Analysis Of FⅧ Gene Defects And Clinical Manifestations In Hemophilia A Patients

ObjectiveThe study aims to further explore the molecular pathogenesis of patients with hemophilia A by analizing the genetic defects and epigenetic dysregulation which possibly contributes to the clinical phenotype diversity of hemophilia A. Which help us deeply understand its’ pathogenic molecular mechanism and find more information to explore the treatment of hemophilia A.Methods24 confirmed hemophilia A patients from Chongqing was randomly selected, whose clinical and laboratory data was collected by retrospective review of medical record or messages got by telephone following-up. LD-PCR(long distance-polimerase chain reaction) and multiple-PCR within two tubes were used to identify the intron 1 inversion and intron 22 inversion respectively. High-throughput sequencing were used to analyze the mutation of the exons of FⅧ gene. And mass spectrometric technology were used to study the promoter region methylation patterns of FⅧ gene for hemophilia A patients and normal control. All detected mutations was compared with Pumbmed、UCSC、Factor Ⅷ Variant Database for identifying new mutation types which haven’t yet been reported. Combining with the mutation data and inversion results, we analyzed the clinical manifestations of the 24 patients. While the methylation in the promoter region between case and control was analysed by F-test.Results1.The number of patients with FⅧ:C<1%,1%~5% and >5% was 13(54.1%),9(37.5%)and 1(4.2%)respectively. There is one case(4.2%) whose FⅧ:C was 8.6% after fresh frozen plasma infusion was classified into intermediate-severe type. The 24 patients’ age ranges from 2 years to 48 years. All the patients’ clinical manifestations were consistent with their severity classification.2. Due to the reason of blood sample’s quality,18 out of 24 samples were qualified for inversion detection test. And intron 22 inversion was found in 7 cases(38.9%), no intron 1 inversion was identified.3.Mutation was not found in 7 of the 24 patients through High-throughput sequencing. 21 mutations(15 types) were detected in the remaining 17 cases. 10 out of the 15 types haven’t been found yet before after comparing with Pumbmed、UCSC and Factor Ⅷ Variant Database.4. The methylation level of the Cp G island in the promoter region between the patients and control group has no significant diference.ConclusionHemophilia A, a deficiency of clotting factor Ⅷ, is a X-linked congenital disorder. Patients who suffer from it result in loss of nomal hemostasis and manifest with spontaneous or induced bleeding depending on the severity of the disease. FⅧ gene is fairly large gene which spans 186 kb. Mutations that cause severe destruction of FⅧ gene often cause severe phenotype such as inversion or nonsense mutation. Although there are multiple genetic defects, defects found to now can not completely explain its’ clinical heterogeneity. And methylation patterns of the CpG island in the promoter region of FⅧ gene is found no relationship with defective FⅧ synthesis. Thus it needs much efforts to study.