Omics, mice, and men: Development of precision transfusion medicine

组学、小鼠和人类：精准输血医学的发展

基本信息

批准号：
9755474
负责人：
Mark T Gladwin
金额：
$ 76万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-18 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9755474
关键词：
ANK1 gene Address Affect African American Age Animal Model Asians Autologous Award Biophysics Biotin Blood Blood Platelets Blood Vessels Blood donor Blood flow CRISPR/Cas technology Canis familiaris Cell physiology Cells Cellular Stress Cellular Structures Collaborations Cryopreservation Development Disease Dyes Endothelium Equilibrium Erythrocyte Transfusion Erythrocytes Evaluation Exhibits Forearm G6PD gene Genes Genetic Genotype Heme Iron Hemoglobin Hemolysis Hispanics Hour Human Human Genome Impairment In Vitro Infection Inflammation Inflammatory Inherited Institutional Review Boards Label Lesion Leukocytes Malaria Modeling Molecular Mus Mutation Nitric Oxide Outcome Oxidation-Reduction Pathologic Pathway interactions Physiological Pneumonia Population Predisposition Progress Reports Proteins Protocols documentation Reactive Oxygen Species Recovery Research Risk Role Sampling Sepsis Shock Sickle Cell Trait Signal Transduction Single Nucleotide Polymorphism Stress Study models TLR4 gene Testing Time Transfusion Transgenic Mice Variant arginase biophysical properties cell age cost density genome wide association study genome-wide high risk human female in vivo innovation male men mortality mouse model novel pressure programs protein expression rare variant response stressor trait transfusion medicine

项目摘要

Over the last two cycles of this RO1 award, our research groups have studied the role of cold storage and post transfusion hemolysis as one of the pathways that define the red blood cell (RBC) storage lesion. As summarized in our progress report, these studies have catalyzed a field of research, encompassing the biophysics that govern hemolysis and microparticle related impairments in NO/ROS equilibrium, studies of the transfusion-recipient physiological and pathological responses, and interactions with unit age in storage. We now focus on a simple observation, that the variability in donor RBC stability and function during cold storage and post-transfusion recovery is more dependent on the donor, than to the actual time the unit is in storage. For example, mouse and human female blood is more stable during storage than male blood, and even more striking, common mutations like sickle cell trait (HbAS) exhibit enhanced storage hemolysis and lower post-transfusion recovery. These observations have informed our overarching hypothesis that evolved variability in genes encoding RBC proteins significantly modulate RBC function and recovery after transfusion. The genetic variability that characterizes RBC protein expression levels and function has evolved in large part as a consequence of endemic malaria, which has exerted significant evolutionary pressure on the human genome. Few studies have explored how these common and rare variants might modulate donor RBCs during storage and post-transfusion, nor how these variants might affect susceptibility to hemolysis in disease. To address this question, in collaboration with the REDS III program, we performed the largest genome-wide association study (GWAS) study to date of over 13,000 healthy human blood donors, comparing high density single nucleotide polymorphisms (SNPs) with RBC hemolysis responses to canonical in vitro osmotic, oxidative and storage stress. These RBC-Omics studies have identified almost 19 candidate SNPs at genome wide significance (P values<10-8) that are associated with enhanced or reduced responses to hemolytic perturbations. In our RO1 renewal we propose to explore the function of these common and rare variant SNPs in vitro in human red cells from recalled donors with identified SNPs (Aim 1), in mouse models of transfusion (Aim 2), and in humans with autologous biotin labeled transfusion-recovery studies (Aim 3). Using validated transfusion models that we have developed over the last 3 years for dye-labeled mouse and human donor RBC transfusions into mice, we will explore post-transfusion recoveries of RBC with conserved (similar sequences in mouse and humans) non-synonymous SNPs implicated to RBC stress hemolysis responses, including SNPs in ANK1, SPTA, G6PD, ESYT2, ITFG3, and PIEZ01. To assess in vivo mechanisms, we will generate transgenic mice with human GWAS SNPs using CRISPR-Cas9. We anticipate that these findings will inform a Precision Transfusion Medicine field, in which RBC donor genotype determines the time limits of blood storage and recovery of cells after storage (i.e. the identification of super donors versus fragile donors).

在 RO1 奖项的最后两个周期中，我们的研究小组研究了冷藏和后期处理的作用输血溶血是定义红细胞 (RBC) 储存病变的途径之一。总结如下在我们的进展报告中，这些研究促进了一个研究领域，包括控制生物物理学的领域溶血和微粒相关的 NO/ROS 平衡损伤，输血受者的研究生理和病理反应，以及与储存单位年龄的相互作用。我们现在关注一个简单的观察，即冷藏期间供体红细胞稳定性和功能的变化输血后的恢复更多地取决于捐赠者，而不是设备的实际存储时间。为了例如，小鼠和人类的女性血液在储存过程中比男性血液更稳定，甚至更引人注目，镰状细胞性状 (HbAS) 等常见突变表现出增强的储存溶血和较低的输血后溶血恢复。这些观察结果为我们的总体假设提供了信息，即基因进化出变异性编码红细胞蛋白可显着调节红细胞功能和输血后的恢复。遗传性表征红细胞蛋白表达水平和功能的变异性在很大程度上是作为地方性疟疾的后果，对人类基因组施加了巨大的进化压力。很少有研究探讨这些常见和罕见的变异如何在储存过程中调节供体红细胞和输血后，以及这些变异如何影响疾病中溶血的易感性。为了解决这个问题，我们与 REDS III 项目合作，进行了最大的全基因组研究迄今为止对 13,000 多名健康人类献血者进行的关联研究 (GWAS) 研究，比较了高密度献血者单核苷酸多态性 (SNP) 与红细胞溶血反应对典型的体外渗透、氧化和储存压力。这些 RBC-Omics 研究已在全基因组范围内鉴定出近 19 个候选 SNP 与溶血扰动反应增强或减弱相关的显着性（P 值<10-8）。在我们的 RO1 更新中，我们建议在人类体外探索这些常见和罕见变异 SNP 的功能来自召回供体的红细胞（目标 1）、小鼠输血模型（目标 2）以及人类进行自体生物素标记的输血恢复研究（目标 3）。使用经过验证的输血模型我们在过去 3 年中开发的用于将染料标记的小鼠和人类供体红细胞输注到小鼠，我们将探索具有保守性的红细胞的输血后恢复（小鼠和小鼠中的类似序列）人类）与红细胞应激溶血反应有关的非同义 SNP，包括 ANK1、SPTA、 G6PD、ESYT2、ITFG3 和 PIEZ01。为了评估体内机制，我们将用人类培育转基因小鼠使用 CRISPR-Cas9 的 GWAS SNP。我们预计这些发现将为精准输血医学提供信息领域，其中红细胞供体基因型决定血液储存的时间限制和储存后细胞的恢复（即识别超级捐助者与脆弱捐助者）。