Detection of Factor VIII Gene Mutations in Sudanese Patients with Hemophilia-A

Abstract

Hemophilia A (factor VIII deficiency) is the most common hereditary disorder of blood coagulation. It is due to the absence or decreased function of coagulation factor VIII, resulting from mutations in the factor VIII gene. The aim of the study was to screen the factor VIII gene mutation among Sudanese patients with hemophilia A. This analytical cross sectional study conducted in Khartoum teaching hospital in patients with hemophilia A who attended to hemophilia center, 72 patients with hemophilia A were selected, 5 ml of blood samples were taken in K2 EDTA for DNA extraction for the molecular studies and 5ml tri sodium citrate for APTT, PT, factor VIII assay and factor VIII inhibitor by manual methods. For the molecular studies a master mix and conventional PCR were used. Twenty primers were used as followed: four for Intron 22 inversion mutations. Four for Intron 1 inversion mutation. The twelve primers were used for screening of six exons sequence by sequencer. For exon screening for mutations 120 PCR products were sequenced and analyzed by BLAST and FASTA in the NCBI web site, in which query and subjects procedure was used. PCR products were tested by agrose gel electrophoreses and gel documentation system. The result showed All the 72 patients were males factor VIII activity less than 1% were 59 patients (81.9%) and 13 patients (19.1%) between 1 - 5%. VIII inhibitor 62 patients (80.9%) were negative while 10 were positive (13.8%). All patients were normal PT and elevated APTT. Two patients out of 72 (2.7%) were positive Intron 1 inversion mutation. For exons sequencing there were deletions mutations in three out of ten samples (30%), in exons 11, 23 and exon 24. About fifty PCR runs were done for intron 22 inversin using conventional LD-PCR, but no products were found except the ladder bands.