Molecular Genotyping and Laboratory Analysis of Hemophilia A in West Bank, Palestine

Abstract

Background: Hemophilia A (HA) is a sex-linked bleeding disorder resulting from deficiency or dysfunction of coagulation FVIII, a plasma glycoprotein that plays an important role in the blood coagulation cascade. HA occurs in one in every 5000 male births. More than 1200 mutations have been identified in the F8 gene and associated with HA. These mutations vary from single nucleotide substitution to gross deletions/insertions and inversions. Intron 22 inversion and intron 1 inversion account for approximately 40–50% and 2- 5% of severe HA cases, respectively. Objectives: Screening for the molecular defects has become a crucial tool in hemophilia care with respect to prediction of the clinical course and safe genetic counseling of relatives. Therefore, the aim of the present study was to genotype the F8 gene and conduct a laboratory analysis of hemophilia A in the West Bank, in order to provide proper diagnosis and management of patients. Methods: A total of 79 HA cases were enrolled (73 males and 6 females) from 49 unrelated families. Hematological and coagulation screening tests were conducted for each patient including: Complete blood count (CBC), partial thromboplastin time (PT) and activated partial thromboplastin time (APTT). Coagulation FVIII activity assay (FVIII: C) was performed by using one-stage clotting assay. High molecular weight genomic DNA was prepared using salting out method. Nested Long Distance Polymerase Chain Reaction (NLDPCR) was performed for detection of F8 gene intron 22 inversion (Inv22) for all severe HA cases as well as carrier mothers, while multiplex PCR was used for detection of F8 gene intron 1 inversion (Inv1). DNA sequencing was used to analyze F8 gene mutations for mild and moderate HA patients as well as for those who were negative for both inv22 and inv1. Results: Depending on both FVIII activity levels and genotyping of F8 gene, 54 (74%) cases were grouped as severe, 9 (12.3%) cases as moderate and 10 (13.7%) of the study cases as mild hemophilia A. Analysis of inv22 by NLD-PCR showed that 57.4% of severe HA patients have this inversion, while analysis of inv1 by multiplex PCR revealed that two patients were positive for inv1 with a percentage of 3.7% among all HA severe cases. One sample has been completely screened and the disease-causing mutation has been identified, namely IV (c.388G>A) that result in substitution of the amino acid glycine at codon 130 by arginine (p.Gly130Arg) in A1 domain of FVIII protein. In another 4 samples, so far 3 harmless SNPs were identified. Only 3.7% HA patients receive prophylactic FVIII replacement therapy and the rest (86.3%) were on-demand treatment. Most patients (76.7%) have a family history of HA and 23.3% have no family history of HA. Conclusion: The frequency of F8 gene inv22 and inv1 were found in 57.4% and 3.7% among severe HA patients, respectively. About 23.3 % of HA patients have no family history and thus the development of HA is attributed to de novo mutations. Further investigation of HA cases by DNA sequencing is necessary to detect F8 gene mutations in cases that were negative for inv22 and inv1.