USHERIN--FUNCTION, EXPRESSION, AND ROLE IN PATHOGENESIS

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Usher syndrome type IIa is possibly the most common genetic cause of syndromic deafness. Homozygotes for this autosomal recessive disorder suffer congenital sensorineural hearing loss with progressive retinitis pigmentosa. Through the efforts of this program, the gene for Usher type IIA was recently identified and found to encode a novel extracellular matrix molecule which we refer to as usherin. Herein we show that usherin is a secreted molecule that localizes to the matrix surroundi8ng the spiral ganglion cell bodies and the perineurium in the cochlea, and to the inter-photoreceptor cell matrix surround the photoreceptor cells in the retina. In this proposal we will explore the functional properties of this novel protein and investigate how its absence leads to the observed pathologies. We will employ a number of approaches to determine its biochemical properties as well as what proteins it interacts with, examining both matrix interactions and potential interactions with integrins. Towards this end, we have cloned the full length cDNAs for both mouse and human into both inducible and non-inducible expression systems. We have developed both human and mouse antibodies using synthetic peptide antigens, and are developing additional antisera using larger fusion peptides. A gene knockout mouse model is under development which will be used to examine the structural and molecular changes associated with the inner ear pathology. Since expression of the gene is restricted to important cochlear and retinal cell types, we have cloned a portion of the promoter and expressed it in Y79 photoreceptor cells. This construct will be subjected to deletion mutagenesis to identify the minimal promoter and mobility shift assays to test for the existence of specific nuclear factors. A transgenic animal will be produced expressing green fluorescent protein from the promoter to assess whether the cloned fragment contains all of the elements necessary for appropriate regulation and specificity.