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Hemophilia Dr. Pupak Derakhshandeh (PhD) Assis. Prof. Med. Sci. of Tehran Univ . Major bleeding disorders (50%) Hemophilia A (factor VIIIdeficiency) Hemophilia B (factor IXdeficiency) Clinically: indistinguishable! 5/23/2017 2 Other bleeding disorders (50%) Factor XI- deficiency Von Willebrand disease (VWB) 5/23/2017 3 Hemophilia (A/B) phenotypic features Severe bleeding and Hemorrhages in the wounds, Joints, ankles, knees, hips & elbows, muscles Hematomas & Bruising 5/23/2017 4 Hemophilia A The enlarged knee join 5/23/2017 5 Hemophilia A The enlarged fore head 5/23/2017 6 Hemophilia A Extensive bruising of the fore arm and hand 5/23/2017 7 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% 5/23/2017 8 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 5/23/2017 9 How is ’Hemophilia’ inherited? 5/23/2017 10 X-Linked Inheritance A pedigree of Hemophilia in European royal families 5/23/2017 11 Disease Etiology Hemophilia A/B Mutation in VIII & IX genus (a key component of the clotting cascade) Incidence: Hemph.A1:5000-10.000 male Hemph.B1: 30.000-100.000 male 5/23/2017 12 Mild Hemophilia in female • Disadvantageous x-inactivation • Rare: Severe disease – Extreme skewing of X-inactivation – Girl with Turner Syndrome – Father: hemophiliac + Mother: Carrier 5/23/2017 13 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) 5/23/2017 14 Trifluorophosphine 5/23/2017 15 Genetics Variability and Penetrance: Fully penetrant PND: Just for severe Hemophilia 5/23/2017 16 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 5/23/2017 17 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! 5/23/2017 18 Antibodies that inactivate Factor VIII in response to treatment • Hemophilia A: – Incidence: 35 % • Inversion in intron 22 • Large deletions • Nonsense mutations – Incidence: 5 % 5/23/2017 • Small deletion • Missense mutations 19 Genetics Hemophilia B (Christmas disease) X-Linked recessive Locus Xq27 34 Kb of DNA 8 exons less severe than Hemophilia A 5/23/2017 20 Molecular Pathology: Hemophilia B Complete/partial gene deletion Point Mutation (67 %): These patients splicing have defective mRNA Short insertions (7 %) Deletion (3 %) Rarely Patients: Anti IX factor antibody 5/23/2017 21 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 5/23/2017 22 Inhibitors Less common in hemophilia B than hemophilia A 5/23/2017 23 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 5/23/2017 24 Carrier detection Intragenic RFLPs Closely extragenic RFLPs For E. BclI, Xbal (Fac.VIII) For E. XmnI, TaqI (Fac.IX) 5/23/2017 25 GENETIC LINKAGE • Mendel's original experiments • "law of independent assortment" • genes are transmitted from parents to offspring independently of one another 5/23/2017 26 Haplotype • Any combination of genotypes can occur • The particular combination present in a given individual is called a haplotype 5/23/2017 27 Inheritance of linked genes d 5/23/2017 28 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 5/23/2017 29 THE POLYMERASE CHAIN REACTION - PCR 5/23/2017 30 The restriction enzyme Hind III 5/23/2017 31 Distance between these two genes is about 8 centi-Morgans 5/23/2017 32 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 a5/23/2017 fluorescent signal 33 MOLECULAR HYBRIDIZATION IS USED TO DETECT SPECIFIC GENES 5/23/2017 34 5/23/2017 35 DNA Extraction from blood Hemophilia A 5/23/2017 36 PCR Analysis from BclI Hemophilia A 5/23/2017 37 PCR - Running 5/23/2017 38 Xbal Polymorphism (Factor VIII-Intron 22) in a family with Hemophilia A 5/23/2017 39 Sequencing 5/23/2017 40 Prenatal Diagnosis (PND) PND (10.-16. w.): CVS (Chorionic Villus Sampling) AF (Amniotic Fluid) 5/23/2017 41 CVS (10.-12. weeks) 5/23/2017 42 AF (15. -16. weeks) 5/23/2017 43 First Treatment Factor VIII / IX Purify/ Prophylactic factor VIII / IX from donor plasma: Again bleeding 5/23/2017 Again enlarged knee joint 44 The major drawback of donor derived factor VIII Contamination by virus Hepatitis B/C Human Immune deficiency Virus (HIV) 5/23/2017 45 Second Treatment Recombinant factor Advantage: No possibility of viral contamination Disadvantage: Generate inhibitor antibody (in minority of patients) 5/23/2017 46 Gene Therapy Hemophilia B Factor IX-containing Adenoassociated 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 5/23/2017 47 Gene Therapy Hemophilia B No untoward effects such as: Germline transmission Anti body again factor IX 5/23/2017 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 5/23/2017 49 Von Willebrand disease (VWD) 5/23/2017 50 VWD • In 1926 a Finnish physician, Dr von Willebrand, discovered a clotting disorder • It is not sex linked • is found in one form or another in over 30 breeds of dogs • It is not hemophilia but it's the most commonly inherited bleeding disease of both people and animals 5/23/2017 51 5/23/2017 52 Von Willebrand Autosomal dominant disorders Highly variable in expression Incidence: 1:10 000 Chromosome 12q As a carrier protein for factor VIII 5/23/2017 53 Disorders of platelet function • Functional disorders of platelets are relatively rare • most of these disorders are mild and may not be recognized early in life 5/23/2017 54 The hallmark of von Willebrand disease • defective adhesive glycoprotein von Willebrand factor 5/23/2017 55 protein VWf • • • • • This factor is a large multimeric protein Synthesized Processed and stored in the the endothelial cells 5/23/2017 56 von Willebrand protein • acts as a carrier • stabilizer of coagulation factor VIII: – From premature photolytic degradation – Concentrates it at site of injury • In the absence of vWf, the factor VIII level is low • In classic hemophilia A, the factor VIII level is low because of a deficiency of factor VIII itself • whereas in von Willebrand disease, the factor VIII level is low because of a deficiency in its carrier protein 5/23/2017 57 von Willebrand disease • relatively mild bleeding disorder • except in the occasional patient who is homozygous for the defect • The bleeding manifestations are predominantly skin-related (ie, easy bruising, epistaxis) • Most bleeding episodes occur following trauma or surgery • In women, menorrhagia is common • Pregnant patients with this disease usually do not have problems 5/23/2017 58 Variants of von Willebrand disease • the common form of von Willebrand disease (type I) – a quantitative deficiency of vWfA • A common variant (type IIA) results from: – functionally defective vWf that is unable to form multimers 5/23/2017 59 • Type III von Willebrand disease is a severe form of von Willebrand disease: – characterized by very low levels of vWf – and clinical features similar to hemophilia A – but with autosomal recessive inheritance – It results from a homozygous state or double heterozygosity 5/23/2017 60 -/y +/+ +/y -/+ +/y +/y -/y +/y -/+ • An RFLP was found to be associated with the X-linked recessive disorder hemophilia A in a particular family. Individual II-1 is affected. Assuming that no recombination has occurred, what is the status of the individuals in generation III with respect to hemophilia A? a. III-1 and III-3 are affected, III-2 is normal, and III-4 is a carrier. • b. III-1 and III-3 are affected, III-2 is normal, and III-4 is not a carrier. • c. III-1 and III-3 are normal, III-2 is affected, and III-4 is a carrier. 5/23/2017 • d. III-1, III-2, and III-3 are affected, and III-4 is a61 carrier. Sickle Cell disorder 5/23/2017 62 Sickle Cell disorder Stuck the red cell in the vessels In children: Spleen, chest, wrists,ankles In adults: hips and shoulders Anemia (Hb 7-8 g/dl) Infections (take antibiotics) Painful crises (6-18 months) Swollen and inflamed (hand/food syndrome) 5/23/2017 63 What are the Complications? • • • • • • • • • pain episodes increased infections bone damage yellow eyes or jaundice early gallstones lung blockage kidney damage and loss of body water in urine painful erections in men (priapism) blood blockage in the spleen or liver (sequestration) • eye damage • low red blood cell counts (anemia) • delayed growth 5/23/2017 64 5/23/2017 65 5/23/2017 66 Prenatal diagnosis I. ARMS-PCR (one mutation) II. PCR-RFLP (one inf. RFLP) III. RDB (one mutation) IV. Sequencing 5/23/2017 67 ARMS-PCR 1 5/23/2017 2 3 4 5 6 7 8 9 10 11 12 13 68 5/23/2017 69 5/23/2017 70 Doesn't cause any health problem α+ / º Thalassemia/Thalassemia α+ / º Thalassemia / HbC α+ / º Thalassemia / HbD α+ / º Thalassemia / HbE α+ / º Thalassemia / HbO Arab α+ / º Thalassemia / HbS 5/23/2017 71 Other combinations HbC / Thalassemia (no problem) HbD / Thalassemia (no problem) HbE / Thalassemia (serious anemia) Hbs / Thalassemia (intermediate-severe) HPFH* / Thalassemia (no problem) *Heriditary persistance of fetal hemoglobin 5/23/2017 72 serious anemia HbH / α+/º Thalassemia HbS / Thalassemia HbS / HbC HbS / HbD HbS / HbE HbS / O Arab 5/23/2017 73 Toxic protein alterations • Usually missense mutations: – Cause structural alteration in mono- or oligomeric proteins – Disrupt normal function – Lead to toxic products or precursors • Sickle cell mutations (hem S, b6Glu>Val)* • * Although : recessive • Coinheritance in cis (hem S+ b23Val>Ile) – Sickling to manifest in the heterozygote! 5/23/2017 74 Prenatal Diagnosis (PND) 5/23/2017 75 Grateful Mrs.Rahmani, Manijeh (MS) Mrs.Attaran, Elham (MS) Mrs.Taeb, Fatemeh (MS) Mr. Babr-Zadeh, Farbod (MS) Mr. Sajedi-Far, MohammadMehdi (BS) 5/23/2017 76