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Hemophilia Dr. Pupak Derakhshandeh (PhD) Assis. Prof. Med. Sci. of Tehran Univ .

Hemophilia Dr. Pupak Derakhshandeh (PhD) Assis. Prof. Med. Sci. of Tehran Univ. Major bleeding disorders (50%). Hemophilia A (factor VIII- deficiency) Hemophilia B (factor IX- deficiency) Clinically: indistinguishable!. Other bleeding disorders (50%). Factor XI- deficiency

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Hemophilia Dr. Pupak Derakhshandeh (PhD) Assis. Prof. Med. Sci. of Tehran Univ .

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  1. HemophiliaDr. Pupak Derakhshandeh (PhD)Assis. Prof. Med. Sci. of Tehran Univ.

  2. Major bleeding disorders (50%) • Hemophilia A (factor VIII- deficiency) • Hemophilia B (factor IX- deficiency) • Clinically: • indistinguishable!

  3. Otherbleeding disorders (50%) • Factor XI- deficiency • Von Willebrand disease (VWB)

  4. Hemophilia (A/B) phenotypic features • Severe bleeding and Hemorrhages in the wounds, Joints, ankles, knees, hips & elbows, muscles • Hematomas & Bruising

  5. Hemophilia A The enlarged knee join

  6. Hemophilia AThe enlarged fore head

  7. Hemophilia AExtensive bruising of the fore arm and hand

  8. Hemophilia A/B • Severe: Level of factor VIII/IX <1% • Moderate: 1% < Level of factor VIII/IX<5% • Mild: 5% < Level of factor VIII/IX <25%

  9. X-Linked Inheritance • Located on the X-Chromosome • More commonly affected males • Heterozygote female will pass the gene to 50% of her sons who will be affected, and to 50% of her daughters who will be carriers for the trait • Affected males pass the gene to all of their daughters and none of their sons • Absence of male to male transmission

  10. How is ’Hemophilia’ inherited?

  11. X-Linked InheritanceA pedigree of Hemophilia in European royal families

  12. Disease Etiology Hemophilia A/B • Mutation in VIII & IX genus (a key component of the clotting cascade) • Incidence: • Hemph.A1:5000-10.000 male • Hemph.B1: 30.000-100.000 male

  13. Mild Hemophilia in female • Disadvantageous x-inactivation • Rare: Severe disease • Extreme skewing of X-inactivation • Girl with Turner Syndrome • Father: hemophiliac + Mother: Carrier

  14. Bleeding in Hemophilia • Primary haemostasis: Normal • Formation of a platelet plug • Secondary haemostasis: Bleeding • Stabilization of the plug by fibrin: • Defective (inadequate amounts of thrombin)

  15. Trifluorophosphine

  16. Genetics Variability and Penetrance: Fully penetrant PND: Just for severe Hemophilia

  17. Genetics Hemophilia A • X-Linked recessive • Locus Xq 28 • 26 Exons / 186 kb DNA / 9 kb mRNA / 2,332 AA • Molecular Pathology: • Over 150 Mutations • 45%:Inversion in intron 22 • Other mutations (55%): • 90-95% Point mutation • 6% deletions

  18. Inhibitors • 5-10 % of patient: • Don’t have a detectable mutation on sequencing of Factor VIII • No native Factor VIII • develop anti-factor VIII Antibodies with therapy • Complicate treatment!

  19. Antibodies that inactivate Factor VIII in response to treatment • Hemophilia A: • Incidence: 35 % • Inversion in intron 22 • Large deletions • Nonsense mutations • Incidence: 5 % • Small deletion • Missense mutations

  20. Genetics Hemophilia B (Christmas disease) • X-Linked recessive • Locus Xq27 • 34 Kb of DNA 8 exons • less severe than Hemophilia A

  21. Molecular Pathology:Hemophilia B • Complete/partial gene deletion • Point Mutation (67 %): • These patients have defective mRNA splicing • Short insertions (7 %) • Deletion (3 %) • Rarely Patients: Anti IX factor antibody

  22. Antibodies that inactivate Factor IX in response to treatment • Hemophilia B: • Incidence: 50 % • Deletions/rearangements • Incidence: 20 % • frameshift • Premature stop • Splice-site mutations • Incidence: 0.0 % • Missense mutation

  23. Inhibitors Less common in hemophilia B than hemophilia A

  24. Mutations in Hemophilia • Nonsense Mutation • Truncated Protein • Severe Hemophilia • Chromosome Inversion (about 45%( • Intron 22 (Xbal) • Severe Hemophilia • Pointmutation in CG sequence • Hot spot • Missense mutation

  25. Carrier detection • Intragenic RFLPs • Closely extragenic RFLPs For E. BclI, Xbal (Fac.VIII) For E. XmnI, TaqI (Fac.IX)

  26. GENETIC LINKAGE • Mendel's original experiments • "law of independent assortment" • genes are transmitted from parents to offspring independently of one another

  27. Haplotype • Any combination of genotypes can occur • The particular combination present in a given individual is called a haplotype

  28. Inheritance of linked genes d

  29. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS • Genes can be mapped by linkage studies with polymorphic markers • which are nucleotide sequences identifiable at specific sites along the genome • Numerous markers have been identified throughout the genome using restriction endonucleases • and so it is possible to construct maps of disease genes in relation to closely linked markers

  30. THE POLYMERASE CHAIN REACTION - PCR

  31. The restriction enzyme Hind III

  32. Distance between these two genes is about 8 centi-Morgans

  33. MOLECULAR HYBRIDIZATION IS USED TO DETECT SPECIFIC GENES • 1. Single-stranded DNA is generated • 2. A probe is a known sequence of part of gene to be identified tagged with a radioactive label • Specific probes are synthesised in the laboratory • 3. The probe hybridizes only to the fragment with the corresponding sequence • This is detected by the label , which gives a fluorescent signal

  34. MOLECULAR HYBRIDIZATION IS USED TO DETECT SPECIFIC GENES

  35. DNA Extraction from bloodHemophilia A

  36. PCR Analysis from BclIHemophilia A

  37. PCR - Running

  38. Xbal Polymorphism (Factor VIII-Intron 22) in a family with Hemophilia A

  39. Sequencing

  40. Prenatal Diagnosis (PND) • PND (10.-16. w.): • CVS (Chorionic Villus Sampling) • AF (Amniotic Fluid)

  41. CVS (10.-12. weeks)

  42. AF (15. -16. weeks)

  43. First Treatment Factor VIII / IX • Purify/ Prophylactic factor VIII / IX from donor plasma: • Again bleeding • Again enlarged knee joint

  44. The major drawback of donor derived factor VIII • Contamination by virus • Hepatitis B/C • Human Immune deficiency Virus (HIV)

  45. Second Treatment Recombinant factor • Advantage: • No possibility of viral contamination • Disadvantage: • Generate inhibitor antibody (in minority of patients)

  46. Gene Therapy Hemophilia B • Factor IX-containing Adeno- associated virus vector • Injection in to skeletal muscle • Very small increase in factor IX level (about 1%) • The requirement f. factor IX Infusion: fell by 50-80% f. 3 Months

  47. Gene Therapy Hemophilia B • No untoward effects such as: • Germline transmission • Anti body again factor IX

  48. Exceptions in hemophilia A • Germ line Mosaicism • Manifesting heterozygotes • (5% of heterozygous females :Low level of F. VIII) • Mild Hemophilia • Mobile Elements (Transposons) • LINEs: From Ch X to Ch. 22

  49. Von Willebrand disease (VWD)

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