FⅧ Gene Mutation Spectrum In Chinese Hemophilia A Patients

FVIII gene mutations spectrum of Chinese hemophilia A patientsBackground and Objectives Hemophilia is a common X-linked recessive bleeding disease which affects 1 in 5000 males. As the prevalence of plasma-derived concentrates and recombinant FVIII products in replacement therapy, the life quality of HA patients improved significantly. A severe complication in the replacement therapy is the development of alloantibodies (inhibitors) against FVIII that can neutralize the substituted FVIII. The development of inhibitors is related to multiple factors including type of FVIII gene mutation, severity of HA, race of patients and exposure days of FVIII products. Although china has vast population of HA patients, the FVIII gene mutation spectrum and prevalence of FVIII inhibitor development was rarely studied. The detection of FVIII gene defect can contribute to the genetic counseling, assessment of risk of FVIII inhibitor and better comprehension of pathogenesis of HA. The aim of this study is to explore the FVIII mutations spectrum in Chinese HA patients and the incidence of FVIII inhibitors. Methods we performed mutation screen using LD-PCR for intron 22 inversion detection, using multiplex PCR for intron 1 inversion detection and directly sequencing of coding area(every exons, boundary of intron and exon,5' promoter and 3' untranslated region ) of FVIII gene which were amplified by PCR. The FVIII: C was detected using a one-stage method and FVIII antibody of patients was assayed using Bethesda method.Results Total 145 HA patients were enrolled in this study: 107 severe, 35 moderate and 3 mild patients. The recurrent mutations in severe patients are inversion of intron 22 and intron 1: the percentage was 53.2% (57/107) and 2. 8% (3/107) respectively. Apart from inversion of intron, 64 mutations were detected: 8 nonsense mutations (5 novel), 7 insertion mutations (3 novel), 12 deletion mutations (8 novel), 4 splice site mutations (2 novel), 34 missense mutations (15 novel). Among 145 patients, 2 were detected with FVIII inhibitor and thus the incidence in patients in this cohort is 1.4%. The genotype of two HA patients were both detected with inversion of intron 22.Conclusion 1. In this paper, we characterize the genotype of 145 patients with HA and showed a similar distribution with those previously reported. 2. The incidence of intron 22 and 2 is 53% and 3% respectively. As being recurrent mutations and can be detected derictly, they play an important role in genetic counseling.3. We reported 33 novel mutations, include 3 nonsense mutations, 3 insertion mutations, 8 small deletion mutations, 2 splice mutations and 15 missense mutations. We further illustrate the potential effects induced by these novel mutations by bioinformatic analysis.4. The incidence of FVIII inhibitors in our cohort is lower than reported previous in other ethnic. It might be partly due to the small patients enrolled in this cohort. The risk factors and impact of ethnic need to be further studied with large cohort. Genetic analysis of an inherited afibrinogenemia family caused by a novel frameshift mutation in FGAObjective To identify the genotype in a Chinese family with inherited afibrinogenemia. Methods Samples of peripheral blood were collected from six members of 3 generations. Coagulation studies including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (Fg, clauss) were tested. Fg was also analyzed using immunoturbidimetry method. DNA of six family members was extracted using a DNA extract Kit. All the exons and exon-intron boundaries of the three fibrinogen genes were amplified using PCR and analyzed by direct sequencing. Results Parents of proband were 3 degree consanguinity. A homozygous c.934₉35insA in FGA, which results in the change of protein p.Ser312fsX42, was detected in proband. The parents, grandmother, maternal grandmother and father's sister were all detected with heterozygous mutation as the same with proband.Conclusion Homozygous c.934₉35insA in FGA is a cause of inherited afibrinogenemia and is a novel mutation being reported.