Study On The Clinical And Molecular Pathogenesis Of A Family With Type 2N/3 Von Willebrand Disease

Objective: von Willeband disease(VWD)is the most common hereditary bleeding disorder,which is caused by the quantitative or qualitative deficiency of plasma von Willebrand factor(VWF).Clinical manifestation includes skin and mucosal bleeding,or severe bleeding after trauma,tooth extraction and surgery.And,the female patients can show an increase in menstrual bleeding.VWD can be classified in three types: type1,type 2 and type 3.Type 2N VWD is a subtype of VWD,which is caused by the reduced affinity of VWF for factor ?(F?)due to the mutation in the FVIII binding site mutations of VWF.Thus this can results in excessive hydrolysis of F? in plasma and severe decreased FVIII in plasma.Type 3 VWD is caused by homozygous or compound heterozygous mutation(null or missense)of VWF gene,which results in complete quantitative deficiency of VWF.The pathogenesis,clinical features and therapy of different types and subtypes of VWD are different.In this experiment,diagnostic test and subtype test of VWD is applied to diagnose.Genetic diagnosis is performed by DNA sequncing the whole VWF gene of the proband and his family members.On basis of the results of muations,the molecular pathogenesis of this VWD family is to elucidated.Methods1 The EDTA-anticoagulated peripheral blood of the proband and her family members was collected.The platelet rich plasma prepared was used to determine the ristocetin-induced platelet aggregation(RIPA)and ADPinduced platelet aggregation.Plasma is used to determine the coagulation parameters,VWF antigen(VWF:Ag),VWF ristocetin cofactor(VWF:RCo),F ? coagulant activity(F?:C),F ? binding capacity of VWF(VWF-F?:B/VWF:Ag ratio)and VWF multimer analysis.These tests are used to phenotype diagnosis.2 Genomic DNA was extracted from the peripheral blood leucocyte of the proband and her parents.To seek the genetic mutation of VWF,the whole genes sequencing of VWF was performed by the next generation sequencing method for DNA.Then,the proband's younger brother was verified by the first-generation sequencing.On the basis of mutation analysis,the molecular pathogenesis of the VWD family is intended to beclarified.Results The APTT were prolonged in proband and her younger brother with 59.3s and 68.9s,respectively.The APTT were normal in their parents.The PT,Fib,TT and AT of the proband and her family members were all normal.Plasma VWF:Ag(15.8%,28.7%),VWF:RCo(16%,30%)and F?:C(2.9%,4.2%)were decreased in different extent.Her parents' above parameters were normal.VWF-F?:B/VWF:Ag ratio of the proband,her younger brother and her mother were decreased(0.13,0.10,0.19),but her father's FVIII binding capacity of VWF(VWF-F?:B/VWF:Ag ratio)is normal.The RIPA was decreased in the proband,her younger brother and her father with 20.0%,27% and 30%,respectively.Her mother's RIPA was normal.In ADP-induced platelet aggregation and multimer analysis,the proband and her family members were normal.Gene analysis in the proband and her family members showed there were two same heterozygous mutations in the proband and her younger brother respectively.One allele of VWF in exon 18 revealed a c.C2372T(p.T791M)mutation,which belonged to a mutation of type 2N VWD and come from their mother.The other one allele of VWF in exon 49 revealed a c.C8052TA(p.Y2684X)mutation,which belonged to a mutation of type 3 VWD and come from their father.Conclusion According to the clinical characteristics of the proband and her family members,as well as the laboratory examination of VWF,the type 2N/3 VWD is considered.And it is confirmed by gene sequencing.VWF gene analysis that the heterozygous mutation is the molecular pathogenesis of VWD in the proband and her younger brother.This heterozygous mutation is composed of a type 2N VWD mutation of p.T791 M from their mother and a type 3 VWD mutation of p.Y2684 X from their father.The type 3 VWD mutation of p.Y2684 X is a novel mutation and has not been reported.