Hemolytic Anemia: Biochemical, Molecular and Proteomic Diagnostics

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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.