Project 3: Contribution of Non-VWF Genomic Variants to Quantitative Von Willebrand Factor Pathologies

项目 3：非 VWF 基因组变异对定量冯维勒布兰德因子病理学的贡献

• 批准号: 10113378
• 负责人: DAVID P LILLICRAP
• 金额: $ 26.16万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10113378
• 关键词: Anabolism; Area; Attention; Binding; Binding Proteins; Biology; Blood; Blood Coagulation Disorders; Blood Platelets; C-Type Lectins; C2 Domain; CRISPR/Cas technology; Cells; Chromatin Structure; Code; Collagen; Consensus; Deletion Mutation; Development; Disease; Distant; Elements; Endocytosis; Endothelial Cells; Factor VIII; Family; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genetic Variation; Genetic study; Genome; Genomic approach; Genomics; Genotype; Hemorrhage; Human; Inherited; Knock-out; Knockout Mice; Knowledge; Ligand Binding; Mediating; Megakaryocytes; Modification; Mus; Nuclear; Nucleic Acid Regulatory Sequences; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Plasma; Polysaccharides; Population; Prevalence; Process; Proteins; RNA Splicing; Regulation; Role; Signal Pathway; Single Nucleotide Polymorphism; Site; Symptoms; System; Testing; Transcriptional Activation; Transgenic Mice; Variant; Vascular Endothelial Cell; base; cell type; cis acting element; cohort; disease phenotype; experimental study; genetic variant; genome wide association study; genomic locus; humanized mouse; in vitro Assay; in vitro Model; in vivo; indexing; member; molecular pathology; mouse model; patient population; programs; promoter; rare condition; rare variant; receptor; receptor expression; receptor mediated endocytosis; study population; syntaxin; syntaxin-2; trafficking; trait; von Willebrand Disease; von Willebrand Factor

PROJECT SUMMARY: PROJECT 3 The pathogenic basis of the inherited bleeding disorder, von Willebrand disease (VWD), represents a spectrum of genetic mechanisms and variants. While the qualitative type 2 forms of VWD are the result of missense substitutions in domains of von Willebrand factor (VWF) involved in ligand binding, the quantitative traits, type 1 and 3 VWD, are associated with many different sequence variants throughout the VWF gene. However, following detailed genotyping of the VWF coding region and splices sites in >500 type 1 and >100 type 3 VWD cases, pathogenic variants have not been identified in ~35% and ~15% of patients, respectively. This evidence, along with repeated results from large genome wide association studies (GWAS), strongly supports the involvement of genes in addition to VWF in the regulation of plasma VWF levels. The overall aim of this project is to explore the role of sequence variants at genetic loci other than the VWF gene as contributors to low VWF pathological states. Three specific aims will be pursued in this project. In each of these aims, we will use genomic information derived from sequencing of a large patient population with type 1, type 3 and “Low VWF” levels in whom prior analysis has failed to identify pathogenic sequence variants. In Aim #1, genetic variability in the 5' upstream region of the VWF locus will be examined to determine the influence on VWF transcriptional activation. Two areas of the VWF regulatory region will be examined: the proximal promoter, where cis-acting elements interacting with the Hippo signaling pathway will be examined, and a distant super- enhancer element ~45 kb upstream of VWF. In Aim #2, we will evaluate the role of sequence variants of three genes involved in VWF synthesis, storage and secretion. Variants of STX-2, STXBP5 and TC2N will be examined in endothelial cell-based in vitro models and in vivo, in KO mouse models for these genes. Lastly, we will investigate several VWF clearance receptors (CLEC4M, Stablin-2 and SCARA5) and regulators of the endocytic process (FCHO2 and RAB5C), all of which have been implicated in the regulation of plasma VWF levels in GWAS studies. These experiments will incorporate the development of new transgenic mouse models to assess clearance phenotypes, and will explore the regions and modifications to VWF that mediate the interaction with its clearance receptors.

项目摘要：项目 3 遗传性出血性疾病冯维勒布兰德病 (VWD) 的致病基础代表了一系列疾病 遗传机制和变异。而 VWD 的定性 2 型形式是错义的结果 参与配体结合的冯维勒布兰德因子 (VWF) 结构域的取代、数量特征、类型 VWD 1 和 3 与整个 VWF 基因中的许多不同序列变体相关。然而， 对 >500 个 1 型和 >100 个 3 型 VWD 中的 VWF 编码区和剪接位点进行详细基因分型 病例中，分别有约 35% 和约 15% 的患者未发现致病变异。这 证据以及大型全基因组关联研究 (GWAS) 的重复结果强烈支持 除 VWF 外，还有其他基因参与血浆 VWF 水平的调节。此次活动的总体目标是 该项目旨在探索 VWF 基因以外的基因位点的序列变异作为促成因素的作用 低 VWF 病理状态。该项目将实现三个具体目标。在每一个目标中，我们将 使用从大量 1 型、3 型和“低”患者群体测序中获得的基因组信息 VWF”水平，之前的分析未能识别出致病序列变异。在目标#1中，遗传 将检查 VWF 基因座 5' 上游区域的变异性，以确定对 VWF 的影响 转录激活。将检查 VWF 调节区的两个区域：近端启动子、 其中与 Hippo 信号通路相互作用的顺式作用元件将被检查，并且一个遥远的超级 增强子元件位于 VWF 上游约 45 kb。在目标#2中，我们将评估三个序列变体的作用 参与 VWF 合成、储存和分泌的基因。 STX-2、STXBP5 和 TC2N 的变体将是 在基于内皮细胞的体外模型和体内 KO 小鼠模型中检查了这些基因。最后， 我们将研究几种 VWF 清除受体（CLEC4M、Stablin-2 和 SCARA5）和调节因子 内吞过程（FCHO2 和 RAB5C），所有这些都与血浆 VWF 的调节有关 GWAS 研究中的水平。这些实验将结合新转基因小鼠模型的开发 评估清除表型，并将探索介导 VWF 的区域和修饰 与其清除受体的相互作用。