[ study of intron 22 inversion type I and II of coagulation factor 8 gene in patients with severe Hemophilia A using IS-PCR technique]

ABSTRACT

Hemophilia A [HA] is an X-linked recessive bleeding disorder. The disease is caused by mutations in the F8 gene. Inversion of intron 22 is the most common causative mutation of severe hemophilia A, which is detectable by southern blot and polymerase chain reaction [PCR] methods. The aim of this study was to determine intron 22 inversion type I and type II inversion in severe hemophilia A patients by inverse shifting-PCR method. This study was performed on 30 patients with severe hemophilia A with less than 1% of normal level activity of Factor 8. After obtaining consent from the patients, genomic DNA was extracted from peripheral blood leukocytes. The extracted DNAs were used as template for IS-PCR amplification after circularization [digestion by BcII and then ligation of the produced fragments by ligase enzyme]. In 30 severe hemophilia A patients, 40% of the patients had intron 22 inversion type I, and 6.6% of the patients had intron 22 inversion type II. The results of this study revealed that recombination between intron 22h-1 within the F8 gene and its copies [22h-2 and 22h-3], which lie in opposite direction to intron 22h-1, respectively cause the inversions of intron 22 type II and type I. Inversion of intron 22 type I is more frequent than type II. Also, by application of IS-PCR as a cost effective method, we could save time and improve the molecular detection of inversion. This method and can be used for detection of carriers and patients and for prenatal diagnosis of hemophilia A disease