PB0796 - Characterisation of 17 Novel Von Willebrand Factor Missense Mutations

Background: Von Willebrand factor (VWF) is a large multimeric glycoprotein that plays an important role in haemostasis. Qualitative or quantitative abnormalities in plasma derived VWF is resultant in Von Willebrand disease (VWD). Previous studies have established a strong association between missense mutations found in the heterogeneous protein and disease-causing mechanisms. A recent cross-sectional familial VWD study (the BRNO-VWD study) identified 17 unrecognised missense mutations in suspected VWD patients in the South Moravian population.

Aims: To investigate the impact of 17 unrecognised missense mutations on VWF expression and function.

Methods: Site directed mutagenesis was used to create 17-novel point mutations (R5K, G39K, D47V, E197K, V343M, A631R, C996S, C1031S, A1250D, E1292D, E1615D, D1691E, T1728S, R1830C, G1890E, T2023A, E2353K) in the pcDNA-FL-VWF expression vector that encodes for full length VWF. Recombinant VWF (rVWF) was expressed in HEK293T cells and VWF ELISA for quantification of protein levels in the media and cell lysate. The physiological function of rVWF was evaluated using static and shear-based assays.

Results: The mutations G39R, D47V, A631T, C996S, G1890E all virtually abolished secretion of VWF, while mutations E1615D and E2353K reduced secretion. Interestingly co-transfection with wtVWF rescued expression to varying extents and mutant monomers could be incorporated into multimers and secreted. Endo H and immunofluoresce staining demonstrated retention of mutant VWF within the ER. Significantly, none of the secreted mutations had abnormal function under shear stress, despite the T1728S and R1830C mutations being located in the A3 domain which contains the major collagen binding site. While the E1615D mutation in the A2 domain was cleaved faster by ADAMTS13 indicative of VWD type 2A.

Conclusion(s): The 17 novel mutations have varying effects on the VWF molecule and demonstrate the heterogenicity of VWD