thalassemia, types and its treatment.pptx

thalassemia, types and its treatment.pptx

1. thalassemia

2. Hemoglobin 4 Heme groups 4 polypeptide chains

3. B B A A heme Hemoglobin structure

4. A A A A A A B G D B D G HbA HbF HbA 2 95-98% ~1% <3.5% Hemoglobins in normal adults

5. Hemoglobin Type Name Components Adult A  2  2 A2  2  2 Fetal F  2  2 Embryonic Portland  2  2 Gower 1  2  2 Gower 2  2  2 Abnormal H  4 Bart’s  4

6. Genetic control of Hemoglobin

7. Normal Hb are tetramers of two α -like and two β -like globin polypeptides. The α -like genes are located on the short arm of chromosome 16 whereas the β -like genes are located on chromosome 11. The pred.Hb in adults is HbA (>95%) and the minor Hbs are HbA2 (2to 3.5%) and HBF(0.3to 1.2%). HBF is the pred. Hb in fetal cells during the latter two trimesters of pregnancy. During the first trimester in utero , embryonic Hbs with different subunit composition are found. The embryonic to fetal switch occurs between 6 and 10 weeks of gestation and the switch from fetal to adult hemoglobin occurs at about the time of birth. Globin gene structure: the coding regions are interrupted by noncoding DNA, known as intervening sequences (IVS) or introns . In α - globin gene, introns are present between the codons for amino acids 31 and 32 and between codons 99 and 100. While in β - globin gene, introns are found between codons 30 and31 and between codons 104 and105

8. Demographics: Thalassemia Found most frequently in the Mediterranean, Africa, Western and Southeast Asia, India and Burma

9. The name thalassemia was coined at the University of Rochester in upstate New York by the Nobel Prize-winning pathologist George Whipple and the professor of pediatrics William Bradford from the Greek thalassa for sea and - emia meaning the blood. Thalassemia was first described by Cooley and Lee in 1925. Most of their patients were of Mediterranean ancestry and a majority of them suffered anemia. Hence this group of anemias are also known as Cooley's Anemia or Mediterranean Anemia.

10. Thalassemia - Defined A family of genetic anemias characterized by a reduced rate of production of one or more globin subunits of hemoglobin ( Hb ) Symptoms are caused by the deleterious effects of the normally produced subunits that are now in excess.

11. Clinical Classification of The Thalassemias Silent carrier ( α , β ) Thalassemia Trait ( α , β ) HbH disease ( α - thal ) Hydrops Fetalis ( α - thal ) Severe β - thal Thal . Major Thal . intermedia Hematologically normal Mild anemia with microcytosis and hypochromia Mod.- Severe hemolytic anemia, icterus and splenomegaly Death in utero Severe anemia, growth retardation, hepatosplenomegaly , BM expansion and bone deformities. Transfusion dependent No regular transfusion requirement.

12. Laboratory Investigation CBC-MCV,MCH,RDW Tests Hemoglobin electrophoresis Cellulose acetate: Alkaline pH Citrate agar: Acid ph HPLC DNA Assay

13. β - Thalassemia Result of point mutations Genotypes: β / β , β / β + , β + / β + , β / β , β + / β , β / β +, silent mutation. B – No B- globin chains are produced B + - some beta chains produced - Decreased

14. β - Thalassemia Major( β / β , β / β + , β + / β + ) Cooley’s Anemia Inheritance of abnormal β - genes Marked reduction or absence of β - chain synthesis

15. Mutated β - gene (s) Normal α , γ , δ genes Normal synthesis of α - chains Reduced synthesis of β - chains Enhanced synthesis of γ -, δ - chains Excess α - chains Precipitation of α - chains ↑ HbF and HbA 2 ↑ Oxygen Affinity ↓ HbA Reduced oxygen delivery Damaged RBC membranes and apoptosis Anemia ↑ Iron Toxicity ↑ EPO BM Expansion Fractures and mongoloid facies P.B. circulation Splenic Hemolysis Bone marrow: phagocytosis Ineffective erythropoiesis Hepatosplenomegaly Pathophysiology

16. Clinical Findings Irritability Pallor Failure to thrive Fever Enlarged abdomen Severe anemia Cardiac failure in the first decade of life Growth retardation Bone changes hepatosplenomegaly

17. Lab studies The CBC count and peripheral blood film examination results are usually sufficient to suspect the diagnosis. In the severe forms of thalassemia , the Hb level ranges from 2-8 g/ dL . MCV and MCH are significantly low, but, unlike thalassemia trait, thalassemia major is associated with a markedly elevated RDW, reflecting the extreme anisocytosis . The WBC count is usually elevated in β thalassemia major; this is due, in part, to miscounting the many nucleated RBCs as leukocytes. Leukocytosis is usually present, even after excluding the nucleated RBCs. A shift to the left is also encountered, reflecting the hemolytic process. Platelet count is usually normal.

18. Laboratory Findings Severe hypochromic microcytic hemolytic anemia Hb level: 2 to 3 g/dl marked anisocytosis and poikilocytosis - target cells, basophilic stippling, normoblasts , polychromatophils and teardrop cells. Reticulocytes elevated to about 10%

19. Peripheral blood smear from a patient with β - thalassemia major

20. “ hair- on- end” appearance

21. In addition to the classic "hair on end" appearance of the skull, which results from widening of the diploic spaces and observed on plain radiographs, the maxilla may overgrow, which results in maxillary overbite, prominence of the upper incisors, and separation of the orbit. These changes contribute to the classic "chipmunk facies observed in patients with thalassemia major

22. Treatment and Prognosis Regular transfusion program Administration of iron-chelating agents Splenectomy Bone marrow transplants Untreated patients die in first or second decade of life Hypertransfusion program leads to iron overload eventually

23. β - Thalassemia Intermedia ( β / β + , β + / β + , β / β ) Homozygous β - Thalassemia: mild clinical form. Heterozygous β - Thalassemia: severe clinical form.

24. Clinical and lab Findings Anemia Mild jaundice Splenomegaly Bone deformities Osteoporosis The hemoglobin concentration is maintained in the range of 6 to 9 g/dl without transfusion. Peripheral blood erythrocytes show changes comparable to those of thalassemia major: significant anisocytosis , hypochromia , target cells, basophilic stippling, and numerous nucleated forms

25. β - Thalassemia Minor ( β / β or β + / β ) Heterozygous inheritance of either β + or β gene with one normal β - gene. Asymptomatic excepts in periods of stress

26. Laboratory Findings Mild anemia with Hb levels from 9 to 14 g/dl Erythrocyte count doubled Discovered incidentally Microcytosis , hypochromia , anisopoikilocytosis , target cells and basophilic stippling Hb electrophoresis: increased HbA 2 from 3.5 to 7%

27. 2 most frequent microcytic anemias are β TT & IDA Discrimination indices to differentiate Mentzers index MCV/RBC >14 – IDA 12-14 - Indetermi <12 - TT Green & King index MCV(2) X RDW / Hb x 100 > 65 – IDA, < 65 - TT RDWI MCVXRDW / RBC IDA>220 & THAL<220 England & Fraser index MCV-RBC-(5XHb) - k, k =3.4 <0 -TT RDW IDA (16.3% +/- 1.8% TT (13.6 +/- 1.6%) RBC count : >5.035 -TT

28. Basis Of Discrimination Indices RBC counts higher in thalassaemia trait than IDA RDW high in IDA G&K showed highest reliability than E&F, RBC count, MI, RDWI Annals of hematology Vol 86, 2007

29. Electrophoresis Principle Separation of haemoglobins with electrophoresis at pH 8.4 (alkaline) and pH 6.2 (acid). Scanning allows quantification of the hemoglobin present, bands are seen by staining. At alkaline pH Hb C, E, A2 and O migrate together to form a single band, Hb S, D and G also co migrate. At acid pH Hb C separates from E and O and Hb S separates from D and G. Hb E and O cannot be separated by electrophoresis neither can Hb D and G.

30. Hemoglobin Electrophoresis Theory Hemoglobin electrophoresis is the movement of hemoglobin proteins in an electric field at a fixed pH. Because the various hemoglobins are comprised of different combinations of globin chains (normal or abnormal), they will demonstrate different degrees of mobility. For an alkaline hemoglobin electrophoresis, a hemolysate is applied to cellulose acetate which is electrophoresed in a buffer at pH 8.4-8.6. At this pH hemoglobin proteins move from cathode to anode.

31. Of the hemoglobins normally present in an adult, Hb A migrates the fastest, followed by Hb F. Hb A 2 moves only slightly from the point of origin near the cathode. Abnormal hemoglobins show the following migration patterns: Hb C migrates with Hb A 2 near the cathode. Hb S lies between Hb A 2 and Hb F. Hb H and Bart's hemoglobin are unstable and very fast moving, with Hb H being the faster of the two. They are located nearer the anode past Hb A .

32. Normal Hemoglobin Electrophoresis This electrophoresis gel displays the migration patterns for a person with normal hemoglobin distribution. Normally Hb A is present in excess of 97% with the remaining being made up of Hb A 2 and Hb F. Here you can see the large band of Hb A with a faint band in the Hb F and Hb A 2 regions. Controls for A and F and A, S, and C are included. (AF and ASC are simply labels for the controls and do not indicate order of migration.)

33. This densitometer tracing corresponds to the pattern for normal distribution of hemoglobin.

34. alkaline hemoglobin electrophoresis in Beta thalassemia Lanes 1 and 2: normal patient specimen Hb A is over 98% with a small amount of Hb A 2 visible Lanes 3 and 4: Beta thalassemia minor Hb A is decreased to 94%, Hb A 2 is increased at 5%, and Hb F is 1% Lanes 5 and 6: Delta-beta thalassemia major No Hb A or A 2 is present, Hb F is 100% Lanes 7 & 8: Delta-beta thalassemia intermedia Hb A is 8.5%, Hb A2 is 3.5% and Hb F is 88% Lane 9: AF control Lane 10: ASC control

35. Electrophoresis Strengths Commercial, widely available, rapid methods used for many years. Gives an estimate of HbA2 level. Identifies some variant haemoglobins which are well characterized.

36. Electrophoresis Disadvantages Labor-intensive. Inaccurate in quantification of low-concentration variants (HbA2) and in detection of fast variants ( HbH , Hb Barts ). The precision and accuracy for Hb A2 using scanning of electrophoretic gels is poor (in comparison to HPLC).  An imprecise test in comparison to other tests now available.

37. HPLC Principle Cation -exchange HPLC can be performed on an automated instrument that can quantify Hb A2, Hb F, Hb A, Hb S, and Hb C. Studies show equivalence or superiority over electrophoresis in terms of identification of variant haemoglobins and quantification of HbA2 level. Negatively charged carboxyl molecules bound to silica make up the cartridge matrix. Positively charge molecules (salt and hemoglobin) bind to the carboxyl groups. Haemoglobin molecules are bound and displaced by increasing salt concentration. Haemoglobin variants separate out due to variation in charge.

38. A2 A2  Suggests <3.5% Normal 3.5-8.0% BTT /(Megaloblastic anemia) 11-15% Hb Lepore 20-40% HbE 30-48% HbD    

39. Low A2 Iron Deficiency Anemia Alpha Thalassemia trait HbH Disease Delta Thalassemia Delta –Beta Thalassemia

40. HbF F  Suggests <2% Normal <5% HPFH/Pregnancy/ Aplastic anemia 2-15% HPFH BTT   Sickle 10-15% Delta Beta Thalassemia >50% Beta Thal Major   HPFH (homozygous)   Delta Beta Thalassemia (homo)

41. HPLC Strengths Method of choice for screening for Hb variants; for quantification of HbA2 + HbF concentrations and mandatory in neonatal screening. Quicker and more sensitive than standard techniques for detecting HbF (in diagnosis of HPFH and monitoring sickle cell anemia). Indeed alkaline gel electrophoresis cannot detect HbF in healthy adults or those with marginally increased Hb F. Can be used to characterize rare haemoglobinopathies not well detected with other methods ( HbRambam ). Established role in the diagnosis of thalassaemia and haemoglobinopathies , including with cord blood samples.

42. DNA Analysis Indicated when the hemoglobinopathy is not confirmed by other methods or when the underlying mutation is important to management. Other techniques lead to a presumptive identification of the hemoglobinopathy only. For genetic counseling, defining the particular mutation or deletion is often required – this is achieved by a variety of molecular techniques. DNA from WBCs, amniocytes , or chorionic tissue may be utilized for diagnosis of various α and β globin chain abnormalities.

43. Southern blot hybridization using restriction enzymes digesting labeled complementary probes define deletional mutations in α and rare β thal . PCR amplifies globin genes and utilizes allele specific primers to detect known globin chain mutations eg HbS , E, D, O + several β thal . PCR can be used to detect unknown mutations. Aims to separate amplified DNA on gels or with HPLC on the principle that different amino acids migrate differently. 3 primary methods – mutation analysis, DNA scanning and DNA sequencing.

44. Amplification refractory mutation system (ARMS) The technique is based on the principle of allele-specific priming of the PCR process, i.e. a specific primer will only permit amplification to take place when its 3’ terminal nucleotide matches with its target sequence. The target DNA is amplified using a common primer and either of two allele specific primers, one complimentary to the mutation to be detected (the b- thalassemia primer) and the other complimentary to normal DNA sequence at the same position.

45. mutations Most mutations that cause β - thalassemia are point mutations in functionally important regions of the β - globin gene. Promoter region mutations. Point mutations within promoter sequences prevent RNA polymerase from binding normally, reducing transcription by 75% to 80%. Some Normal B- globin is synthesized, producing B- - thalassemia . Promoter regions are required for accurate and efficient transcription of genes Three consensus sequences are found in the promoter regions of all globin genes: the CACCC box, the CCAAT box, and the TATA box. Point mutations within these sequences tend to reduce the affinity with which these proteins bind so this leads to reduced gene transcription. The C to T substitution at position -101 in CACCC box results in a mild defect associated with a silent carrier phenotype in heterozygous carriers. Likewise single base substituitions at the indicated positions results in β + - thalassemias .

46. ■ Chain terminator mutations. Two types of mutations cause premature termination of mRNA translation. One creates a new stop codon within an exon ; the second consists of small insertions or deletions that shift the mRNA reading frames and introduce downstream stop codons that terminate protein synthesis ( frameshift mutations). In both cases, synthesis of functional B- globin is prevented by premature chain termination, leading to B" - thalassemia . ■ Splicing mutations. Mutations leading to aberrant splicing are the most common cause of B- thalassemia . Most affect introns ; others are located within exons . Some of these mutations alter normal splice junctions, such that normal splicing does not occur at all. Unspliced mRNA is degraded within the nucleus, and Bo - thalassemia results.

47. alpha - THALASSAEMIA MOLECULAR PATHOGENESIS. α- thalassaemias are disorders in which there is defective synthesis of α- globin chains resulting in depressed production of haemoglobins that contain α-chains i.e. HbA , HbA2 and HbF . The α- thalassaemias are most commonly due to deletion of one or more of the α-chain genes located on short arm of chromosome 16. Since there is a pair of α-chain genes, the clinical manifestations of α- thalassaemia depend upon the number of genes deleted. Accordingly, α- thalassaemias are classified into 4 types: 1. Four α- gene deletion: Hb Bart’s hydrops foetalis . 2. Three α-gene deletion: HbH disease. 3. Two α- gene deletion: α- thalassaemia trait. 4. One α- gene deletion: α- thalassaemia trait (carrier)

48. Alpha thalassemia aa / aa Normal aa / a - Mild microcytosis aa / - - Mild microcytosis a - / - - Hemoglobin H disease - / - - Hemoglobin Barts – Hydrops Fetalis

49. H Hgb H disease Hgb H inclusions (supravital stain)

50. Hydrops fetalis (note gross edema) Hydrops fetalis