HEMOGLOBINOPATHIES

1. HEMOGLOBINOPATHIES Dr.S.Chakravarty MD

2. Specific Learning Objectives • At the end of today’s lecture you should be able to – • Enumerate the various hemoglobinopathies with their associated genetic defects . • Describe the molecular basis and the basis of laboratory diagnosis of Sickle cell anaemia , alpha and beta thalassemiain detail.

3. Hemoglobin structure α β β α heme

4. Hemoglobin synthesis 25% 25% 0.5% 1.5% 48% α α γ δ β α α γ δ β 25% 25% 0.5% 1.5% 48% Chromosome 16 Chromosome 11

5. Hemoglobins in normal adults α β α γ α δ β α γ α δ α HbA HbF HbA2 98% ~1% <3.5%

8. Steps in globin chain synthesis: • Transcription • Modification of mRNA precursor by splicing • Translation by ribosomes & further modifications (i.e. glycosylation)

9. Hemoglobinopathies • An inherited mutation of the globin genes leading to a qualitative or quantitative abnormality of globin synthesis. • Hereditary disorders that can result in moderate to severe anemia

10. Types of Hemoglobinopathies: • Structural hemoglobinopathies • Thallasemias • ThallasemicHb variants • Hereditorypersistance of fetal Hemoglobin • Acquired hemoglobinopathies

11. 1. Structural Hemoglobinopathies • Altered Hb polymerization • Altered affinity • Unstable Hb Variants.

12. 1 (a) Altered Hb polymerization: Sickle cell anemia: (Hbs) • Mutation : Glutamic acid is replaced byvalineat 6th position of Beta chain. • Polymerizes reversibly when deoxygenated to form a gelatinous network of fibrous polymers that stiffen the erythrocyte membrane. • The abnormal hemoglobin is less soluble under decreasing oxygen concentrations and polymerize into crystals that distort the red blood cells into a sickle shape.

13. Sickle Cells

14. A number of factors may precipitate a sickle cell crisis, these include: • Hypoxia • Acidosis • Dehydration • Infections • Severe exercise • Increase physical / physiological demand (Pregnancy, physical exercise) Hb S is less soluble in acidosis and dehydration

15. Hydrophobic valine causes stickiness Molecular and Cellular Events leading to Sickle cell crisis Lippincott’s Illustrated Biochemistry

17. Sickle cell anemia • Hemolysis Anaemia • Occlusion of blood vessels by sickled red cells- SEVERE PAIN( DUE TO TISSUE ANOXIA)

20. Lab Investigations • Complete Hemogram Anemia • Sickling test :- Blood smear prepared after adding reducing agent sodium dithionite See under microscope (NOT SPECIFIC ) • Solubility test :- Hemolysate is prepared in the presence of reducing agent .Opalescence is suggestive of sickle cells. (NOT SPECIFIC ) • Hb electrophoresis- CONFIRMATORY • Southern Blot -CONFIRMATORY

21. Migration of HbS in an electrophoretic field • @ pH 8.6 Glutamic acid carboxy group is –ve charged • Lack of this charge of HbS makes it less negatively charged and decreases the electrophoretic mobility towards positive pole. • However at acidic pH (citrate buffer)HbS moves faster than Hb A Lippincott’s Illustrated Biochemistry

22. Management • Repeated Blood transfusions- MAINSTAY • IRON OVERLOAD is a problem • CHELATION therapy with Desferrioxamine

23. Unstable hemoglobin Variants • CHRONIC HEINZ BODY ANAEMIAS -Unstable Hb variants have a tendency to denature. They tend to form molecular aggregates called Heinz Bodies within cells hemolysis • Hb Köln (Hbβ 98valmet) • Hb Poole – γ chain unstable variant

24. Hb variants with altered O2 AFFINITY • INCREASED O2 AFFINITY • Hb Chesapeake • erythrocytosis (ODC shifts to left ) • DECREASED O2 AFFINITY • Hb Kansas • Cyanosis • Hb M- • Most HbM are produced by substitution of Tyr for proximal/Distal His in the haem pocket of the alpha or ß-chainsThis results in facilitated oxidation of the hemoglobin to yield excess methemoglobin which leads to cyanosis. • Hb M Hyde Park, β92His→Tyr; HbMBoston, α58His→Tyr; HbMSaskatoon, β63His→Tyr; Hb MMilwaukee-1, β67Val→Glu.

25. Thalassemia • Hereditary disorders that can result in moderate to severe anemia • Basic defect is reduced production of globin chains.

26. Found most frequently in the Mediterranean, Africa, Western and Southeast Asia, India and Burma • Distribution parallels that of Plasmodium falciparum Demographics Hb D

27. SymbolismAlpha Thalassemia • Greek letter used to designate globin chain: 

28. SymbolismAlpha Thalassemia /: Indicates division between genes inherited from both parents: / • Each chromosome 16 carries 2 genes. Therefore the total complement of  genes in an individual is 4

29. SymbolismAlpha Thalassemia - : Indicates a gene deletion: -/

30. Classification & TerminologyAlphaThalassemia • Normal / • Silent carrier -/ • Minor -/- --/ • Hb H disease --/- • Bartshydropsfetalis--/--

31. SymbolismOther Thalassemia • Greek letter used to designate globin chain: 

32. SymbolismOther Thalassemia +: Indicates diminished, but some production of globin chain by gene: +

33. SymbolismOther Thalassemia 0 :Indicates no production of globin chain by gene: 0

34. Classification & TerminologyBeta Thalassemia • Normal / • Minor /0 /+ • Intermedia0/+ • Major 0/0 +/+

35. Some mutations lie within promoter regions and typically lead to reduced globin gene transcription. In some cases a single-nucleotide change in one of the exons leads to the formation of a termination, or "stop" codon, which interrupts translation of β-globin messenger RNA (mRNA) and completely prevents the synthesis of β-globin. Such alleles are designated β0.  Mutations that lead to aberrant mRNA processing are the most common cause of β-thalassemia. Most of these affect introns, but some have been located within exons. If the mutation alters the normal splice junctions, splicing does not occur, and all of the mRNA formed is abnormal. Unspliced mRNA is degraded within the nucleus, and no β-globin is made

36. Abnormal associations of otherwise normal subunits. • With severe α-thalassemia, the β-globin subunits begin to associate into groups of four (tetramers) due to the paucity of potential  α -chain partners. • These tetramers of b-globin subunits are functionally inactive and do not transport oxygen. No comparable tetramers of alpha globin subunits form with severe beta-thalassemia. • Alpha subunits are rapidly degraded in the absence of a partner from the beta-globin gene cluster (gamma, delta, beta globin subunits).

38. Excessive RBC BREAKDOWN

39. Signs and Symptoms • Anaemia • Bone changes (hair on end) • Ethnicity: Mediterranean, Africa, Southeast Asia • Hypo-Micro, Poikilocytosis • NRBC’s, reticulocytosis, basophilic stippling • Siderocytes (with repeated transfusions)

40. Thalassemia Blood Smears

41. X-ray of scull in Thalassemia: “Hair-on-end”

42. MRI showing marked widening of the diploic space containing alternating bands (arrows) of hypointensetrabeculae and hyperintense marrow.

44. Beta thalassemia major Male 18 years HEPATOSPLENOMEGALY OVERALL DECREASED GROWTH

45. Special CasesThalassemia • HbLepore:  fusion seen in some types of thalassemia • Hb Constant Spring •  chain with 31 additional amino acids • --/cs • Hereditary persistence of fetal hemoglobin (HPFH) This is usually caused by mutations in the β-globin gene. • Beneficial to patients with sickle cell or thalassemia.

46. Special Cases: Thalassemia • Hb H • 4tetramer and γ4 tetramers • Associated with --/-thalassemia

47. Special Cases: Thalassemia • HbBarts & hydropsfetalis • Barts is a 4 tetramer • Associated with --/-- • Lethal • High concentrations are capable of sickling

48. Primary Laboratory InvestigationThalassemia • Peripheral smear – • Severe cases present with • Microcytosis • Hypochromia • Poikilocytosis • Hb Electrophoresis • DNA studies (PCR + S.blot)

50. Course of Thalassemia • Time of presentation • Related to degree of severity • Usually in first few years of life • Untreated severe thalassemia • --/--: Prenatal or perinatal death • --/- & --/cs: Normal life span with chronic hemolytic anemia