Study Of Pathogenic Mechanism And Genetic Analysis Of Eight Patients With Von Willebrand Disease

Objective:von Willebrand Disease(VWD)is a common autosomal inherited haemorrhagic disease.It has different inheritance patterns according to different typologies.VWD can be classified as six types(1、2A、2B、2M、2N and 3)based on qualitative or quantitative defecency.Accurate diagnosis is vital to the analysis and treatment of patients.Nevertheless,there is a high degree of heterogeneity in the clinical presentation and laboratory test results of VWD patients.Some patients have reduced von Willebrand factor(VWF)levels with bleeding symptoms,but no VWF gene mutations have been detected in gene sequencing.We measured VWF levels and coagulation factor Ⅷ(FⅧ)activity in eight patients with von Willebrand Disease(VWD)by using laboratory method.Next-Generation Sequencing(NGS)was combined with first-generation sequencing(sanger method)to identify pathogenic mutations.Online bioinformatics analysis tools were used to predict whether the novel mutations affected protein function.Applying homology modelling to compare the differences in structural alterations between the novel mutation model and the wild-type model.We could analyse the pathogenic mechanisms of VWD patients those who came to our hospital for consultations by combining these methods.We also expanded the mutation database of VWD and provided strategies for the genetic therapy of VWD in the future.Methods:VWF antigen(VWF:Ag)、 VWF activity(VWF:Ac)and coagulation factor Ⅷactivity(FⅧ:C)were measured by an automatic coagulation analyzer.Ristocetin induced platelet aggregation(RIPA)assay by platelet aggregation analyzer.Assessing the patient’s bleeding presentations by using the ISTH-BAT suggested by Chinese guideline on the diagnosis and management of von Willebrand disease(2022).Screening and confirming the VWF gene mutations in the whole exome by NGS and sanger method respectively.Using the NCBI database to exclude SNPs(single nucleotide polymorphism).Applying VWD online database to identify pathogenic mutation sites and novel mutations.Using bioinformatics tools to make protein conservative analysis,functional impairment prediction and the occurrence of splicing mutation.For instance,SIFT,Poly Phon-2,Mutation Accessor and Mutation Taster.Structural differences between the wild-type and mutation type were compared using SWISS MODEL homology modelling and pymol.Results:Five of the eight patients with VWD included in the study were males and three were females.All eight patients had varying degrees of epistaxis and skin ecchymosis.One of the males had an ankle hemarthrosis.Two cases of VWD type 3,both females,with clinical signs of abdominal bleeding and ruptured corpus luteum haemorrhage,respectively.We found seven pathogenic mutations.Four mutation sites of seven have been reported: c.478G>A(p.Gly160Arg)、c.1945+1G>T、c.2921G>A(p.Trp974X)、c.3614G>A(p.Arg1205His).Three mutations are the first reported domestically and abroad: c.1877＿1878ins CCCT(p.Leu626fs)、c.4373G>A(p.Cys1458Tyr)、c.7361C>A(p.Thr2454Asn).Seven benign mutations: c.1411G>A(p.Val471Ile)、c.1451A>G(p.His484Arg)、c.2365A>G(p.Thr789Ala)、c.2555A>G(p.Gln852Arg)、c.4141A>G(p.Thr1381Ala)、c.4414G>C(p.Asp1472His)and c.4693G>T(p.Val1565Leu).Conclusion:We have diagnosed three patients with VWD-type 1(including two patients with tpye 1C),one patient with type 2M and two patients with type 3 by combining laboratory tests,genetic sequencing and bioinformatics analysis.There was no pathogenic VWF mutation detected in two of eight patients.The combination of laboratory diagnosis and genetic testing is important in the diagnosis of VWD because of the great heterogeneity of bleeding manifestations,laboratory test results and genetic testing in patients with VWD.With the advance of automatic standardised testing and NGS,We were able to Strengthen the diagnosis of VWD and give patients more appropriate treatment by complete laboratory testing data and VWD mutation database.