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http://hdl.handle.net/11375/29035
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DC Field | Value | Language |
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dc.contributor.advisor | Kretz, Colin | - |
dc.contributor.author | Sparring, Taylor | - |
dc.date.accessioned | 2023-10-12T19:53:40Z | - |
dc.date.available | 2023-10-12T19:53:40Z | - |
dc.date.issued | 2023 | - |
dc.identifier.uri | http://hdl.handle.net/11375/29035 | - |
dc.description.abstract | Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder. It is caused by mutations in von Willebrand factor (VWF) that impair its secretion and reduces its ability to capture platelets to sites of blood vessel damage and support proper blood clot formation. Mutations in the VWF C domains can lead to intracellular retention and lower circulating VWF levels associated with VWD. It’s currently not well understood why some mutations lead to decreased VWF secretion, while others do not. Here, we investigate the impact of every possible missense mutation, within the VWF C- terminal cysteine knot (CK) domain, on biosynthesis and cell secretion. Methods: We developed a VWF cell secretion assay with a Bxb1 recombinase-based landing pad system, using HEK293T cells expressing VWF-eGFP and alpha-1 antitrypsin- mCherry. Mutations in VWF that affect biosynthesis and secretion were identified based on cell fluorescence intensity using fluorescence activated cell sorting. Alpha-1 antitrypsin- mCherry remained unmutated and served as a control for individual cell variability. We generated a VWF mutagenesis library that contained every amino acid substitution at each position of the VWF CK domain within full length VWF. We used the protein secretion assay to complete a deep mutational scan (DMS) on the VWF CK domain library to comprehensively define the amino acid changes in VWF that affect its biosynthesis and secretion. Cells were sorted into upper/lower florescence intensity bins and prepared for next generation sequencing to identify mutations that are enriched in cells with impaired secretion. iii PhD Thesis – T. Sparring McMaster University – Medical Sciences Results: Cells expressing VWF variants with known secretion defects associated with VWD had ~ 5 – 100 fold increased eGFP:mCherry mean fluorescence intensity compared to cells expressing WT VWF. The DMS dataset provided information on the impact of 1404 of the total potential 1446 single missense amino acid variants of the VWF CK domain. There were 139 VWF variants classified as high abundance variants associated with impaired secretion. High abundance variants were identified in both ClinVar and gnomAD clinical databases. Patients harboring VWF CK domain mutations, within the Zimmerman Program database, had VWF antigen levels that negatively correlated with DMS abundance scores and Bleeding Assessment Tool scores that positively correlated with DMS abundance scores, supporting clinical relevance of our study. Cells expressing a VWD known secretion variant, had a 2-fold (GRP78) and 4-fold (CHOP) upregulation in expression for markers of endoplasmic reticulum stress compared to cells expressing WT VWF. This variant had alleviated endoplasmic reticulum stress, with 75% – 85% reduction in GRP78 and CHOP expression respectively, in response to 4-PBA chaperone treatment, shedding light on protein misfolding and offer a potential novel treatment option for non- responsive VWD patients. Conclusions: With genome sequencing on the rise, accurate interpretation of coding variants is essential. Functional validation of missense mutations could improve variant significance prediction. Here we developed and optimized a screening platform for the identification of VWF variants that impact secretion. The results of this project will enable large scale DMS assays to functionally annotate VWF variants. | en_US |
dc.language.iso | en | en_US |
dc.subject | von Willebrand factor, von Willebrand disease, Deep Mutational Scan | en_US |
dc.title | DEEP MUTATIONAL SCAN OF THE VWF CK DOMAIN TO DEFINE MUTATIONS ASSOCIATED WITH VWD | en_US |
dc.type | Thesis | en_US |
dc.contributor.department | Medical Sciences (Blood and Cardiovascular) | en_US |
dc.description.degreetype | Thesis | en_US |
dc.description.degree | Doctor of Philosophy (PhD) | en_US |
dc.description.layabstract | Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by mutations in von Willebrand Factor (VWF). VWF plays a crucial role in hemostasis by stabilizing FVIII in circulation and mediating the adhesion of platelets to damaged blood vessel walls, thus promoting effective clot formation. Some VWF mutations disrupt its proper secretion into the circulation, leading to low plasma levels and ultimately impaired blood clotting. In this study, we investigated VWF mutations associated with impaired biosynthesis and secretion, focusing on the VWF CK domain. Using deep mutational scanning we created a library of every possible missense mutation of the VWF CK domain and identified those affecting VWF secretion. Interestingly, these findings correlated with clinical data, linking genetic changes to actual patient outcomes. Moreover, the study revealed cellular stress responses to specific mutations, suggesting potential mechanisms of VWF retention. By decoding the impact of mutations, this research brings us closer to understanding VWF pathophysiology, and improving diagnostics for individuals with VWD. | en_US |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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Sparring_Taylor_202310_PhD.pdf | 17.8 MB | Adobe PDF | View/Open |
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