OC 53.2 - Deep mutational scan of the VWF C domains to define mutations associated with VWD

Background: Von Willebrand Factor (VWF) is a multimeric glycoprotein that recruits platelets to sites of blood vessel injury. Mutations in the VWF C domains can lead to intracellular retention and lower circulating VWF levels associated with the bleeding disorder von Willebrand disease (VWD). It’s currently not well understood why some mutations lead to decreased VWF secretion, while others do not.

Aims: Apply deep mutational scanning to identify every possible missense mutation in VWF that either increase or decrease its secretion from cells.

Methods: We developed a VWF cell secretion assay in HEK293T cells using Fluorescence Activated Cell Sorting (FACS).

Results: HEK293T cells expressing VWF-eGFP variants with known secretion defects have increased mean fluorescence intensity (MFI) (V1822G=1575, C2693Y=1842), compared to WT VWF-eGFP (692), thus validating the screen. The VWF mutagenesis library contains every amino acid substitution at each position of the C1-CTCK domains in full length VWF. HEK293T cells expressing the VWF-eGFP mutagenesis library exhibited a broad distribution of MFI compared to cells expressing WT VWF-eGFP, confirming the presence of variants that affect secretion. Cells were sorted into upper and lower eGFP intensity bins and prepared for high-throughput sequencing to identify VWF mutations associated with impaired secretion. Data analysis of this screen is on-going.
VWF mutations can lead to ER retention, which may cause secondary effects on cell viability. Cells expressing variants with the highest 10% eGFP intensity had upregulation of GRP78, CHOP, and ERP57 expression compared to cells expressing WT VWF. Therefore, missense mutations that cause impaired VWF secretion are associated with increased expression of markers associated with the adaptive and apoptotic unfolded-protein response.

Conclusion(s): Deep mutational scanning using FACS provides a platform to identify all possible coding variants in VWF that impact its secretion. These data will provide a detailed structure/function analysis of VWF in mammalian cells and identify variants associated with VWD.