EICOSANOID BIOSYNTHESIS DEFICIENCY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Primary Hypothesis: The identified patient has a mutation of the cytosolic PLA2 enzyme. Secondary Hypothesis: The identified patient has a defect in regulatory mechanism of cytosolic PLA2. We have identified a patient with significantly low levels of multiple measured prostaglandins, products of the cyclooxygenase pathway, and 12-hydroxy-eicosatetraenoic acid (12-HETE), a product of the lipoxygenase pathway, suggesting a functional PLA2 enzymatic deficiency. We seek to further elucidate the molecular mechanism for these biochemical deficiencies. We will initially quantify serum and urinary metabolites of common cyclooxygenase and lipoxygenase pathways, in addition to functional analysis of platelet aggregation and phospholipid characterization to confirm the molecular defect in this individual. Following confirmation of the enzymatic deficiency, we will assess whether the enzyme is entirely absent from the cells or present in an altered form by performing Western blot analysis of the protein. We will then pursue description on a genetic level. PLA2 regulation in humans involves a complex process including calcium release-regulated activation, phosphorylation, translocation, and multiple other modulatory factors such as cytokines, growth factors, and oncogenes. Because human cytosolic and secretory PLA2 genes have been mapped and functional domains well described, genotyping of these enzymes will assist in pinpointing the mechanism of functional deficiency in our patient and delineating between a defect in the PLA2 enzyme itself or in a regulating mechanism. Defining the deficiency in this patient on molecular and genetic levels will contribute significantly to the knowledge of the role of PLA2 enzymes in humans and clinical consequences of their inhibition.