The Oculocerebrorenal Syndrome of Lowe (OCRL; McKusick 309000) is an X- linked disorder characterized by mental retardation, congenital cataracts, and proximal renal tubule dysfunction. Since the last review of this application, the laboratory isolated a candidate gene (OCRL-1) for OCRL through positional cloning and found strong amino acid sequence homology to an enzyme HUMINP5P, one of at least three inositol polyphosphate-5-phosphatases (InP5P) known to exist in various human tissues. The homology suggests but does not prove that the OCRL-1 protein may be another inositol polyphosphate-5-phosphatase. This application is for continuation of genetic and molecular studies in Lowe Syndrome to address the following questions in the next grant period: 1. Does OCRL-1 encode another InP5P or some other enzymatic activity? 2. Do OCRL patients have a defect in InP5P activity? 3. What can we learn from the mutations in OCRL-1 found in OCRL patients? Research Design and Methods: 1. Characterize the OCRL-1 mRNA reading frame by cloning and sequencing additional cDNA. 2. Identify the enzymatic activity encoded by OCRL-1. Express the OCRL-1 protein to obtain highly purified OCRL-1 protein for enzymatic assay and as antigen for raising antisera. Identify the protein in normal cells by Western blotting and immunoprecipitation. 3. Investigate cells and tissues from normals and OCRL patients to determine whether probands have defects in the OCRL-1 gene and its product. 4. Determine the genomic structure of OCRL-1. 5. Identify mutations in OCRL patients. Examine OCRL patients' DNA by Southern blot for major alterations. Use polymerase chain reaction amplification of cDNA (RT-PCR) for single-stranded conformational polymorphism (SSCP) analysis in OCRL patients and normals and sequence the fragments showing SSCP alterations to search for mutations. Develop primers for all exons and intron/exon boundaries in genomic DNA. If OCRL is indeed a defect in inositol polyphosphate metabolism it represents the first human disease due to an inborn error of inositol metabolism, an important but very complex and incompletely understood area of human metabolism. Elucidation of the biochemical and molecular defects will provide important insights into three disease processes in man: mental retardation, cataract formation, and renal tubular dysfunction.
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