Genotyping of Intron 22 and Intron 1 Inversions of Factor VIII Gene Using an Inverse-Shifting PCR Method in an Iranian Family with Severe Haemophilia A

Abstract Background: Haemophilia A (HA) is an X-linked bleeding disorder caused by the absence or reduced activity of coagulation factor VIII (FVIII). Coagulation factors are a group of related proteins that are essential for the formation of blood clots. The aim of this study was to genotype the coagulation factor VIII gene mutations using Inverse Shifting PCR (IS-PCR) in an Iranian family with severe Haemophilia A. Material and Methods: Genomic DNA was extracted from blood of Iranian family members with severe hemophilia A and then was genotyped using specific primers by Inverse Shifting PCR method and was analyzed by sequencing for all FVIII exons. Results: Sequence analysis of F8 gene revealed two distinct mutations. The first mutation was a C-to-G transition at 3780 position in exon 14, which cause an Asp1240 Glu in the region encoding the B domain of FVIII. It seems that this mutation could be a polymorphism. The second mutation was a 2-bp AA deletion in exon 18 (nt. 5820-5823, del. AA). The patient's mother and sister were also heterozygous for 2bp AA deletion. This deletion caused a frame shift in exon 18 and terminated after 29 amino acids for a premature stop codon. Conclusions: Based on the results, it can be concluded that two IS-PCR and genomic sequencing techniques are robust and low cost method that facilitates the analysis of HA patients and carrier detection.