Identifying Genetic Basis and Molecular Mechanisms in Different Types of von Willebrand Disease (VWD)

Abstract

Von Willebrand disease (VWD) is the most common inherited bleeding disorder. It is caused by quantitative or qualitative defects of the von Willebrand factor (VWF) which has crucial roles in hemostasis. VWD is classified into three primary categories. Types 1 and 3 represent partial and total quantitative deficiency of VWF, respectively. Type 2 is due to qualitative defects of VWF, and is divided into four secondary categories 2A, 2B, 2M and 2N. In this study we explored genotype and phenotype characteristics of a cohort of VWD patients with the aim of dissecting the distribution of mutations in different types of VWD. Mutation analysis of 114 patients diagnosed to have VWD was performed by direct sequencing of the VWF gene (VWF). Large deletions were investigated by multiplex ligation-dependent probe amplification (MLPA) analysis. The results showed a mutation detection rate of 68%, 94% and 94% for VWD type 1, 2 and 3, respectively. In total, 68 different putative mutations were detected. Twenty six of these mutations were novel. In type 1 and type 2 VWD, the majority of identified mutations (74% vs 88.1%) were missense substitutions while mutations in type 3 VWD mostly caused null alleles (82%). In addition, the impact of five detected novel cysteine missense mutations residing in D4-CK domains was characterized on conformation and biosynthesis of VWF. Transient expression of human cell lines with wild-type or five mutant VWF constructs was done. Quantitative and qualitative assessment of mutated recombinant VWF was performed. Storage of VWF in pseudo-Weible-Palade bodies (WPBs) was studied with confocal microscopy. Moreovere, structural impact of the mutations was analyzed by homology modeling. Homozygous expressions showed that these mutations caused defects in multimerization, elongation of pseudo-WPBs and consequently secretion of VWF. Co-expressions of wild-type VWF and 3 of the mutants demonstrated defect in multimer assembly, suggesting a new pathologic mechanism for dominant type 2A VWD due to mutations in D4 and B domains. Structural analysis revealed that mutations either disrupt intra-domain disulfide bonds or might affect an inter-domain disulfide bond. <br /> In conclusion, our study extends the mutational spectrum of VWF, and improves the knowledge of the genetic basis of different types of VWD. The gene expression studies highlight the importance of cysteine residues within the C-terminal of VWF on the structural conformation of the protein and consequently multimerization, storage, and secretion of VWF.