Project 1: Molecular Impact of VWF on Clinical VWD

VWD生物学齐默尔曼项目（ZPB-VWD）的项目1侧重于分子影响 临床VWD的VWF。它整合了两个现有队列- 1）既往回顾性PPG队列 在VWD分子和临床生物学的齐默尔曼项目中诊断，2）前瞻性 R 01队列新兵。在目标1中，对1型、2型和3型VWD受试者进行纵向实验室研究 以及VWF水平降低的组，我们统称为低冯维勒布兰德因子，包括 那些先前可能被标记为“轻度VWD”的患者，VWF <正常，但很少或没有出血。 这些可能被认为是出血的风险因素，但不是疾病。我们不仅要研究 实验室表型随时间的变化，但也用中期出血评估对其进行研究 评分（iBAT），用于半定量评估“新发临床出血”量，因为BAT评估出血 但是分数饱和并且不能量化中期出血。此外，目标1将量化评估 与新基质蛋白（如肌球蛋白）或新功能受体（如血小板GP）的潜在异常结合 IIb-IIIa（2b3a）。在目标2中，将研究2型功能变体以确定诊断的保真度（类型 图2A、2B、2 M和2N）。动物模型 已经/将要开发2型变体，并使用出血和血栓形成试验（尾部）进行比较 出血、模板出血、VenaFlux和激光损伤评估。目标3将包括我们 合并1型或3型VWD或LVWF的队列，并在这些现有队列中添加现有的，类似的 来自金斯顿和都柏林的已定义队列，但无VWF突变，且阵列比较结果为阴性 基因组杂交（aCGH）或多重连接依赖性探针扩增（MLPA）。在我们 队列中，我们有至少3例无VWF序列变异的3型VWD受试者，aCGH为阴性， 没有抗VWF抗体。此外，我们有>40个1型VWD和>180个LVWF受试者样品， NSV和阴性aCGH。因此，我们将首先通过全基因组研究这些1型和3型受试者 在核心B中使用NGS测序的WGS测序。当单个候选基因被识别时， 将对受影响的（AFM）和未受影响的家庭成员（UFM）进行测序，以确定 与表型的联系。然后，候选SV将根据它们是否符合 1)VWF内含子变化（项目1）; 2）涉及碳水化合物或碳水化合物受体（项目2）; 3） VWF编码区外的机制（项目3）;或4）表明表观遗传机制（项目4）。 对于可能确定的常见或科学上独特的原因，将在 核心C和研究的各种项目。通过项目1，我们希望了解这种疾病 导致VWD的机制以及它们如何通过发育的动物模型影响实验室和临床表型 模型，并协助确定1型和3型VWD的基因外原因。

Project 1 in the Zimmerman Program for the Biology of VWD (ZPB-VWD) is focused on the Molecular Impact of VWF on Clinical VWD. It consolidates the two existing cohorts – 1) retrospective PPG cohort previously diagnosed in the Zimmerman Program for the Molecular and Clinical Biology of VWD and 2) the prospective R01 cohort recruits. In Aim 1 laboratory studies are done longitudinally on the Type 1, 2, and 3 VWD subjects as well as the group with reduced VWF levels that we collectively call Low von Willebrand Factor that includes those that might have been previously labeled as “Mild VWD” with VWF <normal but little or no bleeding. These may be considered as having a risk factor for bleeding but not a disease. Not only will we study the changes with time of their laboratory phenotype but also study them with and interim Bleeding Assessment Score (iBAT) to semiquantitatively assess the amount of “new clinical bleeding” since BATs assess bleeding but the score saturates and cannot quantify interim bleeding. Additionally, Aim 1 will quantifiably evaluate potential abnormal binding to new matrix proteins (e.g. myosin) or new functional receptors (e.g. platelet GP IIb-IIIa (2b3a). In Aim 2 type 2 functional variants will be studied to determine the fidelity of diagnosis (types 2A, 2B, 2M, and 2N) between phenotypic assignments locally to those centrally assigned. Animal models of type 2 variants have been/will be developed and compared using bleeding and thrombosis testing (tail bleeding, template bleeding, VenaFlux, and Laser-injury assessment. Aim 3 will include subjects in our combined cohort who have type 1 or 3 VWD or LVWF and to these existing cohorts add existing, similarly defined, cohort from Kingston and Dublin but have no VWF mutation and are negative by Array Comparative Genome Hybridization (aCGH) or Multiplex Ligation-dependent Probe Amplification (MLPA). Within our cohort, we have at least 3 type 3 VWD subjects with no VWF sequence variant, are negative by aCGH, and have no anti-VWF antibodies. In addition we have >40 type 1 VWD and >180 LVWF subject samples with NSV and negative aCGH. Therefore we will first study these type 1 and 3 subjects by whole Genome Sequencing (WGS) using NGS sequencing in Core B. When individual candidate genes are identified, sequencing will be performed on the affected (AFM) and unaffected family members (UFM) to ascertain linkage with phenotype. Candidate SV will then be farmed out to Projects 1-4, based upon whether they are 1) VWF intronic changes (Project 1); 2) involve carbohydrates or carbohydrate receptors (Project 2); 3) mechanisms outside the VWF coding region (Project 3); or 4) suggest an epigenetic mechanism (Project 4). For frequent or scientifically unique causes that might be identified, animal models of these will be produced in Core C and studied in the various projects. Through Project 1 we expect to gain insight into the disease mechanisms causing VWD and how they affect laboratory and clinical phenotypes through developed animal models, and to assist with determining extragenic causes of both type 1 and type 3 VWD.