Identification of Type 2 von Willebrand Disease in Previously Diagnosed Type 1 Patients: a Reappraisal Using Phenotypes, Genotypes and Molecular Modelling

Abstract

SummaryIn 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 genotypes were reanalysed in 30 patients who exhibited discrepant VWF activity/VWF:Ag ratios of less than 0.7. The capacity of VWF to bind to glycoprotein Ib (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 multimeric 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 A1 domain. On reappraisal, using the VWF:RiCo assay all patients demonstrated a disproportionately reduced VWF:RiCo/VWF:Ag ratio, indicative of a qualitative defect, while abnormal ratios were detected in only seven kindreds using the in-house ELISA assay and in only one kindred with the commercial ELISA assay. Eight single amino acid substitutions were found in nine kindreds, four of which were novel candidate VWF mutations and four previously described in association with type 2 VWD. In agreement with the phenotype, the novel VWF mutations were located in the VWF-A1 crystal structure at positions that corresponded to potential type 2M defects. This study underlines the difficulties of correct diagnosis of the subtype of VWD and emphasises the importance of using sensitive phenotypic assays, the relevance of the VWF:RiCo/ VWF:Ag ratio, multimeric analysis and molecular modelling analysis.

Classification and Characterization of Hereditary Types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (Unclassifiable) von Willebrand Disease

“…In addition to the physiological fragmentation of the subunit, the mutated VWF shows the presence of an additional 209-kDa species not observed in normal type 1, 2A, 2B, and 2N VWF (52). The relative loss of large (Table 5) (48,51,53). The laboratory phenotype of the R1315C mutation is characterized by subnormal values for FVIII:C (mean 41 U/dL), low levels for VWF:Ag (mean 20 U/dL), very low or undetectable levels for VWF:RCo of less than 0.10 U/dL, and a decreased VWF:RCo/Ag ratio.…”

“…There is no consensus about the definition of "normal high molecular weight" multimers. Three of the 2M VWD patients showed a quantitative loss of the highest VWF multimers in two studies (46,47) (Table 3), whereas a normal VWF multimeric pattern has been noted in the other 19 2M patients (45)(46)(47)(48) (Table 3). RIPA was not measured in 14, but was uniformly decreased or absent in 8, which constitutes a very characteristic feature of VWD 2M (45)(46)(47)(48) (Table 3).…”

Biologic response to desmopressin in patients with severe type 1 and type 2 von Willebrand disease: results of a multicenter European study

“…16 On the other hand, there is the distinct possibility that these patients with low VWF:RCo/Ag ratio, decreased ristocetin-induced platelet agglutination, and normal or near normal multimeric structure are carriers of a qualitatively abnormal VWF protein in its interaction with platelets and that they should be classified as having type 2M VWD, as recently suggested. [17][18][19] This hypothesis can be only validated by the knowledge of gene defects and by the demonstration that the expressed recombinant proteins have abnormal reactions with platelet membrane glycoproteins. In the absence of these molecular data, the 8 patients with low VWF:RCo/Ag ratios were still kept in the type 1 VWD group, as originally indicated by the participating centers at the time of enrollment.…”

von Willebrand disease R1374C: Type 2A or 2M? A challenge to the revised classification. High frequency in the northwest of Spain (Galicia)

“…Considering that the complete absence of HMW multimers is required for type 2A, Casonato et al [16] and Nitu-Walley et al [4] recommend the inclusion of R1374C in subtype 2M VWD. The reason for this is that some HMWM of VWF are present in this variant, with multimers even larger than those seen in normal VWF.…”