Abstract
von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Type 1 is the most common form of VWD and results in a partial quantitative deficiency of von Willebrand Factor (VWF). The mechanisms underlying type 1 VWD are still not very well understood although reduced VWF secretion and increased VWF clearance have been implicated in causing VWD. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD in order to define the underlying mechanism and explore if SV in a particular domain are mechanistically similar.
We utilized homozygous expression in human embryonic kidney cells (HEK-293T) to study the effect of VWF SV on VWF secretion, intracellular retention, multimerization, and function. Novel SV have been identified throughout the entire VWF protein. We introduced the following variants into a VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Sequence variants were confirmed by Sanger sequencing. Variant VWF cDNA is transfected homozygously into HEK-293T cells. The supernatants and cell lysates from 3 independent transfections are collected and analyzed by ELISA for VWF:Ag and VWF binding to collagen type III (VWF:CB). VWF multimer structure is analyzed by SDS-agarose gel electrophoresis and western blotting. The genotype-phenotype patient data is correlated with the data from expression studies to explore a model to predict the impact of the SV on the VWD phenotype.
Variants V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F demonstrated secretion comparable to that of wild type (WT)-VWF. In contrast, VWF variants R960P and C2701Y showed reduced VWF secretion (<50% of WT) with increased VWF in the cell lysate. VWF variants W199X, C524Y, G994D, C996W, Q1353X, E1660X, C2199Y, and C2754Y demonstrated a complete absence of secreted VWF. Not unexpectedly, homozygous expression of stop codon variants W199X, Q1353X, and E1660X demonstrated no VWF in the cell lysate. However, non-secreted VWF variants C524Y, G994D, C996W, C2199Y, and C2754Y showed intracellular retention with detectable VWF in the cell lysate. SV occurring at cysteine residues (C524Y, C996W, C2199Y, C2701Y, and C2754Y) all had reduced secretion and increased intracellular retention, consistent with altered conformation leading to increased intracellular chaperone interaction and proteasomal degradation. VWF binding to collagen is dependent on the presence of high molecular weight multimers (HMWM). VWF:CB/VWF:Ag is used to predict multimer structure with VWF:CB/VWF:Ag < 0.7 indicative of loss of the HMWM. VWF variants V86M, M947V, R1763Q, Q2256H, P2524L, C2701Y had VWF:CB/VWF:Ag ≥ 0.7 consistent with normal multimer structure, while variants R960P, R1204W, T2282I, A2569E, and C2693F had VWF:CB/VWF:Ag < 0.7 indicating abnormal multimer structure.
47.3% of the 19 VWF variants studied had normal VWF secretion, 10.5% had reduced secretion with increased intracellular retention, and 26.3% revealed absent secretion with intracellular retention. Variants with a premature stop codon did not synthesize VWF at all. Some SV had normal secretion and multimerization (V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F) implying that the VWD phenotype in these patients results from yet unidentified mechanisms and may not be associated with these SV. Reduced plasma survival is unlikely as these patients had normal VWFpp/VWF:Ag level consistent with normal VWF clearance. Among the VWF variants with normal or decreased secretion, 45.4% had reduced VWF:CB/VWF:Ag consistent with abnormal multimer structure. Heterozygous expression, as observed in the patient, is expected to normalize these multimerization defects. The decreased or absent secretion observed for 52.7% of the variants studied correlates with the patient phenotype, indicating reduced secretion is the mechanism underlying these patients' type 1 VWD phenotype. No domain-specific correlation of VWF secretion or multimer abnormality was observed.
In summary, VWF expression studies confirmed the causative nature of many, but not all of the novel sequence variants identified in type 1 VWD subjects in the Zimmerman Program.
Disclosures
No relevant conflicts of interest to declare.
A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient
