Osteogenesis imperfecta (OI) is the mildest and most common inherited form of brittle bone disease. Little is known about its etiology except that dermal fibroblasts from most affected individuals produce about half the normal amount of type I procollagen. My data suggest that OI type I results from mutations that affect the expression of COLlAl, the gene encoding the pro-alpha1(I) chain of type I procollagen. The specific aims of this project are to identify and characterize COLlAl mutations that result in OI type I, to determine the mechanisms by which type I collagen synthesis is altered, and to understand why some COLlAl mutations result in mild disease while others produce lethal or severe phenotypes. The long-term objective of this project is to broaden our understanding of collagen gene regulation and its role in producing a clinical phenotype. Mutation identification is the first step in achieving the long-term objectives. Mutations that affect initiation of transcription (promoter and enhancer mutations) will be identified by a variety of mismatch techniques, including denaturing gradient gel electrophoresis, chemical cleavage and single-strand conformational polymorphism electrophoresis, using as substrate, genomic DNA that has been amplified by the polymerase chain reaction (PCR). Splicing defects that result in exon skipping will be approached by analysis of PCR amplified COLlAl cDNA for the presence of heteroduplex formation, and by mismatch techniques using amplified genomic DNA fragments that span donor and acceptor splice sites. Translational mutations that lead to a shortened or elongated protein, will be identified at the protein level by cell-free translation, and at the molecular level by mismatch techniques with amplified cDNA as substrate. Major structural rearrangements will be identified by restriction endonuclease analysis. Mutant alleles from each mutation category will be sequenced to determine the changes that alter COLlAl gene expression, and relevant functional assays will be used to assess how a given mutation alters type I procollagen production. Finally, a mutation map will be established and compared with that already generated for the more than 70 characterized mutations from individuals with lethal and deforming OI.