Regulators of von Willebrand Factor Levels

冯维勒布兰德因子水平的调节器

• 批准号: 10278447
• 负责人: PUDUR JAGADEESWARAN
• 金额: $ 29.7万
• 依托单位: UNIVERSITY OF NORTH TEXAS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10278447
• 关键词: Adhesives; Adult; Affect; Anticoagulants; Biological Assay; Blood Coagulation Disorders; Blood Coagulation Factor; Blood Platelets; CRISPR/Cas technology; Candidate Disease Gene; Chromosome Mapping; Complement; Counseling; Data; Development; Diagnosis; Disease; Disease model; Eligibility Determination; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Equilibrium; Ethylnitrosourea; Etiology; Factor VIII; Feedback; Fish Diseases; Fishes; Genes; Genetic; Genetic study; Glycoproteins; Goals; Hemorrhage; Hemostatic function; Heterozygote; Human; Individual; Inherited; Injury; Knock-out; Knowledge; Laboratories; Lasers; Lead; Measures; Messenger RNA; Methods; Modeling; Molecular; Mutation; Neuraminidase; Pathway interactions; Patients; Phosphotransferases; Physiological; Plasma; Plasma Proteins; Play; Population; Production; Proteins; RNA-Binding Proteins; Rapid screening; Regulation; Regulator Genes; Research; Risk; Risk Factors; Role; Signal Transduction; Site; Structure; Technology; Thrombosis; VWF gene; Zebrafish; experience; genome sequencing; genome wide association study; genome wide screen; genome-wide; glycosyltransferase; high throughput screening; improved; innovation; knock-down; mouse genetics; mutant; novel; screening; thrombotic; trait; transcription factor; von Willebrand Disease; von Willebrand Factor; whole genome

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.

摘要 血管性血友病因子（VWF）是一种多聚体糖蛋白，在血管性血友病中起粘合剂的作用。 在损伤部位，血管内皮下的血小板，并稳定血浆中的因子VIII。不足 VWF导致血管性血友病（VWD），这是最常见的遗传性出血性疾病 在人类身上。VWF基因中的许多突变引起VWD已被表征。尽管如此 在VWD的遗传学研究进展中，只有有限的信息是关于引起VWD的修饰基因， I型VWD，其中患者具有相当低的VWF水平。同样，高风险的个人 VWF水平存在血栓形成的风险。因此，在参与基因的知识方面存在空白。 调节VWF水平。鉴定修饰剂/调节剂，解释 1型VWD是理解1型VWD的机制所必需的。同样，调控基因 高VWF水平可能有助于缓解血栓发作。我们最近创建了 斑马鱼VWD模型，用于通过使用杂合子的各种基因的功能敲低来鉴定基因 VWD鱼。对于这个提议来说，斑马鱼最重要的优势是它能够迅速地适应 使用我们实验室开发的piggyback knockdown方法筛选基因， 修饰控制VWF水平的基因。这次筛选应该为我们提供了 这些基因在斑马鱼中已经得到了功能验证。这些结果应 可用于对来自I型VWD患者的相应基因进行测序。他们也应该 补充I型VWD患者的全基因组测序工作， 对应于上述斑马鱼筛选中鉴定的基因的序列。同样，这些结果 将有助于预防血栓形成的风险。我们在这个提案中的目标将是确定新的 控制VWF水平的因素。 为了鉴定参与改变Vwf水平的新基因，我们提出了一个特定的目标。在这 目的：建立一种检测斑马鱼Vwf修饰基因的筛选方法， 通过ELISA测定Vwf水平。随后，我们将敲除一个已知的基因， 修饰基因的VWF在人类中，并确认该试验的效用，在筛选基因控制 人VWF水平。然后，我们将全面执行基因组范围内的基因敲除， 可能调节斑马鱼的Vwf水平。这一目标的结果将确定迄今为止未知的 调节Vwf水平的因素。我们还将为选定的基因产生突变斑马鱼， 进行结构-功能研究。以上对斑马鱼修饰基因的鉴定将是有用的 作为引起I型VWD和高VWF水平的候选基因。此外，全基因组 在此开发的敲除策略将提供研究其他生理途径的方法。