Analysis Of Intron 1 And Intron 22 Inversion In Factor Ⅷ Gene In Patients With Haemophilia A

Background HemophiliaA(HA) is caused by factorâ…§( Fâ…§) gene defects,which is common inherited bleeding disorder and resulted from deficiency and functional abnormality of coagulation factor. It is a classic example of the X-linked recessive inheritance and the incidence is about in five to ten thousand males. The Fâ…§gene located at the end of the long arm of X chromosome, at Xq28. The Fâ…§gene is 186kb long and contains 26 exons and 25 introns. Gene mutation of Fâ…§gene is variegated and highly particular. The severe HA is often caused by the mutation of deletion, inversion and insertion of the fragment of DNA and intron 22 inversion were detected in Fâ…§gene in near 50% severe patients with HA in 1993 report. In recent study found that Intron 1 inversion was a another important molecular pathogenic factor which could result in the severe HA except for intron 22 inversion. The gene inversion may be a higher risk factor to result in Fâ…§antibody in HA patients and the Fâ…§antibody was one of the most severe complication in HA patients. The dead rate was high and the treatment was troublesome in HA patient which were detected the Fâ…§antibody in clinic. In our study the directly gene diagnosis were performed in Fâ…§gene in 81 HA patients and 11 doubtful carriers in 6 patient's family, by the detected firstly of the intron 1 inversion and the intron 22 inversion.Objective To perform testing of the intron 1 inversion and the intron 22 inversion in Fâ…§gene in patients with HA and family, then to investigate the contribution of the gene intron inversion in Fâ…§gene toward the pathogenesis of HA and to investigate the relationship of gene intron inversion with Fâ…§antibody. Some doubtful carriers in some patient's family background were detected with directly gene diagnosis in order to identify the role of the genetics control in HA family.Methods The concentration of Fâ…§(Fâ…§: C) and Fâ…§antibody were detected with one stage method and Nijmegen method in 157 HA patients. Meantime, 81 unrelated patients with HA were directly detected by multiplex PCR and long-distance PCR in search of intron 1 inversion and the intron 22 inversion in Fâ…§gene. And survey of some patient's family background was conducted.Results Among 157 patients with HA, 129 cases were severe, 20 moderate and 8 mild. 5 moderate and mild cases which were diagnosed by Fâ…§: C were found to be intron 22 inversion. 5 moderate and mild cases were rechecked the Fâ…§: C and the Fâ…§: C of these cases were all below the 1% , then these 5 cases were diagnosed finally and were severe cases. Among 81patients which were tested with gene inversion, 65 cases were severe, 13 moderate and 3 mild. In 81 unrelated patients with HA, 3 severe cases were found to be intron 1 inversion which accounted for 4.6% of total 65 severe cases; Fâ…§antibody of 1 case which had intron 1 inversion was to be positive. Two female members in a HA family which had patient with intron 1 inversion were detected in which 1 case was carrier and 1 case was not carrier. In 81 unrelated patients with HA, 25 severe cases were found to be intron 22 inversion which accounted for 38.5% of total 65 severe cases; Fâ…§antibody of 1 cases which had intron 22 inversion were to be positive. Nine female members in 5 HA families which had patient with intron 22 inversion were detected in which 7 cases were carriers and 2 cases were not carriers.Conclusions Intron 1 inversion was a another important molecular pathogenic factor which could result in the severe HA except for intron 22 inversion. Intron 1 and 22 inversion may be a higher risk factor to result in Fâ…§antibody in HA patients. Intron inversion testing could be not only directly used for gene detection in patients and family but also used to rectificate the deviation of experiment grouping in HA patients. LD-PCR is a directly gene diagnosis method for intron 22 inversion. The method of LD-PCR need not use the radicisotope and the application of this method could detect simply, rapidly, accurately and correctly. Above all the fortes the method could be make extensive use in clinic.