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  (thalassa) meaning the sea

3. Prevalence of thalassaemia in Hong Kong Chinese  -thalassaemia5%  -thalassaemia3.1%

4. Prevalence of thalassaemia in Hong Kong Chinese  -thalassaemia (-- SEA )  -thalassaemia deletion90%  -thalassaemia codons 41-42 (-CTTT)  0 45% IVSII-654 (C  T)  0 20% nt-28 (A  G)  + 16% codon 17 (A  T)  0 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  -globin chains in adults

12.  -globin gene mutations Deletional (common) -- SEA -  3.7 -  4.2 Non-deletional (rare) Hb CS Hb QS codon 30 deletion Hb Q-Thailand Hb Westmead  2 codon 31  2 codon 59 Others

13. Prevalence of thalassaemia in Hong Kong Chinese (MCV < 80 fL)  -thalassaemia (-- SEA )  -thalassaemia deletion90%

14. Single  -globin gene deletion and triplicated  -globin gene Prevalence –6% for –  3.7 and –  4.2 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  anti-3.7 and  anti-4.2 Hb 13.5 g/dL, MCV 85.5 fL, MCH 28.7 pg

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

16. Molecular diagnosis of  -thalassaemia Clark & Thein, Clin Lab Haematol 26: 159-76; 2004 Deletions –Gap PCR –Southern blotting Non-deletional mutation: on specifically amplified  2 or  1 genes –Restriction digest –ARMS-PCR –ASO –Direct sequence analysis

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

18. LIS internal control (2350 bp) -α 3.7 (2022/2029 bp ) α2 (1800 bp) -α 4.2 (1628 bp) -- SEA (1349 bp) water αα/-- SEA ladder -α 3.7 /-- SEA -α 4.2 /-- SEA αα/αα blank Multiplex PCR for 3 commonest  -thalassaemia deletion

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

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

23. Multiplex ARMS for the 3 commonest non-deletional  2-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  -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.  -thalassaemia phenotypes  -thalassaemia trait Aymptomatic Hypochromic microcytic red cells High HbA 2 Variable  HbF Genotype: simple heterozygotes for  -thalassaemia alleles

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

35.  -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  -globin mutation Co-inheritance of  -thalassaemia or triplicated  -globin genes Genetic determinant(s) for enhanced  -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 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

42. Southern blot hybridization with  -probe

43. PCR-based mutation detection  -multiplex PCR  -thalassaemia PCR

44. The spectrum of  -thalassaemia alleles in Chinese

45. Genotype phenotype correlation in  0 /  0 thalassaemia

46. Genotype phenotype correlation in  0 /  + thalassaemia

47. Homozygous  0 /  0 and compound heterozygous  0 /  + thalassaemia

48. Clinical phenotype of  + /  + thalassaemia

49. Clinical phenotype of HbE /  -thalassemia

50. Molecular pathology of  -thalassaemia

51. Thalassaemia intermedia: family study 1

52. Thalassaemia intermedia: family study 2

53. Thalassaemia screening using MCV and MCH cutoff

54. Co-inheritance of  -thalassaemia determinants significantly ameliorates the phenotype of severe  -thalassaemia Yes  0 /  0 homozygotes + two  -globin gene deletion or non-deletional  -globin gene mutation  + -thalassaemia homozygotes or compound heterozygotes  single  -globin gene deletion No  0 /  0 homozygotes + single  -globin gene deletion

55. Co-inheritance of  -thalassaemia determinants significantly ameliorates the phenotype of severe  -thalassaemia Points to note: Molecular heterogeneity of  -thalassaemia and  -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  -thalassaemia ameliorates severe  -thalassaemia only in the presence of at least one   -thalassaemia allele)

56. Co-inheritance of  -thalassaemia in severe  -thalassaemia

58. Conclusion The co-inheritance of (-- SEA )  -thalassaemia (SEA) deletion ameliorates the clinical phenotype of  0 /  + but not necessarily  0 /  0 -thalassaemia in Chinese patients

59. Co-inheritance of  -thalassaemia in severe  -thalassaemia Implications 1. Detection of SEA deletion in couples at risk of offspring affected by  0 /  + -thalassaemia (~ 8 / year) 2. At prenatal diagnosis, a genotype of  0 /  + -thalassaemia + SEA deletion is predictive of thalassaemia intermedia, but the same cannot be said for  0 /  + -thalassaemia alone or  0 /  0 -thalassaemia + SEA deletion

60. Triplicated  -globin gene in  -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

61. Triplicated  -globin gene in  -thalassaemia heterozygotes

62. Distinction from simple  -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

63. Triplicated  -globin gene in  -thalassaemia heterozygotes

65. Genetic basis for phenotypic variation in the Chinese Severity of  -thalassaemia mutation  0 /  0 severe  0 /  + 2/3 severe; 1/3 intermedia  0 /  +++ intermedia   /  + intermedia (mild) Concurrent  -thalassaemia SEA deletion ameliorate  0 /  + only but not necessarily  0 /  0 Triplicated  -globin gene in  -thalassaemia heterozygotes Often associated with thalassaemia intermedia phenotype

66. Genetic basis for phenotypic variation in the Chinese Determinants of HbF production –XMnI G  -promoter polymorphism: inconsistent effect –Familial determinants of high HbF remains to be defined

67. Effect of XMnI G  -promoter polymorphism

68. Genotype phenotype correlation in  0 /  0 thalassaemia

69. Genetic determinants of high HbF

70. A   -HPFH: nt -196 C→T SubjectSex/AgeHb (g/dL) MCV (fL) MCH (pg) HbA 2 (%) HbF (%) HbH bodies α-genotypeβ-genotype IndexF/428.261.321.84.534.9Negativeζζζαα/ζζααβ 41/42(-CTTT) /β A Elder brother 1 M/5211.859.920.35.80.8Negativeζζζαα/ζζααβ 41/42(-CTTT) /β A Elder brother 2 M/4611.458.319.24.845.3Negativeζζζαα/ζζααβ 41/42(-CTTT) /β A Elder Sister F/4812.691.529.92.213.3Negativeζζαα/ζζααβ A / β A DaugtherF/1310.560.219.65.70.8Negativeζζαα/ζζααβ 41/42(-CTTT) /β A Son of elder brother 2 M/1212.362.318.35.61.5Negativeζζαα/ζζααβ 41/42(-CTTT) /β A Note: All subjects are negative for XmnI G γ-polymorphism

71. Genetic modifiers of single gene disorders Primary modifiers Secondary modifiers Tertiary modifiers

72. Hyperbilirubinaemia Jaundice Gall stones

73. 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

74. UGT1A1 alleles in Chinese Hsieh S-Y et al, Am J Gastroenterol 96: 1188 - 1193, 2001

75. Detection of UGT1A1 polymorphisms UGT1A1 promoter genotype –direct sequencing of PCR product Gly71Arg mutation at exon 1 –PCR restriction analysis of MspI cleavage site

76. 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

77. Prevalence of UGT1A1 polymorphisms (TA) 7 = 25 cases (19.6%); G71R = 34 cases (26.8%) MajorIntermedia (TA) 7 homozygous 02 (TA) 7 heterozygous14 (2)9 (1) G71R homozygous42 G71R heterozygous24 (2)4 (1)

78. Predictors of bilirubin level

79. Predictors of gall stones

80. Genetic haemochromatosis and iron overload in  -thalassaemia Homozygosity for HFE alleles C282Y and H63D –predisposes to iron overload in  -thalassaemia Prevalence in Chinese patient cohort AlleleFrequency C282Y0% H63D1.3% S65C0%

81. 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

82. Transferrin receptor-2 (TFR2) polymorphisms Allelic frequency PolymorphismPatientsControlp-value exon 5 I238M 7.1%4.7%0.24 IVS16+251 -CA24.5%22.2%0.54

83. TFR2 polymorphism and iron overload in transfusion independent  -thalassaemia intermedia

84. 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

85. Conclusions Disease severity explainable by nature of  -thalassaemia mutation and interacting  -thalassaemia Problem of discordant phenotype in  0 /  + Genetic modifiers may play in role in modulating phenotype (especially complications)