Gene Diagnosis And Study Of Mechanism In Patients With Hemophilia A

Part One Gene Diagnosis and Study of mechanism In SevereHemophilia A Patients【Objectives】Hemophilia A(HA) is a recessive X-linked inherited disorder with an incidence in males of 1:50000 and rare in females. HA patients have the symptoms of hemarthrosis、deep-muscle hematomas、intracranial bleeding with mild or no antecedent trauma. Disease severity is classified as mild, moderate, or severe based on residual clotting factor activity(>5, 1to5, and<1 IU/d L, respectively). Although the factor VIII(FVIII) concentrate replacement therapy can strongly reduces HA-related mortality and disability, its cost is high for HA patients, especially for severe hemophilia A. So gene diagnosis is an important method to detect the carrier to reduce the birth rate in patients with severe hemophilia A.【Methods】Blood samples of 43 male patients with severe HA who were diagnosed in our hospital from 2009 to 2014 were collected. We screened the presence of intron 22 and intron 1 by Long distance-PCR(LD-PCR) and multiple PCR.Other mutations were studied with polymerase chain reaction(PCR) and direct sequencing, the trail effects were compared with normal sequenced of FVIII gene using Chromas to find the mutation.【Results】All of the 43 severe HA patients, intron 22 inverson was detected in 21 HA patients,accounts for 48.8% of total cases.Except intron 22 inversion, 21 mutations were detected in the other 22 HA cases, including 6 nonsense mutations, 5 missense mutations, 2 splice sitemutations and 9 frame mutations. Meanwhile, fourteen kinds of mutation have been reported. Seven mutation were found for the first time,including c.214G>T(E72X),c.215T>C( F73S), IVS5+2G > A( splicing), c.2519C>G(S840X), c.3180 del A(V1061Xfs),c.5639 del A(N1880Tfs*3),c.6572 del A(N2191Mfs*18).【Conclusions】There is obvious heterogeneity in FVIII gene mutation and the intron 22 inversion is still the main way of mutation of severe cases in our study. The analysis of pathological mechanism of hemophilia A provided the foundation for further studies to the function of coagulation factor VIII. Gene diagnosis is a rapid、safe、reliable method for carriers diagnosis and prenatal diagnosis.Part two The detection and mechanism of factor VIII inhibitor insevere hemophilia A patients【Objectives】Hemophilia A(HA) is an X-linked recessive bleeding disorder due to a deficiency or functional deficit of coagulation VIII. At present, factor VIII(FVIII) replacement therapy is the main method of treatment for HA patients. Although replacement therapy can strongly reduce HA-related mortality and disability, it also can lead to FVIII inhibitor development. FVIII inhibitor is a kind of antibody which can neutralize the activity of FVIII. Such inhibitor can lead to decreased effectiveness of FVIII replacement therapy, increase disability and death rate. With the development of the hemophilia A treatment, the incidence of FVIII inhibitor is growing in HA patients, especially in patients with severe HA who have serious bleeding phenotype and need more frequent treatment. There are many reasons for inhibitor formation, including environment factors and genetic factors.The aim of our study is to investigate the elements associated with the FVIII inhibitor development in severe hemophilia A patients.【Methods】We have collected the clinical data of 43 hemophilia A patients who were diagnosed in our hospital from 2009 to 2014, including the age of initial treatment、exposure day. Meanwhile all plasma samples of the HA children were collected and were measured by Nijmegen-modified Bethesda assay simultaneously to detect the inhibitor. We also detect the FVIII gene mutation types of the 43 HA patients. Finally, results through the chi-square test and Logistic regression analysis.【Results】All of the 43 severe HA patients, FVIII inhibitor was detected in nine cases(20.9%), and the median titer of the inhibitor is 0.82 BU. Difference of the gene types between inhibitor positive and negative inhibitor patients was not statistically significant.The same results were found in other elements(the age of initial treatment, exposure day).【Conclusions】In summery, the reason of FVIII inhibitor development is not complete known. How to reduce the incidence of inhibitor is still a problem and should be further studied. As treatment options evolve, stratification of patients by inhibitor risk will be of major clinical significance, from both a clinical and health-economical perspective, to individualize and optimize treatment. Part three A novel missense mutation, p.Phe314 Cys, in F9 gene resultsin hemophilia B in female patient【Objectives】Hemophilia B(HB) is rare in females and only a few cases have been reported.In this study, we report a woman with hemophilia B. According to analyze the phenotype and genotype of the case, we try to investigate the molecular mechnism of hemophilia B in female.【Methods】A female patient complained of intracranial hemorrhage because of head injure, with a histories of menorrhagia, spontaneous retroperitoneal hematoma and abnormal postpartum bleeding. Coagulation parameters were detected in the proposita、her parents and her daughter who were not consanguineous marriage, including APTT、PT、FVIII:C 、FIX:C、FVIII:C and FIX inhibitor. The antigen of FIX in plasma was evaluated by western blotting. In order to explore the molecular mechanism, all exons of F9 gene were sequenced after PCR amplification. An expression plasmid carrying c.1108T>G mutation was constructed with site-directed mutagenesis method based on the wide-type(WT) F9 expression plasmid, and was transiently transfected into HEK293 cells. The conditional media and the cell lysates were analyzed by western blotting. The X chromosome inactivation pattern(XIP) was determined by PCR analysis of highly polymorphic dinucleotide repeats in the ZNF261 gene.【Results】The coagulation parameters of the proposita showed a prolonged APTT(62.2s), markedly decreased FIX:C(2%), but PT and FVIII:C in normal range. The inhibitor of FIX was negative in plasma of proposita. However, all coagulation parameters of her parents and her daughter were normal. A novel unreported missense point mutation(in exon 8 of FIX gene, c.36029T>G, p.Phe314Cys) was found in heterozygous state in the proposita, but this mutation wasn’t found in her parents and daughter. Also we found that protein level of FIX in plasma of the proposita was the same as the normal person by western blotting. The expression experiment in vitro revealed that the conditional media of F9 WT and the conditional media of F9 MT contain the similar protein level of FIX. Meanwhile the same result is found in the cell lysates. According to assess the X-inactivation, we found that the proposita’s maternally derived X chromosome was inactivated mostly while the majority of her paternal derived one kept active.【Conclusions】Here we identified a female patient with hemophilia B, due to a novel missense mutation(c.36029T>G, p.Phe314Cys) in FIX gene. Located in catalytic domain of FIX, this mutation impaired the FIX activity, but didn’t affect the synthesis and secretion of FIX. Futhermore this mutation in FIX gene didn’t be detected in her parents and the X-chromosomes derived from her mother were inactivated, we postulated that this mutation may originate from her father’s spermatogonium, all of which finally resulted in phenotype of hemophilia B in proposita.