Expression Profiling of Ehlers-Danlos Fibroblasts

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DESCRIPTION (provided by applicant): The Ehlers-Danlos syndrome (EDS) is a generalized connective tissue disorder caused by defects in fibrillar collagen metabolism. Diagnosis in EDS is complicated by overlap in the clinical features of the several described forms and by locus heterogeneity. We hypothesize that dermal fibroblasts derived form EDS patients will display alterations in gene expression that are characteristic of the mutant gene responsible for EDS in each patient. These expression patterns can be identified by microarray technology and will be used to guide mutational analysis in EDS and to identify new candidate genes. In Aim 1, changes in gene expression will be defined for fibroblast cell lines derived from patients with mutations in the COL5A1 and COL5A2 genes causing classical EDS; patients with mutations in C0L3A1 causing vascular type EDS; and patients with mutations in tenascin-X causing a recently described recessive variant of classical EDS. Cell lines will be characterized either by a small number of genes whose mis-expression is specifically associated with one of the EDS genes, or by global patterns of expression using a clustering algorithm. In Aim 2, the ability of expression profiling to direct mutational analysis will be tested. Expression profiling will be carried out on cell lines not previously studied. Cell lines that demonstrate expression changes characteristic of known EDS genes will be subjected to mutational analysis by direct sequencing of the appropriate gene. Expression profiling may also lead to the identification of new candidate genes in EDS. It is expected that some genes will be consistently mis-regulated in all or most of the cell lines carrying known mutations. While these genes are not useful for distinguishing between known mutations, they are themselves good EDS candidate genes. In Aim 3, these genes will be tested for their ability to disrupt collagen deposition in vitro, which is a cellular marker for the EDS phenotype. To do this, adenoviral vectors will be created that express 0.5 to 1.5 kb of antisense sequence derived from the candidate gene as the 3' untranslated region of LacZ. These constructs allow us to monitor infection of wild-type fibroblasts and produce potent inhibition of gene expression. Collagen deposition will be analyzed by immunohistochemistry and metabolic labeling.