Urine podocyte and podocyte GL3: novel screening tools for phenotype assessment and treatment efficacy in Fabry disease

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Project Summary-Abstract: Fabry disease is caused by deficiency of a lysosomal enzyme a-galactosidase-A, that leads to accumulation of the enzyme substrates and mostly globotriaosylceramide (GL3). Since Fabry disease pathogenesis is closely linked to GL3 accumulation, establishing a diagnosis of Fabry disease requires demonstration of a- galactosidase-A deficiency and accumulation of GL3 in cells/organs. Newborn screening can identify infants with a-galactosidase-A deficiency. However, there is no non-invasive assay for detection of GL3 accumulation in key cells. This is a challenge for deciding who needs treatment and when treatment needs to be started. Fabry nephropathy is a major complication and the second cause of mortality in patients with Fabry disease. Podocytes play pivotal role in kidney failure in Fabry patients. Our study will use state-of-the-art imaging flow cytometry techniques to develop standardized protocols for quantifying podocytes in the urine as an indication of Fabry nephropathy. In addition, our assay will measure GL3 in urine podocytes. We will develop standard protocols using a Fabry podocyte cell line that we have developed using CRISPR/Cas9 approach. We will calibrate our GL3 measurement technique using correlative electron microscopy and unbiased stereology. We will test our protocols in urines collected from patients with Fabry disease with correlations with urine function and relevant clinical data. Development of this test will be an important addition to screening Fabry patients for early detection of disease complication and treatment.