Osteogenesis imperfecta (OI) is a clinically, genetically and biochemically heterogeneous disorder characterized by bone fragility and other connective tissue abnormalities. In the vast majority of affected individuals the clinical condition results from mutations in the two genes (COL1A1 and COL1A2) that encode the chains of type I procoliagen. The objectives of the studies proposed in this application are to characterize the mutations in the collagen genes that give rise to different forms of OI, to determine the relationship among the nature of the mutation, the gene in which it is located, and the clinical phenotype, to determine how mutations alter the intracellular and extracellular behavior of molecules that contain abnormal chains, to identify the parent of origin of new dominant mutations, and to determine the extent of mosaicism for mutations in collagen genes among parents of first affected individuals. For mutations that alter chain structure, the site of the mutation will be approximated by peptide mapping, the precise location of the mutation will be determined by single-base mismatch chemical cleavage or single stranded conformational polymorphisms, the appropriate region of cDNA from fibroblasts or genomic DNA will be amplified and either sequenced directly or cloned into M13 and selected clones will be sequenced. For mutations that alter the expression of collagen genes, the abnormal allele will be identified by the absence of expressed polymorphic markers from the mRNA synthesized from that gene (in heterozygotes) and the structure of the mutation identified and characterized following search of the gene using base mismatch chemical cleavage of single stranded conformational polymorphisms to detect regions of difference. The effects of abnormal chains in type I procollagen molecules will be determined by examinining the kinetics of secretion and the effects of RER binding proteins on secretion, the efficiency of secretion of molecules with one or more than one abnormal chain, the effect-,of these molecules on molecular structure at the EM level, the thermal stability of molecules, and the ability of molecules to participate in fibrillogenesis. Finally, the extent of mosaicism will be determined by examination of tissues from parents and measuring the presence of the mutant allele by digestions with appropriate restriction enzymes or by allele-specific oligonucleotide hybridization. These studies should help to understand the molecular basis of OI, and how mutations are translated to phenotype. Finally, they have implications for the pathogenesis of more common disorders of bone formation.
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