Clinical Analysis And Molecular Characterization In Von Willebrand Disease Patients

Part One Clinical analysis of162patients with von WillebranddiseaseObjective von Willebrand disease is known as the most common inherited bleedingdisorder, which is caused by genetic defects in the von Willebrand factor(VWF), resultingin quantitative deficiencies or qualitative abnormalities of VWF. The patients have thesymptoms of mucocutaneous bleeding, epistaxis, gastrointestinal bleeding and unremittingbleeding after injury. Female suffers menorrhea often.The aim of this study is to explorethe epidemiological feature, clinical characters and therapeutic regimens for vonWillebrand disease(VWD). Methods We have analyzed the clinical data and laboratorytests results of162VWD patients in our center. These tests are generally required forscreening, diagnosis and subtyping. Results There are76males and86female amongthese patients. The mean age were7.2(2.0~41.0) and20.7(5.0~48.0) years respectively.86patients(53.1%) were identified to be type1VWD,34patients(21.0%) were type3VWDand42patients(25.9%) were type2VWD. Among the type2VWD patients,33patientswere type2A,4patients were type2M,5patients were type2B. Eighty-sevenpatients(53.7%) had a definite family history of bleeding tendency. The most common andspecific bleeding symptoms were easy bruising (61.7%), epistaxis(53.7%), prolongedbleeding after surgery or minor injury(53.1%). Menorrhagia(66.3%) was common infemale patients. The analysis of Vicenza bleeding scores in all patients showed that only56(34.6%) patients had abnormal bleeding scores. FVIII/VWF concentrates andcryoprecipitate were applied in45patients(27.8%). Desmopressin(DDAVP) was used onlyin8patients. Eight female patients need oral contraceptives jointly to control menorrhagia.Hysterectomy had to be performed in two female patients with VWD. Oral iron therapywas applied in25female patients complicated with anemia. Conclusion VWD is a common congenital bleeding disorder with heterogeneous characters, it is necessary toscreen, identify, classify accurately this disease, so as to apply effectively individulizedtreatment. Part Two Laboratory diagnosis and molecular characterizationof six von Willebrand disease patientsObjective von Willebrand factor(VWF) is a non-enzymatic plasma glycopmtein,synthesized in endothelial cells and megakaryocytes, that plays a key role in primaryhaemostasis by potentiating platelet adhesion to damaged vascular endothelium and byacting as the carrier protein to factor VIII(FVIII). von Willebrand disease(VWD) is aheterogeneous bleeding disorder characterized by a variety of quantitative(type1and3)and qualitative(type2) abnormalities of VWF. Multiple tests are generally required toensure accurate diagnosis and subtyping in clinical laboratories. Exploring the molecularmechanisms of VWD will help gain more insight into relationship of structure and functionof VWF and add a lot to the diagnosis and treatment. Our study of this part was to revealthe molecular pathogenesis of the6VWD patients, investigate the functional andquantitive changes of VWF protein resulting from gene mutations, explore the relationshipbetween phenotype and genotype. Methods A panel of methods were employed forclinical diagnosis of the six VWD patients and genetic test was applied for molecularanalysis. We use PLT, APTT and Fg for screening; VWF:Ag, VWF:RCo and FVIII:C fordiagnosis; and RIPA, VWF:CB, multimer assay and VWF:FVIIIB for subtyping. Thegenome DNA were collected from each patient. All regions of VWF gene, includingfifty-two exons and the flanking sequences, were amplified by PCR．The products of PCRwere sequenced by the dideoxy chain termination and the trial effects were compared withnormal sequences of VWF gene using Chromas to find the mutations. Results Three type2A and three type3VWD patients were diagnosed. Conventional PCR was carried out toamplify the VWF gene of the six patients. Seven causative mutations[4814A>C(Y1605S),752A>G(E248G),4789C>T(R1597W),421G>A(D141N),3379+1G>A, 2480G>A(C827Y),3897delT] in VWF gene were identified and four[4814A>C(Y1605S),752A>G(E248G),2480G>A(C827Y),3897delT] of them were first reported. Y1605Smutation which was rightly located on the ADAMTS13cleavage site(p.Y1605-M1606)may unfold the normally buried p.Y1605-M1606bond, leading to enhanced sensitivity toADAMTS13cleavage. The E248G mutation which located on D1domain ofVWF-propeptide disturbed the multimerization by dominant negative effect. R1597W isa common mutation in type2A VWD. It surrounded the ADAMTS13cleavage site whichhas been proved to enhance access to the normally buried p.Y1605-M1606bond, resultingin accelerated clearance of HMW and intermediate multimers. One type3VWD patientwas found to bear homozygous mutations,3379+1G>A, at the slice site of intron25.Splice site analysis revealed aberrant transcription which was responsible for significantreduction in VWF:Ag level. The C827Y mutation, located in the D’ domain, disturbed theconformation of the VWF subunit,1eading to hampered multimerization and accelerateddegradation. The defect in combination with the decrease of VWF production caused bythe3897delT mutation was considered to give rise to the phenotype of type3VWD. Onlyone heterozygous mutation, D141N, was found in a type3VWD patient while furtheridentification and reseach need to be performed.Conclusion In this part, we diagnosed three type2A patients and3type3VWD patients.The sequencing results of VWF gene in the6patients enrich our knowledge on molecularcharacterization of type2A and type3VWD patients while expression studies were askedto clarify these causative mutations.