1. Products of haemopoiesis

2. ABNORMALITIES IN THE HEMOPOIETIC SYSTEM CAN LEAD TO HEMOGLOBINOPATHIES HEMOPHILIA DEFECTS IN HEMOSTASIS/THROMBOSIS HEMATOLOGICAL MALIGNANCY

3. MUTATIONS AND DNA VARIOUS TYPES OF MUTATIONS CAN OCCUR LEADING TO DISEASE PHENOTYPE POINT MUTATIONS INSERTIONS OR DELETIONS TRANSLOCATIONS COMPLEX CHROMOSOMAL REARRANGEMENTS

4. Sickle cell disease

6. Thalassemia The thalassemias are a diverse group of genetic blood diseases characterized by absent or decreased production of normal hemoglobin, resulting in a microcytic anemia of varying degree The alpha (  ) thalassemias are concentrated in Southeast Asia, Malaysia, and southern China. The beta (  ) thalassemias are seen primarily in the areas surrounding Mediterranean Sea, Africa and Southeast Asia.

7. The β-like globin chains are controlled by a gene cluster on chromosome 11 in which the different genes are arranged in the order 5’-ε-Gγ-Aγ-ψβ-δ-β-3’. The α-like gene cluster is on chromosome 16, p13.3, and the genes are arranged in the order 5’-ζ-ψζ-ψα2- ψα1-α2-α1-θ-3’.

8. Temporal globin expression

9. Temporal Globin expression  globin expression is rather stable in fetal and adult life, because it is needed for both fetal and adult hemoglobin production  globin appears early in fetal life at low levels and rapidly increases after 30 weeks gestational age, reaching a maximum about 30 weeks postnatally  globin molecule is expressed at a high level in fetal life ( 6 weeks) and begins to decline about 30 weeks gestational age, reaching a low level about 48 weeks postgestational age.  globin appears at a low level at about 30 weeks gestational age and maintains a low profile throughout life.

10. Genetics of Thalassemia

11. Types of Thalassemia  thal: excess of  globins, leading to formation of  globin tetramers (  4 ) that accumulate in the erythroblast, leading to ineffective erythropoiesis. Two types of mutations, the β0 in which no β globin chains are produced and β+, in which some β chains are produced but at a reduced rate.  thal : excess of  globins, leading to the formation of  globin tetramers (  4 ) called hemoglobin H. Results in hemolysis, generally shortening the life span of the red cell. Hemoglobin H-Constant Spring disease is a more severe form of this hemolytic disorder. Most severe form is  thalassemia major, in which fetus produces no  globins, which is generally incompatible with life.

15. Thalassemia Prevention Preventive programs in (i) public education, (ii) population screening, genetic counseling and prenatal diagnosis have been very effective in reducing the birth rate of β- thalassemia major. Combination of hematological and molecular techniques offers the most reliable and accurate strategy for β- thalassemia prenatal diagnosis Development of molecular techniques not only made it possible to offer prenatal diagnosis at an early stage of the pregnancy but they can help to resolve diagnostic problems.

17. HAEMOPHILIA X LINKED RECESSIVE DISORDER HAEMOPHILIA A – MUTATIONS IN FACTOR VIII GENE HAEMOPHILIA B – MUTATIONS IN FACTOR IX GENE SIMPLE AND COMPLICATED MUTATIONS THE FLIP TIP MUTATION

19. Hemophilia Mutations Deletions Point mutations Flip tip mutations

20. F8A E1 E22 E23 E26 F8A E1 E22 E23 E26 TEL CEN TEL INVERSION 22 E1E22E23 E26 A B C THE IVS 22 MUTATION IN HAEMOPHILIA A. F8B

23. Activated Protein C and Factor V The function of protein C is to inactivate factor Va and factor VIIIa The first step in this process is the activation of thrombomodulin by thrombin. Subsequently, protein C combines with thrombomodulin in order to produce activated Protein C (APC) Activated protein C can then degrade factor Va and factor VIIIa

24. Factor V Leiden Factor V Leiden is a genetically acquired trait that can result in a thrombophilic (hypercoaguable) state resulting in the phenomenon of activated protein C resistance (APCR) Over 95% of patients with APCR have factor V Leiden.

25. Activated Protein C and Factor V Leiden When one has factor V Leiden, the factor Va is resistant to the normal effects of activated protein C, thus the term activated protein C resistance The result is that factor V Leiden is inactivated by activated protein C at a much slower rate (see Figure 3), thus leading to a thrombophilic (propensity to clot) state by having increased activity of factor V in the blood

27. Prevalence of FVL Factor V Leiden is seen more commonly in the northern European populations About 4-7% of the general population is heterozygous for factor V Leiden. About 0.06 to 0.25% of the population is homozygous for factor V Leiden. The factor V Leiden mutation is relatively uncommon in the native populations of Asia, Africa and North America. In contrast, in Greece and southern Sweden, rates above 10% have been reported.

28. Prothrombin and Deep Vein Thrombosis Prothrombin is the precursor to thrombin in the coagulation cascade Thrombin is required in order to convert fibrinogen into fibrin, which is the primary goal of the coagulation cascade The gene has a mutation at position 20210, hence the disorder being referred to as prothrombin mutation 20210 The prothrombin gene mutation is seen more commonly in the Caucasian population. About 1-2% of the general population is heterozygous for the prothrombin gene mutation

29. Relative Risk of Venous Thrombosis Normal Risk1 Use of OCP4 FVL heterozygous5-7 + OCP30-35 Homozygous80 + OCP>100 Prothrombin heterozygous3 + OCP16

30. Leukaemia, the current hypothesis Defect in maturation of white blood cells May involve a block in differentiation and/or a block in apoptosis Acquired genetic defect Initiating events unclear Transformation events involve acquired genetic changes Chromosomal translocation implicated in many forms of leukaemia

31. Chronic Myeloid Leukaemia Malignancy of the haemopoietic system Transformation of the pluripotent stem cell 9;22 translocation giving rise to the Philadelphia (Ph’) chromosome Creation of a leukaemia specific mRNA (BCR-ABL) Resistance to apoptosis, abnormal signalling and adhesion

32. Clinical Course: Phases of CML Chronic phase Median 4–6 years stabilization Accelerated phase Median duration up to 1 year Blastic phase (blast crisis) Median survival 3–6 months Terminal phase Advanced phases

33. Cytogenetic Abnormality of CML: The Ph Chromosome 12345 6781011912 131415161718 19202122xY

34. The Ph Chromosome: t(9;22) Translocation 22 bcr abl Ph ( or 22q-) bcr-abl FUSION PROTEIN WITH ELEVATED TYROSINE KINASE ACTIVITY 9 9 q+

35. abl t(9;22) Translocation bcr-abl fusion gene Philadelphia chromosome 9 22 bcr p210 bcr-abl p190 bcr-abl ALL CML Chromosome 22 11314bcr Chromosome 9 2-111c-abl ALL breakpoint CML breakpoint bcr-abl Gene and Fusion Protein Tyrosine Kinases 9+

36. Prevalence of the Ph Chromosome in Haematological Malignancies Leukaemia% of Ph+ Patients CML95 ALL (Adult)15–30 ALL (Paediatric)5 AML2 Faderl S et al. Oncology (Huntingt). 1999;13:169-184.

37. P210 stimulates signal transduction in CML cells

38. Wortmannin LY294002 Farnesyl transferase inhibitors (SCH 66336) Imatinib

39. ACUTE LEUKEMIA Translocation is a major mechanism Involves genes whose normal function is to control cell division, haematological development etc These genes are known as master genes MLL, AML1 Mutatation of these genes through translocation leads to leukemia

40. MLL Promiscuous partner

42. AML1 21q AML1-ETO t(8;21) T(3;21) TEL-AML t(12;21) Loss of transactivation domain critical to t(8;21) and t(3;21) abnormalities Inv (16)

43. Molecular Mechanisms of AML1 action

44. AML1 21q AML1-ETO t(8;21) T(3;21) TEL-AML t(12;21) Loss of transactivation domain critical to t(8;21) and t(3;21) abnormalities Inv (16)

45. What is AML1 Subunit of a multifactorial transcription factor known as Core Binding Factor AML1 is also known as Core Binding FactorA It has homology to the drosophila developmental gene “runt” in its DNA binding region Also has a transactivation domain at its carboxy terminus

47. What does AML 1 do? Binds DNA Binding site for AML1 is a core enhancer that is located at the 5’ control region of genes that are involved in controlling lineage differentiation T cell receptor, myeloperoxidase, IL3, GM-CSF, CSF1 AML1 plays a pivotal role in hemopoietic differentiation by orchestrating expression of appropriate lineage specific genes

48. What do translocations involving AML1 do? T(8;21) Generates AML1-ETO fusion T(3;21) generates AML1-EVI1, AML1- EAP1 or AML1-MDS1 All of the above involve replacement of the transactivation domain These new fusion proteins can no longer activate AML1 binding sites in lineage specific genes

49. Molecular Mechanisms of AML1 action

50. Inversion 16 Here AML1 is not involved However the other member of the Core Binding Factor complex (CBFb) is mutated Net result is a pertubation of transcription of genes with AML1 binding sites

51. Inversion 16 and AML

52. Molecular Mechanisms of AML1 action

53. Summary Master genes such as AML1 and MLL control lineage specific gene expression, thus orchetrating lineage specific development of hemopoiesis Mutations in these genes disrupt this control, thus leading to aberrant hemopoiesis and development of leukemia

54. APML MOLECULAR GENETICS M3 FORM OF AML NON RANDOM CHROMOSOMAL ABNORMALITY t(15;17) IN 95% OF CASES RARa GENE ON CHROMSOME 17 PML GENE ON CHROMOSOME 15 t(11;17); t(5;17)

56. MOLECULAR MEDICINE INTO ACTION PRESENCE OF RARa CRITICAL TO THE TREATMENT OF THIS DISEASE STANDARD CHEMOTHERAPY ONLY PARTIALLY EFFECTIVE TREATMENT WITH RA REMOVES DIFFERENTIATION BLOCKADE

57. ALL TRANS RETINOIC ACID

59. NON HODGKINS LYMPHOMA B CELL FOLLICULAR LYMPHOMA t(14;18)(q21;q14) BCL 2 AND IMMUNOGLOBULIN GENES INVOLVED DYSREGULATION OF BCL 2 FAILURE OF APOPTOSIS

64. Summary Molecular changes implicated in haemoglobinopathies Factor VIII and Factor IX in Haemophilia Factor V leiden and Prothrombin in Deep vein Thrombus Molecular abnormalities in Leukemia, particularly translacations CML, a paradigm for malignancy Mutations in master genes disrupt control of hemopoiesis leading to development of leukemia Knowledge of molecular changes can influence diagnosis, prognosis and treatment