A Study On Diagnosis Of Hemophilia A

Objects：Hemophilia is a common hemorrhagic disease which is mainlydue to the absence of thromboplastin. The most common one is hemophiliaA(HA). Hemophilia A, also called hereditary antihemophilic globulindeficiency or FⅧ:C deficiency，is an X-linked recessive genetic disease.Approximately85%congenital bleeding disorders belong to HA. Theincidence of HA in male live births is approximately1/5000, female aremainly carriers and patients are rare in women. The function of coagulationfactor Ⅷ deficiency or lack of content caused by the coagulation factor genemutations is the main cause of the disease occurred. The clinicalmanifestations is spontaneous repeated or after minor trauma on the wholebody bleeding in childhood. Except for the skin, mucous membrane bleeding,bleeding in muscles and joints are the important feature of HA. Repeated jointbleeding often leads to joint deformity, and even bleeding in vital organs canbe life threatening. Severity of HA is primarily associated with FⅧ activity.The order of severity of HA is depending on the FⅧ:C. The FⅧ:C of severeHA is less1%, and the moderate is at1%～5%.And the FⅧ:C of the lighttype is at5%～25%.The other,s FⅧ:C is at25～45%,which is calledsubclinical HA.At present, there is no effective curative treatment for HA. To sustain thelives of patients, the only methods are to rely on alternative therapies orsymptomatic treatment. To meet the clinical fertility requirements of HAsuspicious carriers, the most effective way is to implement prenatal geneticdiagnosis for screening out the sick fetus to reduce the birth rate in childrenwith hemophilia. The prenatal diagnosis for HA is mainly to detect the riskmale fetuses and the carrier of female fetuses. As one method for screeninghigh risk fetuses, the activity assay of FⅧ in cord blood have been to do somefurther research and the assay result can not be used as the diagnosis evidence. It showed that inversion of intron22in FⅧ gene is a significant type of factorFⅧ mutation, and it lead to about30%~40%severe HA. And another reasonfor other5%severe HA is inversion of intron1mutation in FⅧ gene, whichis discovered recently. So, when gene diagnosis was done, the inversion ofintron22or intron1were the firstly be detected to determine if the twomutation hotspots were involved into HA. After the two mutation hotspotswere excluded, the sequencing test for whole FⅧ gene was the preferredmethod for finding the risk mutation sites.In this study, in order to determine if the HA patients were severe, theactivity of coagulation factor FⅧ:C from HA patients was detected. Then, theinversion of intron22and intron1were analyzed for the severe HA. Finally,whole FⅧ gene sequencing test was done to try to find other mutation for thesevere HA patients who carry the inversion of intron22or1mutation. In short,the objective of this study is to establish the methods for clinical diagnosis HA,including testing for inversion of intron22and1mutation in FⅧ gene, aswell as other mutations.Methods：The concentration of FⅧ (FⅧ: C) were detected with onestage method in33HA patients. Detection of Factor Ⅷ gene inversionamong33patients with hemophilia A by long distance-PCR and0.6%agarosegel electrophoresis. Multiple-PCR were used to detect intron1inversionsrespectively.Seeking other mutations with sequencing technology.Results：All of the33HA are severe,which were diagnosed by FⅧ: C.Intron22inversion was detected in13HA patients, which accounted for about39.4%of total detected patients.And intron1inversion was detected in1HApatients, which accounted for about3%of total detected patients.Did not findother mutation by sequencing technology.Conclusions：There are many kinds of hemophilia,and the coagulationfactor Ⅷ activity is the confirmed diagnosis test of HA.The intron22inversion and the intron1inversion are the most important pathogenesis.It cancurtail expenditures if we making sequencing after the intron22inversion andthe intron1inversion test.We can detect the intron1inversion by multiple-PCR,which is simple and convenient.We can use the longdistance-PCR in clinical because of it high-performance and doing withoutradioactive source.