999

27. Multiplex mini-sequencing screen Wang W et al, Clin Chem 49: 800 – 803, 2003

30. Molecular screening of non-deletional a-globin gene mutations by denaturing HPLC Guida V et al, Clin Chem 50: 1242 – 1245, 2004

31. Thalassaemia array Chan K et al, BJH 124: 232 – 239, 2004

32. Thalassaemia array

33. b-thalassaemia phenotypes b-thalassaemia trait • Aymptomatic • Hypochromic microcytic red cells • High HbA2 • Variable  HbF • Genotype: simple heterozygotes for b-thalassaemia alleles

34. b-thalassaemia phenotypes b-thalassaemia major • Onset < 1 year • Transfusion dependent • Many complications • Markedly HcMc RBC • Nucleated reds • Majority HbF • Genotypes: homozygous or compound heterozygous for b-thalassaemia alleles

35. b-thalassaemia syndromes

36. 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

37. Why study genotype phenotype relationship? • Genetic counselling • Management decisions

38. Genetic factors affecting disease severity • Nature and severity of b-globin mutation • Co-inheritance of a-thalassaemia or triplicated a-globin genes • Genetic determinant(s) for enhanced g-globin chain production

40. Mutation detection by dot blot hybridization

41. Detection of five -thalassaemia mutations by ARMS 1 2 3 4 5 6 7 8 Internal control -28 17 43 71-72 654 Internal control 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 BlankControl

42. Southern blot hybridization with a-probe

43. PCR-based mutation detection a-multiplex PCR db-thalassaemia PCR

44. The spectrum of b-thalassaemia alleles in Chinese

45. Genotype phenotype correlation in b0/b0 thalassaemia

46. Genotype phenotype correlation in b0/b+ thalassaemia

47. Homozygous b0/b0 and compound heterozygous b0/b+ thalassaemia

48. Clinical phenotype of b+/b+ thalassaemia

49. Clinical phenotype of HbE / b-thalassemia

50. Molecular pathology of b-thalassaemia