This is a renewal application to continue studies related to X-linked hypophosphatemic rickets. In the previous funding period, the principal investigator and a world-wide consortium, the hyp consortium, successfully cloned the gene (PEX) for this disorder. In addition, the investigative team has continued to produce experimental data suggesting that the hypophosphatemia of the disorder is due to extrarenal production of a hormone or metabolic factor that inhibits Na+ -dependent phosphate transport. The principal investigator now proposes to extend their knowledge regarding the PEX gene which is responsible for XLH. He will use SSCP technology and direct sequencing to identify and localize point mutations in patients with XLH in order to define the crucial epitopes of the PEX protein. In addition, he will use cDNA from the PEX gene to isolate the murine counterpart which will permit us to study in depth its tissue expression and physiologic regulation under a variety of conditions. As a complement to these studies, he will conduct experiments designed to identify and characterize the humoral factor upon which the PEX protein likely acts and which effects abnormal Na+-Pi cotransport in the kidney. The investigator will assess the biological characteristics of the Pi transport inhibitory activity present in the conditioned medium from immortalized hyp-mouse hepatocytes. In addition, he will make detailed efforts to assure that the factor produced in vitro acts as phosphatonin in vivo. Moreover, the investigator will use standard techniques to isolate the protein factor and perform structural analysis. Finally, he will assess what elements of the hyp-mouse phenotype are phosphate independent, but PEX dependent. The investigator will compare various phenotypic characteristics in hyp-mice with those in NaPi-2 gene knockout mice. Collectively, these investigations will provide new data regarding Pi homeostasis, as well as the pathogenesis of XLH and related vitamin D resistant diseases. Such advances will potentially improve the therapeutic strategies for a variety of human disorders.
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