Hemolytic Anemia: Biochemical, Molecular and Proteomic Diagnostics

遗传性溶血性贫血，包括酶缺陷和红细胞膜蛋白缺陷，是一组异质性疾病，对诊断提出了持续的挑战；测试仍然是耗时且高度专业化的。红细胞酶紊乱的诊断挑战体现在（即使在最有经验的实验室）在超过3/4的检查病例中无法建立诊断。假定的膜蛋白缺陷的诊断挑战是不同的——大多数病例最终可能与特定的突变有关，但所涉及的程序是高度专业化的，昂贵且耗时。在全世界范围内，提供这种诊断服务的实验室很少，而且存在着将来无法获得这种专门知识的危险。在本提案中，我们利用三个实验室的专业知识，以便将新的诊断方法与传统的酶学和膜蛋白分析进行比较。两种新的诊断方法将应用于溶血性贫血患者的血液样本：1)使用2D差异凝胶分析（DIGE）比较患者与正常对照的红细胞蛋白质组；2)测量患者样本与对照组的活性氧损伤。在本项目开始时，我们将使用档案临床样本，通过分析已知缺陷的样本来验证蛋白质组学方法。然后，我们将应用这些方法来鉴定用于评估的新诊断患者样本中蛋白质表达的变化。我们还将确定ROS生成增加或对蛋白质的氧化损伤是否是溶血性疾病的共同特征，以及ROS生成/氧化损伤水平与溶血严重程度之间是否存在任何关系。所有样品都将进行标准的酶研究，以便我们可以将蛋白质组学与目前的诊断方法进行比较。由于蛋白质组学具有鉴定与这些疾病发病机制有关的新蛋白质/基因的潜力，在这项研究的后期，我们希望我们的大部分努力致力于表征我们发现在患者红细胞中与对照组相比表达水平改变的蛋白质（及其相应基因）。公共卫生相关性：溶血性贫血：生化、分子和蛋白质组学诊断本应用程序探索了帮助遗传性溶血性贫血诊断和评估的新方法。这些疾病的已知原因包括编码几种红细胞酶和红细胞结构蛋白的基因突变。诊断方法是不完善的，因为通常没有发现病变，目前的诊断方法既昂贵又复杂。我们将探索使用无偏倚的蛋白质组学方法来查明在没有分子诊断的患者中存在异常数量的蛋白质（新蛋白质或预期蛋白质的丢失）。我们还将研究氧化损伤是否是这类贫血发生的一个因素。我们正在进行这些研究，试图为我们目前无法提供具体诊断的一大群患者找到这种疾病的原因。

DESCRIPTION (provided by applicant): Hemolytic Anemia: Biochemical, Molecular and Proteomic Diagnostics The hereditary hemolytic anemias, comprising both enzyme deficiencies and red cell membrane protein defects are a heterogeneous group of disorders that present a continuing diagnostic challenge; testing remains time consuming and highly specialized. The diagnostic challenge in red cell enzyme disorders is exemplified by a failure (even in the most experienced laboratories) to establish a diagnosis in more than 3/4 of cases examined. The diagnostic challenge in presumed membrane protein defects is different-most cases can eventually be related to specific mutations, but the procedures involved are highly specialized, costly and time consuming. Throughout the world, there are very few laboratories offering such diagnostic services, and there is a danger that this expertise will be unavailable in the future. In this proposal, we have draw upon the expertise of three laboratories in order to compare new approaches to diagnosis with conventional enzymology and membrane protein analysis. Two novel approaches to diagnosis will be applied to blood samples from patients with hemolytic anemia: 1) comparison of the red cell proteome of patients versus normal controls using 2D difference gel analysis (DIGE); and 2) measurement of damage due to reactive oxygen species in patients samples versus controls. At the beginning of this project, we will use archival clinical samples to validate proteomic methods by analyzing samples with known defects. We will then apply these methods to identify changes in protein expression in samples from newly diagnosed patients sent for evaluation. We will also determine whether increased ROS production or oxidative damage to protein are a common characteristics of hemolytic disorders and if there is any relationship between the level of ROS production/oxidative damage and the severity of hemolysis. All samples will undergo standard enzyme studies in parallel so that we may compare proteomics against current methods for establishing a diagnosis. Because proteomics has the potential to identify novel proteins/genes involved in the pathogenesis of these disorders, in the later years of this study, we expect much of our effort to be devoted to characterization of proteins (and their corresponding genes) that we find expressed at altered levels in patient red cells versus controls. PUBLIC HEALTH RELEVANCE: Hemolytic Anemia: Biochemical, Molecular and Proteomic Diagnostics This application explores new approaches to aid the diagnosis and evaluation of hereditary hemolytic anemia. Known causes of these disorders include mutation in genes encoding several red cell enzymes and red cell structural proteins. Diagnostic approaches are imperfect because often no lesion is found, and current diagnostic methods are both expensive and complicated. We will explore the use of an unbiased proteomic approach to pinpoint proteins that are present in abnormal amounts (new proteins, or loss of expected proteins) in patients without a molecular diagnosis. We will also examine whether oxidative damage is a factor in development of this type of anemia. We are pursuing these studies to try to find the cause of this disorder for the large group of patients for whom we can currently provide no specific diagnosis.