1. 1Hemoglobinopathies Dr Pupak Derakhshandeh, PhD Ass Prof of Medical Science of Tehran University

2. 2Hemoglobinopathies Disorders of Hemoglobin

3. 3 5 % of world population: carrier for genes, important disorders of hemoglobin

4. 4 Structure and function of hemoglobin  Oxygen carrier  In vertebrate: red blood cells  Four subunits:  2α- and 2  -chains

5. 5Hemoglobin

6. 6 Each Subunits  Globin: Polypeptide chain  Heme : Prosthetic group (Iron-Containing pigment) Heme + Oxygene Oxygene transporting

7. 7 Normal adult hemoglobin HbA:  2 α globin chain (141 AA)  2  globin chain (146 AA)  α22 α22 α22 α22  Equal length

8. 8 Normal adult hemoglobin HbA2:  2 α globin chain  2  globin chain     

9. 9 Normal adult hemoglobin HbF:  2 α globin chain  2 γ globin chain  α2γ2 α2γ2 α2γ2 α2γ2

10. 10 Normal adult hemoglobin

11. 11 Globin genes synthesis Lessons from the thalasemia. Nature Reviews, Genetics, volume2, 2001

12. 12Thalassemia  Onset: Childhood  Hypo chromic / Microcytic anemia  Low level of MCV / MCH Mean corpuscular volume (MCV) Mean corpuscular hemoglobin (MCH)   -Thal: Elevated HbA 2 ( α 2  2 ) HbF ( α 2 γ 2 ) HbF ( α 2 γ 2 )  α -Thal: Normal HbA 2, HbF

13. 13

14. 14 Thalassemia Minor

15. 15  Thalassemia major

16. 16  Thalassemia major

17. 17 Thalassemia Minor Thalassemia minor is an inherited form of hemolytic anemia that is less severe than thalassemia major.Thalassemia minor is an inherited form of hemolytic anemia that is less severe than thalassemia major. This blood smear from an individual with thalassemia shows small (microcytic), pale (hypochromic), variously-shaped red blood cells.This blood smear from an individual with thalassemia shows small (microcytic), pale (hypochromic), variously-shaped red blood cells. These small red blood cells (RBCs) are able to carry less oxygen than normal RBCs.These small red blood cells (RBCs) are able to carry less oxygen than normal RBCs.

18. 18 Hematological values & a-Thalassemia Molecular diagnosis of hemoglobin disorders, Clin. Lab. Haem. 2004, 26, 159–176     

19. 19 Thalassemia Major

20. 20 an inherited form of hemolytic anemia red blood cell (hemoglobin) abnormalities the most severe form of anemia the oxygen depletion in the body becomes apparent within the first 6 months of life Thalassemia major

21. 21 If untreated, death usually results within a few years Note the small, pale (hypochromic), abnormally-shaped red blood cells associated with thalassemia major The darker cells likely represent normal RBCs from a blood transfusion

22. 22 Diesease  Autosomal recessive  Deficiency: Synthesis of α/  - globin  Origin: Mediteranean, African, Iranian, Indian, Southeast Asian  Resistant to malaria

23. 23 Prevalence of  -Thalassemia  ~ 1.5 % in Africans and African Americans  ~ 30 % in Sardinia

24. 24 Pathogenesis of  -Thalassemia  In adequate Hb production  Reduced MCV/MCH  Unbalanced accumulation of  globin subunits  Ineffective Erythrocyt  200 different mutations  In Iran over 70 mutations !

25. 25 Prenatal diagnosis  I. ARMS-PCR (22 common mut.)  II. PCR-RFLP (9 inf. RFLPs)  III. RDB (60 mut.)  IV. Sequencing

26. 26 ARMS-PCR

27. 27 PCR-RFLP 1 2 3 M 4 5 6 7

28. 28  globin mutations 1.Transcriptional mutations (  + )  In promotor regulatory elements  -101(silent)  -92 (silent)  -88  -30

29. 29  globin mutations 2. RNA-Processing (  º )  Splice junction  IVSI-1 Cd30  IVSI-2  IVSI-3’ end del 25bp  IvsI-130  Consensus splice sites (  º/ + )  IVSI-5  IVSI-6  IVSII-844

30. 30  globin mutations  Cryptic splice sites in Introns (  + )  IVSI-110  IVSII-745  Cryptic splice sites in exons  Cd 26 (HbE)  Cd 121 (HbD panjab/O Arab)

31. 31  -Thalassemia major  Onset: 6 months  Severe hemolytic anemia  Hb level< 7 g/dl  Skin: pale  Growth retardation  don’t eat or sleep well  Hepatosplenomegaly  Bone marrow expansion:  Make more red cells  Expantion in face and skull  Spleen: destroy of young red cell  80% of untreated patients: † by 5 y.  Treatment: Cardiac/Hepatic: † by 30 y.  Transfusion +Chelation > 30y.

32. 32  -Thalassemia

33. 33 Peripheral Blood Smear (1) Normochrome Normocyte MCV  MCH  MCV  MCH 

34. 34 Peripheral Blood Smear (2) Hypochrome Microcyte MCVMCHMCVMCH

35. 35 Defected globin chains

36. 36 Prevalence of α-Thalassemia  0.01 % in non malarial areas ig. UK, Japan ig. UK, Japan  ~ 49 % in Southwest Pacific Islands

37. 37

38. 38 α globin mutations  Deletions: 80-85 % of αThalassemia  Del: 3.7 kb (most frequent)  Del: 4.2 kb  α 2 InsI-5bp deletion (α Hph1 α)  α 2 InCd T>C (α Nco1 α)  α º Variant:  --MED  --CAL  --SEA

39. 39  -Thalassemia Trait -  –Hemoglobin is with in the reference range. –Reticulocyte count is within the reference range. –Mean corpuscular volume (MCV) is 75-85 fL. –Mean corpuscular hemoglobin (MCH) is 26 pg.

40. 40 a-Thalassemia Alpha1 thalassemia minor (--/  ) –Hemoglobin is within the reference range. –Reticulocyte count is within the reference range. –MCV is 65-75 fL. –MCH is 22 pg.

41. 41 Hemoglobin H disease Peripheral smear from a patient with hemoglobin H disease showing target cells, microcytosis and hypochromia. Morphological abnormalities are similar to those observed in beta thalassemia. In alpha2 thalassemia (silent trait) only mild microcytosis is observed.

42. 42 HbH disease Hemoglobin H disease –Hemoglobin is 7-10 g/dL. –Reticulocyte count is 5-10%. –MCV is 55-65 fL. –MCH is 20 pg. –The peripheral blood smear shows small misshapen red cells, hypochromia, microcytosis, and targeting. –Brilliant cresyl blue stain demonstrates hemoglobin H inclusion bodies.

43. 43 HbH disease  Functional α globin : 1  α:  globin ratio : 0.3  Hb level: 7-9 g/dl  Genotype: --/-α  HbH Inclusion (Heinz body): Many  Moderate anemia  Hepatosplenomegaly  Galstones, infection, folic acid deficiency

44. 44 Hydrops fetalis –Hemoglobin is 4-10 g/dL. –MCV is 110-120 fL. –The peripheral blood smear shows severe hypochromia, and nucleated red blood cells.

45. 45 Hydrops fetalis  Functional α globin : 0  α:  globin ratio : 0.0  Genotype: --/--  HbH Inclusion (Heinz body): Present  Severe anemia  Heart defect/fatal in utero/ shortly after birth

46. 46 haemoglobinopathies Reduced synthesis of globin chains (Thalassaemia) Synthesis of a structurally abnormal Hb variant Thalassaemia β-thalassemia ( over 200 point mutations) a-thalassemia (over 80 deletions & point mutations)

47. 47 a-globin genes cluster Chromosome16p13.3 Blood,Vol 91, No 7 (April 1), 1998: pp 2213-2222 Exon IExon IIExon III Intron IIntron II

48. 48 Alpha-Thalassemia inheritance AutosomalrecessiveAutosomalrecessive

49. 49 1) alpha-globin gene deletions approximately 90% of mutations approximately 90% of mutations 2) alpha-globin point mutations approximately 10% of mutations approximately 10% of mutations  -Thalassemia mutations

50. 50 a-thalassemias phenotype  + or  -thalassemia 2   + or  -thalassemia 2 - non-functional one  -globin gene (-  ) - non-functional one  -globin gene (-  )   o or  - thalassemia 1 - non-functional both  -globin genes - non-functional both  -globin genes (--)

51. 51 a + -Thalassemia Archives of Iranian Medicine, Vol 4, No 4, October 2001 Rightward Leftward

52. 52 a 0 -Thalassemia Blood,Vol 91, No 7 (April 1), 1998: pp 2213-2222 > 300 kb Southeast Asia Philippines Thailand Chinese Mediterranean Greece, Turkey ~ 30 kb ~ 20 kb ~ 23 kb (cd51  1)

53. 53 a-globin genes affected production a-globin chains GenotypeOutcome 0100%  Normal, healthy 175%  Silent carrier, healthy 250%  or  Carrier, a-thalassaemia trait, Mild hypochromic microcytic anemia 325% --/-  Hemoglobin H disease, mild to severe hemolytic anemia 40% - -/- - Hemoglobin Bart's, hydrops fetalis Haemoglobinopathies and clotting disorders. Vol. 36, No. 10, October 2007 Outcome of deletions a-globin genes

54. 54 HbH Disease

55. 55 Method detection a-Thalassemia mutations Salting out DNA extraction - human whole blood - human whole blood PCR-Based Strategies - Gap-PCR - Gap-PCR - Multiplex Gap-PCR - Multiplex Gap-PCR DNA Sequencing

56. 56 Gap-PCRGap-PCR Molecular diagnosis of hemoglobin disorders, Clin. Lab. Haem. 2004, 26, 159–176

57. 57 Primers Multiplex PCR (2) Chong SS, et al. Single tube multiplex-PCR screen for common deletional determinants Of a-thalassemia. Blood 2000;95:360–362.

58. 58 Primers DNA Sequencing NameSequence 5'-3' 1 M13S 13 5´ - CGA CGT TGT AAA ACG ACG GCC AGT CGC CAG CCA ATG AGC GCC - 3´ 2S6 5´ - TCC ATT GTT GGC ACA TTC CG - 3´ 3S8 5´ - TGT CCA CGC CCA TGC TGG CAC - 3´ American Journal of Hematology 74:99–103 (2003)

59. 59 Hba1 Sequence

60. 60 Hba2 Sequence

61. 61 Gap-PCR

62. 62 MED Mutation 1. Positive control 2. Marker 200bp 3. Negative control TimeTemperatureCycle 5 minute941 30 second94 35 30 second60 120 second72 7 minute721

63. 63 Multiplex PCR

64. 64 Multiplex PCR TimeTemperatureCycle 5 minute941 60 second94 35 60 second60 120 second72 7 minute721 1) Smart taq DNA Polymerase 2) AMS Buffer 3) DMSO (5-10%) 4) Mgcl2 (50 mM) (2 mM) 5) dNTP (10mM) (0.2 mM) 6) Primers (10pm) (0.3 µl) 7) ddH20 8) Template

65. 65 Result

66. 66 Multiplex PCR (1)

67. 67 Multiplex PCR (2)

68. 68 Multiplex PCR (3)

69. 69 Multiplex PCR (4)

70. 70 Patient samples Male RBC : 5.81 MCV :75.9 MCH :25.6 HbA2 :2.6 Female RBC : 5.41 MCV :75.8 MCH :25.0 HbA2 :2.7

71. 71 Patient sample HbH -a 3.7 /-(a) 20.5 -a 3.7 /-(a) 20.5

72. 72 Mutations in Khorasan province

73. 73 Sequencing Khorasan province -  2: 7 sample (4 point mutation) -  1: 7 sample (2 point mutation) Khoozestan province -  2: 4 sample (4 point mutation) -  1: 7 sample ( all normal) 950 bp PCR Product  S8 Primer  2 S6 Primer

74. 74 Normal Seq. AATAAA Mutant Seq. AAAAAA

75. 75 Normal  2 IVSII-55 CGC>CTC

76. 76 Normal Normal  2 IVS II-24 TCT>TTT

77. 77 Khorasan & Khoozestan province RBCHbHctMCVMCH HbA 2  5.53 + 0.51 13.52 + 1.45 41.73 + 3.61 73.96 + 6.09 24.48 + 2.23 2.96 + 1.21  MED  000000    5.68 + 0.43 14.07 + 1.14 42.98 + 3.49 75.45 + 5.23 25.31 + 1.25 2.66 + 0.5     5.74 + 0.56 13.1 + 0.94 41.25 + 3.97 71.9 + 2.89 22.9 + 1.54 2.83 + 1.57     5.81 + 0.64 13.97 + 1.57 42.23 + 3.75 71.2 + 2.38 23.47 + 0.76 2.37 + 0.55    5.73 + 0.7 14.33 + 1.52 43.45 + 2.78 75.9 + 4.49 24.95 + 0.59 2.28 + 0.56    000000

78. 78 Relationship of MCV to mutations

79. 79 Relationship of MCH to mutations

80. 80 Treatment  Blood transfusion (3-4 weeks for life)  Iron accumulation in body  Remove the iron: Desferal:  Infused under the skin (8-12 h/6 times a week)  Bone marrow transplantation  A sib brother or sister  HLA matched

81. 81Treatment Avoid iron supplementation. It contributes to iron overload Administer folate supplementation to provide adequate amounts of the vitamin for increased utilization resulting from the hemolytic process and high bone marrow turnover rate. Provide prompt attention to infection, especially in children who have had a splenectomy. Administer blood transfusions only if necessary. If chronic transfusion is needed (hemoglobin H disease), iron chelation therapy should be considered to avoid iron overloading.

82. 82 Surgical Care Hemoglobin H disease –Perform a splenectomy if transfusion requirements are increasing. –Surgical or orthodontic correction may be necessary to correct skeletal deformities of the skull and maxilla due to erythroid hyperplasia.

83. 83 Sickle Cell disorder

84. 84 Sickle Cell disorder  Stuck the red cell in the vessels  In children: Spleen, chest, wrists,ankles  In adults: hips and shoulders  Anemia (Hb 7-8 g/dl)  Infections (take antibiotics)  Painful crises (6-18 months)  Swollen and inflamed (hand/food syndrome)

85. 85 What are the Complications? pain episodes increased infections bone damage yellow eyes or jaundice early gallstones lung blockage kidney damage and loss of body water in urine painful erections in men (priapism) blood blockage in the spleen or liver (sequestration) eye damage low red blood cell counts (anemia) delayed growth

86. 86 The combination of hemoglobinopathies Doesn't cause any health problem:  α + Thalassemia / α + Thalassemia (-a/-a) HbH disease:  α º Thalassemia / α + Thalassemia (--/-a) Hydrops fetalis:  α º Thalassemia / α º Thalassemia (--/--)

87. 87 Doesn't cause any health problem  α + / º Thalassemia/  Thalassemia  α + / º Thalassemia / HbC  α + / º Thalassemia / HbD  α + / º Thalassemia / HbE  α + / º Thalassemia / HbO Arab  α + / º Thalassemia / HbS

88. 88  Thalassemia /  Thalassemia Caused severe health problem!

89. 89 Other combinations  HbC /  Thalassemia (no problem)  HbD /  Thalassemia (no problem)  HbE /  Thalassemia (serious anemia)  Hbs /  Thalassemia (intermediate-severe)  HPFH* /  Thalassemia (no problem) *Heriditary persistance of fetal hemoglobin

90. 90 Doesn't cause any health problem  HbC / HbC  HbC / D, E, O Arab, HPFH  HbD / HbD  HbD / C, E, O Arab, HPFH  HbE / HbE  HbE / C, D, O Arab, HPFH

91. 91 Doesn't cause any health problem  HbO Arab / HbO Arab  HbO Arab/ C, E, D, HPFH  HPFH / HPFH  HbH /  Thalassemia !   Thalassemia major/α +/º Thalassemia!   Thalassemia major / HbC, D

92. 92 serious anemia  HbH / α +/º Thalassemia  HbS /  Thalassemia  HbS / HbC  HbS / HbD  HbS / HbE  HbS / O Arab

93. 93 Prenatal Diagnosis (PND)

94. 94 ThankS for Your Attention Attention