The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited connective tissue disorders characterized by tissue fragility. More than 10 types of EDS have been described and mutations in more than six genes does not account for all the known types. One of the most severe forms of the syndrome, EDS type IV, results from mutations in the COL3A1 gene that encodes the chains of type III procollagen. Fewer than 10 mutations in this gene have been identified so that the relationship between mutation and phenotype is unclear. The objective of studies proposed in this application is to characterize the mutations in the COL3A1 gene that result in the EDS type IV phenotype in 70 probands and members of their families, to determine the rate of mosaicism for these mutations among unaffected parents of affected probands, to determine the parent of origin of mutations and, for mosaic individuals, to determine the grandparent of origin of the mutation searching for potential effects of allele imprinting on mutation, to determine the effects of mutations on folding of molecules that contain one or more abnormal chains, and to analyze the effects these mutations have on intracellular and extracellular processing of the abnormal molecules. In addition, mutations in the COLlA2 gene [that encodes the pro(alpha 2(I) chains of type I procollagen] that result in EDS type VII and in a rare form of EDS type II will be characterized. Mutations in the COL3A1 or COL1A2 gene will be located at the protein level by peptide mapping and the position confirmed by base mismatch chemical cleavage or analysis of single-stranded conformational polymorphisms. The target region of the cDNA will be amplified by PCR and either sequenced directly or cloned into M13 prior to sequence determination. The nature of the mutation will be confirmed in genomic DNA from the proband and in other affected family members, when available. The rate of folding of molecules that contain abnormal chains will be studied by they rate at which they acquire protease resistance during pulse-chase experiments, and by examining the rate at which interchain disulfide bonds form in the amino-terminal propeptide of type III procollagen. The nature of proteins that delay transfer of abnormal molecules from the rough endoplasmic reticulum will be examined by co-precipitation and cell-fractionation experiments. These studies will increase our understanding of disorders that result in skin and vascular fragility, and provide models for aberrant molecular folding and intracellular behavior of abnormal collagen molecules.
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