Expression of 14 von Willebrand factor mutations identified in patients with type 1 von Willebrand disease from the MCMDM-1VWD study

Abstract

Summary. Background: Candidate von Willebrand factor (VWF) mutations were identified in 70% of index cases in the European study ‘Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease’. The majority of these were missense mutations. Objectives: To assess whether 14 representative missense mutations are the cause of the phenotype observed in the patients and to examine their mode of pathogenicity. Methods: Transfection experiments were performed with full‐length wild‐type or mutant VWF cDNA for these 14 missense mutations. VWF antigen levels were measured, and VWF multimer analysis was performed on secreted and intracellular VWF. Results: For seven of the missense mutations (G160W, N166I, L2207P, C2257S, C2304Y, G2441C, and C2477Y), we found marked intracellular retention and impaired secretion of VWF, major loss of high molecular weight multimers in transfections of mutant constructs alone, and virtually normal multimers in cotransfections with wild‐type VWF, establishing the pathogenicity of these mutations. Four of the mutations (R2287W, R2464C, G2518S, and Q2520P) were established as being very probably causative, on the basis of a mild reduction in the secreted VWF or on characteristic faster‐running multimeric bands. For three candidate changes (G19R, P2063S, and R2313H), the transfection results were indistinguishable from wild‐type recombinant VWF and we could not prove these changes to be pathogenic. Other mechanisms not explored using this in vitro expression system may be responsible for pathogenicity. Conclusions: The pathogenic nature of 11 of 14 candidate missense mutations identified in patients with type 1 VWD was confirmed. Intracellular retention of mutant VWF is the predominant responsible mechanism.

Expanded phenotype–genotype correlations in a pediatric population with type 1 von Willebrand disease

“…For example, it could be inferred that a normal PP/Ag ratio in association with a significant reduction in absolute VWFpp level might be an indication of defective VWF synthesis or release. In our cohort, this combination was observed in association with both nonsense mutations, a small insertion mutation, a splicing mutation, and several missense mutations, including all mutations identified within the propeptide domains (exons [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. Furthermore, the differentiation between type 3 and severe type 1 VWD is not always straightforward, as standard VWD assays lack precision at very low levels of VWF:Ag.…”

“…Previous clinical studies have indeed made both such in vivo observations in subsets of patients with type 1 VWD; however, the relative contributions of these mechanisms in the majority of patients are unknown [8][9][10]. Recently, further insight has been gained through in vitro expression studies, in which the potential pathogenicity of 14 candidate missense mutations was examined through transfection of cell lines in culture [11]. Although these experiments confirmed that intracellular retention of VWF appears to be an important mechanism in type 1 VWD, this approach does not assess the influence of factors external to the VWF locus on the phenotypic expression of a given mutation.…”

Intracellular Storage and Regulated Secretion of Von Willebrand Factor in Quantitative Von Willebrand Disease

“…Through these mechanisms the pathogenic nature of those mutations in VWD are unraveled. Because of the lack of WPB formation in most expression systems that have been extensively applied (21,41), the current concept of the pathogenesis of VWF mutations identified in VWD are mainly based on the expression data on the constitutive secretion, synthesis, and multimer patterns of VWF. However, the main source of VWF in the circulation are the endothelial cells that synthesize and store VWF into WPBs and secrete VWF into the circulation through basal and regulated pathways (8,9).…”

Common and rare von Willebrand factor (VWF) coding variants, VWF levels, and factor VIII levels in African Americans: the NHLBI Exome Sequencing Project

“…40 Based on in vitro analysis and expression of recombinant p.Arg2287Trp missense variant, it was suggested to be a causative mutation due to a mild reduction in the amount of secreted VWF. 41 Type 2M VWD is characterized by a decreased VWF activity to antigen ratio and defective binding of VWF to platelets due to mutations in the VWF A1 domain. A lower VWF activity to antigen ratio and increased prevalence of the type 2M phenotype has been reported in AAs.…”