TENASCIN-X AND THE EHLERS-DANLOS PHENOTYPE

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The Ehlers-Danlos syndrome (EDS) is a group of connective tissue disorders characterized by hyperextensible skin and joints, vascular fragility, and poor wound healing. The syndrome results from defects in fibrillar collagen biosynthesis or deposition and mutations in several collagen genes are found in EDS subtypes. However, the gene(s) responsible for the majority of classical EDS cases remains unknown. Recently, the PI's laboratory identified two unrelated patients with EDS due to deficiency of tenascin-X (TN-X), a large matrix glycoprotein. While this finding demonstrates the first known function of any tenascin, it is not yet known what fraction of EDS is accounted for by TN-X mutations, how TN-X causes this phenotype, or what other functions of TN-X are obscured by redundancy between TN-X and other tenascins. These questions form the basis of this proposal. In aim 1, TN-X mutational analysis will be performed in EDS patients with known TNX protein deficiency, in a second cohort of EDS patients in whom collagen mutations have been excluded, and a third unselected EDS cohort. TN-X secretion by cultured fibroblasts will be evaluated, and the mutations present on each TN-X mutant allele will be identified by Southern blotting, PCR and the protein truncation test. These data will refine our clinical understanding of TNX deficiency and establish an algorithm for diagnosis. We postulate that TN-X is a matricellular protein that regulates matrix deposition through simultaneous interactions with matrix and cellular receptors. Aim 2 will identify integrin and non-integrin TN-X receptors and TN-X binding proteins. In this aim, specific binding of radiolabeled TN-X to myoblasts and dermal fibroblasts will be demonstrated. TNX binding integrins will be identified by TNX affinity chromatography, followed by immunoprecipitation with anti-integrin antibodies. Non-integrin TN-X binding proteins will be identified through a yeast two-hybrid screen. The involvement of specific receptors in mediating TN-X induction of matrix synthesis and anti-adhesion will be demonstrated. Interaction of TN-X with secreted, proteins will be verified in solid phase binding assays and the role of such interaction in regulation of matrix deposition investigated. In aim 3, the TN-X deficient phenotype will be recapitulated in mice by targeted disruption of the TN-X gene. Morphology, biomechanical properties of connective tissues and wound healing of TN-X deficient mice will be compared with wild type littermates. Functional redundancy among tenascins will be evaluated subsequently by creation of TN-X/C double knockout and TN-X/C/R triple knockout mice.