Osteogenesis imperfecta is a clinically heterogeneous disorder that affects more than 1/20,000 individuals. The phenotypic range is from lethal in the perinatal period, to a mild increase in fracture frequency. More than 90% of affected individuals have mutations in the genes that encode the chains of type I collagen, the major structural protein of bone. The objectives of studies proposed in this application are to determine how these mutations perturb molecular assembly, intracellular transport, secretion, and extracellular processing and; to identify the mechanisms by which, and the intracellular locations at which, cells recognize abnormal proteins and initiate strategies to prevent their secretion. The yeast two-hybrid system will be used to identify novel proteins that interact with portions of the type I procollagen molecule, and those genes isolated and characterized. Antibodies to the newly identified proteins, as well as antibodies to type I procollagen, HSP47, GRP78 and GRP94, and prolyl-hydroxylase (PDI) will be used to determine which proteins interact with the abnormal molecules synthesized by cells from patients with different classes of mutations in the COLIAI and COLIA2 genes. Additional mutations will be identified that occur at CpG sites in the regions that encode glycine residues in the triple helix of both chains of type I procollagen, by analysis with restriction endonucleases of those known sites (36 in the COLIAI gene and 27 in the COLIA2). Further additional mutations that occur in large families with variable expression, and those for which a parent is mosaic, will be sought to elucidate the basis for variable expression. Finally, the kinetics of MRNA processing for mutations that affect splicing will be identified. These studies are intended to increase the ability to understand the phenotypic effects of mutations, and identify sites along the processing pathway where intervention could ameliorate the phenotypic effects of mutations in collagen genes.
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