The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited connective tissue disorders characterized by tissue fragility and abnormal skin. Clinical, biochemical and molecular genetic characterization has identified more than 8 distinct types of EDS among which the molecular basis of EDS type IV (mutations in the COL3A1 gene that encodes the chains of type III procollagen), EDS type IV (mutations in lysyl hydroxylase), EDS type VII (mutations in the COL1A1 and COL1A2 gene, and in procollagen N-proteinase), and EDS type IX (mutations in the copper-transporter ATPase) have been identified. The mutations in the more common EDS types I, II and III remain undetermined and although a number of mutations have been identified in the COL3A1 gene that results in the EDS type IV phenotype, the mechanisms by which these mutations are translated into the phenotype are unclear.
The aims of the studies proposed in this application are to identify and characterize mutations in the COL3A1, COL5A1 and COL5A2 genes that encode the chains of type III and V collagens in individuals with EDS type IV and types I and II, respectively, to determine the mechanisms by which mutations in the COL3A1 gene affect mRNA processing and protein transport and secretion. The mutations in the COL3A1, COL5A1 and COL5A2 genes will be sought following synthesis of cDNA from cultured fibroblasts and analysis of large overlapping fragments of cDNA synthesized by PCR. The mutation bearing fragments will be identified by SSCP or heteroduplex analysis and the mutations will be identified by direct sequence analysis, using strategies familiar to the laboratory. Defects in nuclear transport of abnormal COL3A1 mRNA will be analyzed is several ways. The site of mRNA accumulation will be mapped by in situ hybridization and binding proteins identified by gel shift assays to examine nuclear extracts for the presence of binding proteins that recognize premature termination codons in the retained mRNA species. Antibodies to candidate proteins which recognize partially folded collagen intermediates, including prolyl hydroxylase, lysyl hydroxylase, GRP78, GRP94, HSP47 and others will be used to identify mechanisms by which abnormal proteins are recognized and their secretion inhibited. These studies should help to understand the molecular mechanisms by which mutations are expressed as pehnotypes and provide models for additional genetic disorders of the skin and other tissues.
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