NOVEL PATHWAYS IN ISCHEMIC STROKE IN SICKLE CELL ANEMIA

项目摘要 卒中是镰状细胞性贫血(SCA)的一种破坏性并发症，在所有未发生中风的患者中，约11%会发生中风 干预。在患有SCA的儿童患者中，这些卒中大多是缺血性的，与动脉有很强的联系 血管病变。在SCA患者中，有一个很强的可遗传成分影响卒中的发展。 然而，我们对相关基因的了解是有限的。我们小组最近完成了一个无偏见的基因组 应用外显子全序列对SCA中卒中的广泛关联性研究。我们确定并验证了两个 GOLGB1(Y1212C)和ENPP1(K173Q)基因的特殊错义突变对卒中的保护作用 在SCA中。我们的研究目标是确定GOLGB1和ENPP1作为血管调节因子的功能作用 内皮损伤和缺血性卒中风险。 我们先前观察到：a)GOLGB1水平影响单核细胞和内皮细胞的高尔基体结构 细胞；b)GOLGB1 Y1212C变异与SCA分离的单核细胞中更致密的高尔基体有关 C)ENPP1影响血管表面的血小板聚集。从这个初步数据来看，我们 假设1)GOLGB1影响高尔基体结构，高尔基体结构进而调节血栓前状态的分泌输出 可能影响SCA中中风的因素，如von Willebrand因子(VWF)；2)ENPP1影响 血栓形成可以直接通过血小板聚集，也可以间接通过促炎症的ADP的水解。 我们计划通过分析患者样本中GOLGB1和ENPP1的功能来检验这些假设 与SCA合作。我们将从大量接受SCA治疗的患者中分离出原代单核细胞和内皮细胞 德克萨斯儿童医院的护理。我们将通过追求三个目标来检验我们的中心假设：目标1：检查 GOLGB1在镰状细胞性贫血高尔基复合体形态和功能中的作用--我们将使用高分辨率 GOLGB1在维持单核细胞高尔基体结构和蛋白质转运中的作用 来自SCA患者的内皮细胞；目标2：确定ENPP1活性在镰状细胞性贫血中的作用-我们将 检测ENPP1是否调节SCA患者的血小板聚集和血栓形成。我们会 检测ENPP1酶的血浆和内皮活性；目的3：确定GOLGB1和GOLGB1的作用 微流控血管系统体外模型中的ENPP1-我们将结合RNA表达和蛋白质组学 以确定受我们候选基因影响的内皮通路。 我们提议的研究将表征我们的两个基因及其遗传变异对功能的影响 在SCA患者样本中。这将有助于我们了解中风和脑梗塞的病理生理机制。 有可能为预防或治疗SCA患者中风的药物治疗提供靶点。

Project Summary Stroke is a devastating complication of sickle cell anemia (SCA) that will occur in ~11% of all patients without intervention. In pediatric patients with SCA, most of these strokes are ischemic and strongly linked with arterial vasculopathy. There is a strong heritable component that affects development of stroke in SCA patients. However, our understanding of the genes involved is limited. Our group recently completed an unbiased genome wide association study of stroke in SCA using whole exome sequencing. We identified and validated two particular missense mutations in the GOLGB1 (Y1212C) and ENPP1 (K173Q) genes as protective against stroke in SCA. Our study goal is to define the functional role of GOLGB1 and ENPP1 as modulators of vascular endothelial injury and ischemic stroke risk. We have previously observed that: a) GOLGB1 levels affect Golgi structure in monocytes and endothelial cells; b) The GOLGB1 Y1212C variant is associated with more compact Golgi in monocytes isolated from SCA patients; and c) ENPP1 influences platelet aggregation on vascular surfaces. From this preliminary data, we hypothesize that 1) GOLGB1 affects Golgi structure, which in turn regulates secretory output of pro-thrombotic factors such as von Willebrand factor (vWF) that can affect stroke in SCA; and 2) that ENPP1 affects risk of thrombosis either directly via platelet aggregation or indirectly through hydrolysis of the pro-inflammatory ADP. We plan to test these hypotheses by analyzing the function of GOLGB1 and ENPP1 in samples from patients with SCA. We will isolate primary monocyte and endothelial cells from a large cohort of SCA patients receiving care at Texas Children’s Hospital. We will test our central hypotheses by pursuing three aims: Aim 1: Examine role of GOLGB1 in form and function of Golgi complexes in sickle cell anemia - We will use high resolution imaging to determine the role of GOLGB1 in maintaining Golgi structure and protein trafficking in monocyte and endothelial cells from SCA patients; Aim 2: Determine the effect of ENPP1 activity in sickle cell anemia - We will test whether ENPP1 modulates platelet aggregation and thrombus formation in patients with SCA. We will measure plasma and endothelial activity of the ENPP1 enzyme; Aim 3: Determine effects of GOLGB1 and ENPP1 in a microfluidic in vitro model of vasculature - We will combine RNA expression and proteomic profiling to identify endothelial pathways affected by our candidate genes. Our proposed studies will characterize the functional impact of our two genes and their genetic variants in SCA patient samples. This will benefit our understanding of the pathophysiological pathways of stroke and potentially provide targets for drug therapy to prevent or treat stroke in SCA patients.