Friday, April 26, 2024
4/26/2024
ABSTRACT Von Willebrand Factor (VWF) is a multimeric glycoprotein that acts as an adhesive in the subendothelium to the platelets at the injury site and stabilizes factor VIII in the plasma. Deficiency of VWF results in von Willebrand disease (VWD), which is the most prevalent inherited bleeding disorder in humans. Many mutations in the VWF gene causing VWD have been characterized. Despite this progress in genetic studies on VWD, only limited information is available on modifier genes that cause type I VWD, where the patients have considerably lower VWF levels. Similarly, individuals with high VWF levels are at risk for thrombosis. Thus, there is a gap in the knowledge of the genes involved in regulating the levels of VWF. Identification of modifiers/regulators that explain the molecular basis for type 1 VWD is needed for understanding the mechanisms of type 1 VWD. Likewise, regulatory genes for high VWF levels might help alleviate the thrombotic episodes. We have recently created the zebrafish VWD model to identify genes by functional knockdowns of various genes using heterozygote VWD fish. The most important advantage of the zebrafish for this proposal is its amenability to rapidly screen for genes using the piggyback knockdown method developed in our laboratory for the regulatory genes that modifier control the VWF levels. This screening should provide us with the list of the regulatory genes that would have been functionally validated in zebrafish. These results should be useful in sequencing the corresponding genes from patients with type I VWD. They should also complement the whole genome sequencing efforts of type I VWD patients in taking a first look at the sequences corresponding to genes identified in the above zebrafish screening. Similarly, these results will be useful in counseling for the risk of thrombosis. Our goal in this proposal will be to identify novel factors that control the levels of the VWF. To identify novel genes involved in modifying the levels of Vwf, we propose one specific aim. In this aim, we will establish a screening protocol for identifying Vwf modifier genes in zebrafish by assaying for the Vwf levels by ELISA. Subsequently, we will knockdown a known gene that is already found as a modifier gene for VWF in humans and confirm the assay's utility in screening for the genes controlling human VWF levels. Then we will comprehensively perform genome-wide knockdowns of genes that may regulate the Vwf levels in zebrafish. The results from this aim will identify the hitherto unknown factor(s) that regulate the Vwf levels. We will also generate mutant zebrafish for selected genes to pursue structure-function studies. The above identification of modifier genes in zebrafish will be useful as candidate genes causing the type I VWD and high VWF levels. Furthermore, the genome-wide knockdown strategies developed here will provide an approach to study other physiological pathways.
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