title: Functional analysis of three recombinant A1-VWF domain mutants in comparison to wild type and plasma-derived VWF facilitates subtyping in type 2 von Willebrand disease.
abstract: Phenotypic diagnosis of VWD, in particular type 2, is challenging. Molecular diagnosis may fail to provide clarity since mutations within a short stretch of the same domain may cause various phenotypes, and since even experts will ascribe different subtypes to similar mutations. We assessed diagnostic difficulty in VWD by investigating five cases where phenotypic data was unclear. We identified 3 novel mutations within the A1 domain of the VWF gene: L1460F (2 related patients), Y1363C (1 patient), E1389K (2 related patients). These were not found in 100 normal individuals or documented in the VWF mutation database. Detailed functional analysis of recombinant mutants included VWF multimers, VWF:Ag, VWF:RCo, VWF:CB, and Platelet-VWF binding studies, and results assessed against recombinant WT and plasma derived (pd) VWF. Multimer analysis showed clear loss of HMW VWF with E1389K only, consistent with coincident low relative CB/Ag ratio. VWF-platelet binding studies using two independent approaches showed enhanced activity for L1460F, but reduced activity for E1389K and Y1363C. A novel finding was that WT rVWF showed enhanced platelet binding in RIPA analysis compared to pdVWF with this being dependent on the dilution material used. Through these extensive studies, we assigned L1460F to type 2B, E1389K to 2A, and Y1363C to 2M VWD. Thus, although molecular analysis is not required to classify VWD patient subtypes, a thorough and combined phenotypic, genotypic and functional analysis will assist assignment of the VWD subtype.
authors.affiliation: Department of Pathology and molecular Medicine, Queen's University, Kingston, Ontario, Canada.
authors.forename: Rouzbeh
authors.initials: R
authors.lastname: Chegeni
authors.forename: Linda
authors.initials: L
authors.lastname: Vickars
authors.forename: Emmanuel J
authors.initials: EJ
authors.lastname: Favaloro
authors.forename: David
authors.initials: D
authors.lastname: Lillicrap
authors.forename: Maha
authors.initials: M
authors.lastname: Othman
mesh_terms: D001792:Blood Platelets / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D056728:von Willebrand Disease, Type 2 / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014841:von Willebrand Factor