Von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Von Willebrand factor's (VWF) complex intracellular processing may result in variable VWF expression which requires exploring the underlying mechanism related to individual variants. The mechanisms underlying type 1 VWD are not fully understood, although reduced secretion and increased clearance have been implicated. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program in order to (1) define the underlying mechanism, (2) explore if mutations in a particular domain are mechanistically similar, and (3) correlate expression study data to patient phenotype.
Homozygous and heterozygous expression in mammalian cells was used to study the effect of SV on VWF secretion, intracellular retention, function, and multimerization. Novel SV identified throughout the entire VWF protein were introduced into VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Variant VWF cDNA was transfected homozygously and cotransfected heterozygously 1:1 with wild type (WT) VWF pCIneo into Human Embryonic Kidney cells (HEK293T). Supernatants (sup) and cell lysates from 3 separate transfections were collected and analyzed by ELISA for VWF:Ag, VWF binding to platelet glycoprotein 1b (VWF:GPIbM), VWF binding to collagen type III (VWF:CB). Typically VWF:GP1bM/VWF:Ag > 0.7 indicates normal platelet binding and VWF:CB/VWF:Ag > 0.7 indicates normal multimer structure. VWF multimer structure is analyzed by SDS-agarose electrophoresis and western blotting with differential staining of the VWF secreted from variant and WT alleles.
V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, and A2569E demonstrated secretion of VWF in sup comparable to that of WT-VWF both when transfected homozygously and hetereozygously, normal VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and normal multimer structure. Two of the stop codon SV W199X and E1660X were not expressed homozgyously (as expected) but demonstrated normal secretion, function, and multimerization when cotransfected 1:1 with WT. Homozygous expression of R960P and C2693F demonstrated reduced VWF secretion, VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and abnormal multimerization. Heterozygous transfection with WT improved VWF secretion, but not multimer defects,reflected by a reduced VWF:CB/VWF:Ag. Homozygous expression of C524Y, G994D, C996W, Q1353X, C2199Y, C2701Y, and C2754Y resulted in absent or greatly reduced secretion with increased intracellular retention. Heterozygous expression resulted in reduced VWF secretion (<50% of WT) for all 7 variants. Q1353X, C2199Y, C2701Y, and C2754Y had normal VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag, and multimer structure, but consisted primarily of secreted WT-VWF. G994D had VWF:CB/VWF:Ag ˂ 0.7 and reduced high molecular weight (HMW) multimers. Co-expression of C524Y and C996W with WT-VWF demonstrated nearly absent VWF secretion, indicating a strong dominant negative effect. The very low VWF:Ag levels precluded assessment of VWF function and multimerization.
The VWF:CB/VWF:Ag ratio < 0.7 proved a reliable indicator of multimer structure. No clear domain-specific correlation of VWF secretion or multimer abnormality was observed. Co-expression results of Q1353X, C2199Y, C2701Y, C2754Y, C524Y, G994D, and C996W are consistent with the patient's phenotype, except that patients with C2701Y, and C524Y had slight defect in multimers. In contrast, the in-vitro G994D showed low VWF:CB/VWF:Ag and abnormal multimers but were normal in the patient. C2693F showed consistently abnormal multimer structure in both in vitro and in the patient while R960P had abnormal multimer structure in vitro only.
In sum, 52.6% of the 19 SV theoretically are not causative of the VWD phenotype observed in the patients. These subjects had normal VWF propeptide/VWF:Ag suggesting reduced plasma survival is unlikely to be the causative mechanism. The VWD mechanism in these subjects remains undefined. Variants R960P, C2693F, and G994D (15.7%) behaved like Type 2 VWD SV with loss of HMW multimers. We identified 31.6% of the 19 VWF SV as likely to cause the type 1 VWD phenotype with reduced secretion.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.