Laboratory Diagnosis And Molecular Characterization Of A Novel Mutation Of Von Willebrand Factor In Type 2A Von Willebrand's Disease

Von Willebrand disease (vWD) is a hemorrhagic disease due to von Willebrand factor (VWF) deficiency or dysfunction. According to etiology, vWD can be divided into hereditary and acquired. Hereditary von Willebrand's disease is mainly autosomal dominant inheritance, only a small number is recessive inheritance. Different classifications and subtypes of vWD have different molecular pathogenesis. Studies of the molecular pathogenesis of vWD may help to further understand the relationship between structure and function, and these are important for the clinical diagnosis, treatment and prognosis.Objective:Our aim is to develop systematic methods for clinical laboratory diagnosis of vWD, and to investigate molecular pathogenesis in a vWD pedigree.Method:VWF:Ag (VWF antigen), VWF:CB, VWF: Rco, RIPA (ristocetin induced platelet aggregation) and VWF multimer analysis were used to diagnose the patient and his family. The 52 exons and the exon-intron boundaries of VWF gene of the proband were amplifed by polymerase chain reaction (PCR) and sequenced. Sequencing results were compared with sequences NM₀00552.3 by Chromas software. According to the detected mutation, we constucted an expressing mutant plasmid containing full length cDNA of VWF using site-directed mutagenesis. Human embryo kidney 293 (HEK293) cells were transiently transfected with plasmid pSVVWF (wild type) and pSVVWFdel6 (mutant) using lipofectamine 2000. Cell lysate and conditioned medium were collected after 48 hours. Antigen levels of recombinant VWF were measured by an enzyme linked immunosorbent assay (ELISA), and VWF multimer analysis were used to detect multimer profile of conditioned medium using polyclonal rabbit anti-human VWF antibody. Results:1. The proband had markedly reduced levels of VWF:Ag (20.5%), RIPA (6.1%), and VWF:CB (3.8%) and slightly reduced levels of plasma FVIII:C (25%) when compared to values of pooled normal plasma. His plasma vWF lacked the high molecular weight multimers (HMWMs), but the multimer pattern of his platelet vWF was similar to that of normal controls.2. Plasma VWF:Ag, VWF:RCo, FVIII:C, RIPA and VWF:CB of four affected family members were also decreased significantly. And plasma VWF multimers lack HMWMs, while patelet VWF were normal.3. The sequencing of genomic DNA identified a heterozygous 6 bp deletion within exon 28 of the VWF gene, resulting in the deletion of D1529-V1530 of the VWF A2 domain. This mutation was not found in 68 unrelated normal individuals. Similar findings were observed in his mother and three other family members of three generations.4. The antigen levels in conditioned medium from HEK293 cells tranfected with pSVVWFdel6 (mutant) was significant reduced (26.89±4.64%) compared with wild type pSVVWF. VWF multimers showed, in conditioned medium from HEK293 cells the mutation led to significant lack of high molecular weight multimers (HMWMs) However, in cell lysate, the VWF level of mutant and wild type plasmid had no significant differences.Conclusion:1. We diagnosed a type 2A von Willebrand's disease family.2. We identified a novel VWF mutation, which was a heterozygous 6 bp deletion within exon 28, deleting amino acids D1529-V1530 in A2 domain while keeping the remaining protein in-frame.3. The expression experiments of recombinant VWF showed that the mutant might disturb the synthesis of VWF. The absence of HMWMs in plasma could be possibly due to the mutant of VWF gene which retented in cellular and prevented release to plasma..