Identification of type 2 von Willebrand disease in previously diagnosed type 1 patients. a reappraisal using phenotypes, genotypes and molecular modelling

In order to investigate the possibility that qualitative type 2 defects in von Willebrand factor (VWF) occurred in patients previously diagnosed with quantitative type 1 von Willebrand disease (VWD), the phenotypes and genoty pes were reanalysed in 30 patients who exhibited discrepant VWF activity/VW F:Ag ratios of less than 0.7. The capacity of VWF to bind to glycoprotein I b (GpIb) was reassessed using the ristocetin co-factor activity (VWF:RiCo) assay compared to an in-house and a commercial ELISA assay (based on a mAb directed against the GpIb binding site on VWF). This was supplemented by mu ltimeric analysis and the amplification and sequencing of a 936 bp fragment of exon 28 of the VWF gene with the aim of identifying mutations in the Al domain. On reappraisal, using the VWF:RiCo assay all patients demonstrated a disproportionately reduced VWF:RiCo/VMF:Ag ratio, indicative of a qualit ative defect, while abnormal ratios were detected in only seven kindreds us ing the in-house ELISA assay and in only one kindred with the commercial EL ISA assay. Eight single amino acid substitutions were found in nine kindred s, four of which were novel candidate VWF mutations and four previously des cribed in association with type 2 VWD. In agreement with the phenotype, the novel VWF mutations were located in the VWF-A1 crystal structure at positi ons that corresponded to potential type 2M defects. This study underlines t he difficulties of correct diagnosis of the subtype of VWD and emphasises t he importance of using sensitive phenotypic assays, the relevance of the VW F:RiCo/VWF:Ag ratio, multimeric analysis and molecular modelling analysis.