Thesis (Ph.D.) - Cairo University - Faculty of Medicine - Department of Pediatrics

Background: Hemophilia A is caused by mutations in the gene encoding coagulation factor VIII (FVIII). In severe Hemophilia A (sHA), two inversions are responsible for approximately 50% of the genetic alterations (intron 22 and intron 1 inversions) and the other mutations are extremely diverse. Development of inhibitor among children with sHA is one of the major complications of treatment affecting around 30% of the patients. Certain F8 mutations might be a risk factor for inhibitor development. Objectives: Our aim was to detect the genetic alterations present in the FVIII gene (F8) in Egyptian children with sHA and to correlate the identified mutations with the risk of inhibitor development.Subjects & methods:Thirty eight male with sHA attending the outpatient clinic in Cairo University children{u2019}s hospital were recruited.The inhibitor status was explored using the Nijmegen Modification of the Bethesda Assay. Recruited children genotype was disclosed following UK guideline for the molecular analysis of hemophilia A. We initially detected the presence of the intron 22 inversion and intron 1 inversion by performing inverse PCR. Pedigrees with negative screening results were then analyzed by confirmation sensitive gel electrophoresis followed by direct sequencing. Results: The causative mutation was identified in 32 patients 15 patients with inhibitors and 17 without had molecular disclosure). Whereas the DNA failed to be amplified for 6 patients (5 with inhibitors and one without) despite repeated attempts of extraction. Intron-22 and -1 inversions were detected in 15 (46.9 %) children and 0% of patients respectively. 33 % of interon-22 inversions had inhibitors. Besides these two mutations, 9 different mutations were identified in 17 children, including 10 splicing mutations, 5 missense and 2 frameshift.Three novel mutations were identified, all were missense mutations. Forty seven percent of the patients with identified mutations developed inhibitors against exogenous FVIII

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