Haemophilia B And Von Willebrand Molecular Pathogenesis Studies

ObjectiveThe hemophilia B (HB), which is caused by the mutations in the factorâ…¨gene, is known as an X-linked recessive disease and occurs in about 1:30000 male live births. At present, due to lacking of the eradicative therapy, the gene diagnosis and detection of the carriers, which is an effective method to prevent the infant patients to be born, block the transmission of harmful gene and improve the population quality, should be actively carrying out. The aim of this study is to diagnosis the propositi and carriers in the gene level and explore the molecule pathogenesy of HB.Methods1. The three unrelated HB families gave informed consent to be included in the study. The genome DNA were collected from each propositus and the family member.2. The polymorphisms of the six STR loci were detected by polymerase chain reaction with multiple reaction system and the fluorescently-labeled primers.3. For the propositi and doubtful carriers, all regions of Fâ…¨gene, including all exons and the flanking sequences, were amplified by PCR using the primer sequences which were devised with Primer five. The products of PCR were sequenced by the dideoxy chain termination using ABI 3700 sequencer, the trial effect was compared with normal sequences of Fâ…¨gene using Chromas for finding the mutations.Results1. 9 doubtful carriers were been found through the allele analysis. But confirmed by the direct sequencing, the doubtful carriers of the first and third family did not have the same gene defects as the propositi, the gene defects were spontaneous mutations. To the second family's carrier, the heterozygous mutation was found in the corresponding locus of the propositus, the gene defect of the propositus was transmitted from his mother.2. 3 missense mutations were identified in Fâ…¨gene of the propositi when compared with normal sequence. G22119A was identified in exon 6 of the first family's propositus, it existed in the shearing situs of the flanking sequences, affected the normal physiologic function of Fâ…¨. G7932C (Glu8Asp) was identified in exon 2 of the second family's propositus, it impacted Fâ…¨binding with phospholipids. T32685C (Cys336Arg) was identified in exon 8 of the third family's propositus, it affected possibly the synthesis and secretion of Fâ…¨intracellular.Conclusions1. The defect of Fâ…¨gene is the molecular pathogenesis of HB.2. Combination analysis of multiple STR loci could be an effective and simple method for indirect diagnosis of the carriers in the HB family. However, there is possible to misdiagnose with the genetic linkage analysis for the families without the family history of HB.3. Gene sequencing is one of the straightest and rigorousest method for the diagnosis of HB. ObjectiveThe von Willbrand disease, which is known as a common inherited bleeding disorder with the prevalence about 10/100000, is caused by genetic defects in the von Willebrand factor (vWF), resulting in quantitative deficiencies or qualitative abnormalities of vWF. The patients have the symptoms of mucocutaneous bleeding, hemorrhinia, gastrointestinal bleeding and unremitting bleeding after injury, female suffers menorrhea often. The aim of this study is to explore the molecular pathogenesis of vWD, investigate the functional and quantitive changes of vWF protein resulting from gene mutations, research the relationship between phenotype and genetype.Methods1. The blood samples were collected from the three unrelated patients with vWD. Through detecting the vWF:Ag, Fâ…§:C, RIPA and vWF multimers to determine the diagnosis and the subtype of vWD.2. The genome DNA were collected from each patient. All regions of vWF gene, including fifty-two exons and the flanking sequences, were amplified by PCR. The products of PCR were sequenced by the dideoxy chain termination, the trial effects were compared with normal sequences of vWF gene using Chromas for finding the mutations.3. The expression vectors, carrying the mutations found in the vWF gene of the patients, were constructed by site-directed mutagenesis. 4. Cos-7 cells were transfected with pSVvWF3467, pSVvWF4738, pSVvWF6424 and wild type plasmid. The recombinant proteins and normal vWF were detected by ELISA and analysed by SPSS 13.0.Results1. Comparing with pooled plasma, vWF:Ag,Fâ…§:C,RIPA of the patients were decreased.2. The vWF multimers assay disclosed that the vWF multimers of the first patient was absence completely, the high molecular weight multimers was disappeared in the plasma of the second patient, the distribution and structure of vWF was normal in the plasma of the third patient.3. The heterozygous missense mutations were identified in vWF gene of the patients. C6424T (L2142F) mutation was identified in exon 37 of the first patient, C4738G (L1580V) mutation was identified in exon 28 of the second patient, C3467T (T1156M) mutation was identified in exon 26 of the third patient. The first one, a novel mutation, was not reported previously in the international literature.4. The expression experiment in vitro revealed that the antigen levels of vWF T1156M, vWF L1580V and vWF L2142F in media were lower than wild type vWF. In the corresponding transfected Cos-7 cells lysates, the antigen levels of vWF T1156M and vWF L1580V were similar to the level of wild type vWF, however, the antigen level of vWF L2142F was 32.7% of wild type vWF.Conclusions1. The defect of vWF gene is the molecular pathogenesy of vWD.2. The T1156M substitution had a definite effect on the level of vWF expression and secretion in vitro experiments, being substantially less than the secretion of wild type VWF. The space conformation of vWF was changed by the L1580V mutation, the change leaded that the protein had increased sensitiveness with the vWF-cleavring protease. The L2142F mutation resulted in a decreased expression of the recombinant vWF protein, however, the decreased degree is not consistent with the clinical symptom and the laboratory examination report of the patient. The result supposed that the patient might be with a complex heterozygote, there might be an undiscovered morbigenous mutation in the introns or the regulation sequence.