1. Haemoglobin Disorders in Pakistan Brig Nuzhat Salamat FCPS (Haem), CTM Classified Pathologist

3. WHO Pakistan: Children less than 5 years of age having Hb less than 11 gms/dl

4. Symptoms May be asymptomatic. Fatigue. Shortness of breath. Left upper quadrant pain (if there is associated splenomegaly). Right upper quadrant pain (secondary to cholelithiasis in haemolytic anaemia). Failure to thrive, poor feeding. Intermittent infections. Symptoms related to underlying disease pathology - eg, acute pain in sickle cell crises and chronic/recurrent diarrhoea, which can suggest a malabsorption syndrome such as coeliac disease.

6. (WHO 1999)

8. Haemoglobin Disorders in Pakistan β-Thalassaemia α-Thalassaemia  -Thalassaemia Abnormal Haemoglobins  Hb-S  Hb-E  Hb-D Miscellaneous Hb disorders

9. Abnormal Haemoglobins in Pakistan (Ahmed 1998)

10. β-Thalassaemia Trait in Pakistan Disorder:Ethnic group:Type of study:Place:Subjects:Methods used:Carriers:95% CIReference:  -thalassaemia PathanPopulation basedPeshawar129CEM Electrophoresis5 (3.9%)0.56-7.24%Stern et al, 1968  -thalassaemia Mixed, all groupsPopulation basedKarachi1224CEM Electrophoresis17 (1.4%)0.73-2.04%Hashmi and Farzana 1976  -thalassaemia ?Population basedParis67CEM Electrophoresis2 (3.0%)1.08-7.08%Coquelet et al, 1983  -thalassaemia Mostly PunjabiHospital basedLahore437CEM Electrophoresis42 (9.6%)6.83-12.36%Latif 1983  -thalassaemia Mostly PunjabiPopulation basedLahore300CEM Electrophoresis5 (1.6%)0.24-3.16%Hameed and Chaudhry 1984  -thalassaemia ?Hospital basedKarachi256CEM Electrophoresis16 (6.3%)3.24-9.16%Ihsanullah et al, 1985  -thalassaemia ??London??? (6%)?Modell and Berdoukas 1984  -thalassaemia Mixed, all groupsPopulation basedRawalpindi500CEM Electrophoresis27 (5.4%)3.42-7.38%Khattak and Saleem 1992a @  -thalassaemia PunjabiPopulation basedRawalpindi245CEM Electrophoresis8 (3.3%)1.14-5.46%Khattak and Saleem 1992a @  -thalassaemia PathanPopulation basedRawalpindi201CEM Electrophoresis16 (8.0%)4.26-11.74%Khattak and Saleem 1992a @  -thalassaemia MixedHospital basedRawalpindi1000CEM Electrophoresis39 (3.9%)2.7-5.1%Hassan et al, 1997  -thalassaemia Mostly PunjabiPopulation basedLahore320CEM Electrophoresis3 (0.94%)0.12-2.0%Khan and Hayee 1986  -thalassaemia MixedPopulation basedRawalpindi500CEM Electrophoresis12 (2.4%)1.06-3.74%Zuhur-ur-Rehman et al, 1991

11. β-Thalassaemia in Pakistan (Ahmed 1998)

12. Registered Cases of Thalasaemia in Pakistan (Ahmed 1998)

16. Thalassaemia Syndromes Thalassaemias are a heterogenous group of genetic disorders of haemoglobin synthesis that result from a reduced rate of production of one or more of the globin chain(s) of haemoglobin.

17. Abnormal Haemoglobins Defective quality of haemoglobin differentiates them from the quantitative reduction in globin chain synthesis in thalassaemia syndromes. The structurally abnormal haemoglobins are mostly produced in normal amounts. However, some abnormal haemoglobins, Hb-E and Hb- Knossos, for example, are also associated with reduced globin chain production.

18.  -Thalassaemias:  o -Thalassaemias  + -Thalassaemias Deletion Non-deletion With  -chain Hb variants With  -chain Hb variants With  -Thalassaemia  -Thalassaemias:  o -Thalassaemias  + -Thalassaemias With  -chain Hb variants With  -chain Hb variants With  -Thalassaemia  -Thalassaemia: (  ) o -Thalassaemia ( A  ) o -Thalassaemia (  ) o -Thalassaemia  -Thalassaemia  -Thalassaemia Hereditary Persistence of Fetal Haemoglobin (HPFH): Deletion (  ) o -HPFH Non-deletion Linked to  -globin gene cluster Unlinked to  -globin gene cluster Thalassaemia Syndromes

19. Abnormal Haemoglobins Sickling Disorders:  With  -Thalassaemia  With  -chain Hb variants  With  -chain Hb variants Other Structural Variants (Hb-E, C, D etc.):  With  -Thalassaemia  With  -Thalassaemia Unstable Haemoglobins: Haemoglobin-M: Altered Affinity Haemoglobins:

22. Reduced haemoglobinization Ineffective erythropoiesis Expansion of marrow cavities Hypersplenism Haemodilution

23. Complete Blood Counts

24. Peripheral Blood Film

25. Electrophoresis Size of the molecule Charge on the molecule Pore size of the medium Voltage and Current Buffer composition and concentration

26. Hb-Electrophoresis Cellulose Acetate Membrane Cellogel Agarose Gel Agar Gel Polyacrylamide Gel

27. Cellulose Acetate Membrane Hb-Electrophoresis (pH 8.7) Slow moving  Hb-A 2 /E/C Intermediate  Hb-D/S/Lepore  Hb-F  Hb-A Fast moving  Hb-H/J/Barts

28. Quantitative Estimation of Hb Fractions By  Chromatography  Elution after Electrophoresis  Densitometry?? For  Hb-A 2  Others??

29. Miscellaneous Investigations Sickling Test Heinz Body Test Test for Unstable Haemoglobins Test for Methaemoglobin

30. -Thalassaemia Trait Typical form Silent -thalassaemia trait Interaction with -thalassaemia Interaction with structural Hb variants

31. Typical -thalassaemia Trait

32. RDW in Thalassaemia

33. Inheritance of Thalassaemia

34. Mean Pre-transfusion Hb in Thal Major in Pakistan (Ahmed 1998)

35. Different classes of beta thalassaemia mutations

36. Classes of mutations of beta thalssaemia

37. (Suhaib Ahmed et al, BJH 1996) β-Thalassaemia Mutations in Pakistan

38. Distribution of Thalassaemia Genes in Pakistan Index families 183/591 (31%) Control Families 0/397 (0%) General Population (5%) (Ahmed et al, 2002)

39. Survival Pattern of Thal Major in Pakistan (Ahmed 1998)

40.  -Thalassaemia in Pakistan -  3.7  /   8.3% -  3.7  / -  3.7  2.0% -  4.2  /   0.2% Anti -  3.7    /   0.9% -  SEA -  SEA / -  3.7  ? Non deletional  -thal? Ahmed 1998; Khan et al, Hemoglobin 2003; 27: 161-6.

41. Alpha thalassaemia

42.  -Thalassaemia in Pakistan Sr.FamEthnicSexAgeTransSplHbTRBCMCVMCHHbFHbA 2 MutationXmn-I Heterozygous ( A  ) o /Nor) 1. PathanF30No-11.55.1573.222.315%2.4% ( A  ) o /Nor -/+ 2. PunjabiM33No-13.45.6178.323.917%2.9% ( A  ) o /Nor -/+ 3.2.PunjabiF28No-10.44.4872.123.215%2.6% ( A  ) o /Nor +/+ 4.3.PunjabiF27No-12.55.6771.622.012%2.8% ( A  ) o /Nor -/+ 5.3.PunjabiM6No-11.35.9164.019.112%2.4% ( A  ) o /Nor -/+ 6.3.PunjabiF4No-10.55.4166.219.414%2.3% ( A  ) o /Nor -/+ Mean11.65.3770.921.713%2.6% Homozygous ( A  ) o /( A  ) o 7.2.PunjabiM8No+10.55.5063.319.1100%Nil ( A  ) o /( A  ) o +/+ 8.3.PunjabiM33No+10.56.3065.120.0100%Nil ( A  ) o /( A  ) o +/+ 9.3.PunjabiF2Yes++5.02.2771.822.070%*1.3% ( A  ) o /( A  ) o +/+ 10.4.PunjabiF34No++11.24.4076.925.4100%Nil ( A  ) o /( A  ) o +/+ 11.5.BohraF10No++10.35.3270.119.0100%Nil ( A  ) o /( A  ) o +/+ 12.6.BohraM29No++10.75.1770.620.7100%Nil ( A  ) o /( A  ) o +/+ Mean9.74.8269.621.0100%Nil Compound heterozygous ( A  ) o /IVSI-5 (G-C) 13.6.BohraF3Yes++8.03.872.321.320%*1.3% ( A  ) o /  o -/+ (Ahmed & Anwar 2005)

43. Thalassaemia Intermedia Mild/Silent alleles Co-incidental α-thalassaemia trait Co-incidental structural variants Xmn-I polymorphism δβ-Thalassaemia

44. Wide range of clinical presentation Primary modifiers Secondary modifiers Tertiary modifiers Environmental

45. Cause of Thalassaemia Intermedia: n:Mean age: At 1 st transfusion: At examination: Xmn-I +/+ genotype146 years13 years  + -mutation 63 years8 years  + -mutation and suspected coincident  -thal 611¼ years18 years Unidentified thalassaemia mutation27½ years12½ years Confirmed coincident  -thalassaemia 29½ years13½ years Suspected coincident  -thalassaemia 99½ years16 years Total397 years14 years Thalassaemia Intermedia in Pakistan (Ahmed 1998)

46. -Thalassaemia Silent carrier:-/ -Thalassaemia Trait:-/- or --/ Hb-H Disease:--/- Hydrops Fetalis:--/--

47. Diagnosis of -Thalassaemia Blood counts Hb-H inclusions Electrophoresis PCR

48. Structural Hb Variants Hb-D/S/Lepore Hb-A 2 /E/C

49. Unstable Haemoglobin Disorders

51. Hb-M Cyanosis Haemolytic anaemia Electrophoresis Spectroscopy

52. Polymerase Chain Reaction (PCR)

53. Molecular Methods in Diagnosis Mutation Analysis Specific methods ARMS Dot Blot & Reverse Dot Blot Sequencing Non specific methods DGGE SSCP Linkage analysis

54. ARMS

55. Applications in Thalassaemia Prenatal Diagnosis Diagnosis in previously transfused patients Silent thalassaemia alleles Distinction between structural variants Thalassaemia intermedia α-thalassaemia β-Thalassaemia carriers in certain situations Rare thalassaemias Donor chimerism studies after SCT

56. β- Thalassaemia mutations in Pakistan

57. Multiplex ARMS PCR for Thalassaemia (Ahmed et al, Prenatal Diagnosis 2000) Primer ID: Mutations Pooled: Amplified Product size: AD-1Fr 8-9 (+G) IVSI-5 (G-C) Fr 41-42 (-TTCT) IVSI-1 (G-T) Del 619bp 215 bp 285 bp 439 bp 280 bp 242 bp AD-2Cd 5 (-CT) Fr 16 (-C) IVSI-1 (G-T) Cd 30 (G-C) Cd 30 (G-A) IVSII-1 (G-A) 205 bp 238 bp 280 bp 634 bp AD-3Cd 15 (G-A) Cap+1 (A-C) 500 bp 567 bp

58. Identify Couple at Risk Pregnancy Fetal Sampling Lab DiagnosisAffected FetusNormal Fetus Termination of Pregnancy Prenatal Diagnosis

59. Prenatal Diagnosis by ARMS Mutation 1. Father 2. Mother 3. CVS 4. CVS 5. -ve Normal 6. CVS 7. CVS 8. +ve 9. -ve 10. Blank

60. Misdiagnosis in PND (<0.5%) Maternal Contamination in Fetal Sample PCR Failure Clerical Mistakes Meotic Crossover in linkage analysis Non paternity (Ahmed S. 2007, Submitted to Prenatal Diagnosis)