OIM--A MURINE REPLICA OF HUMAN OSTEOGENESIS IMPERFECTA

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Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue which predominately affects bone. Recent investigations indicate that the majority of OI cases are the result of mutations affecting the metabolism of the alpha 1 or alpha 2 chains of type I collagen. These mutations presumably disrupt collagen fiber formation in the extracellular matrix and/or impair the interaction of these abnormal fibers with other protein and proteoglycan components of the skeletal matrix. OI is also important because it may provide insight into the pathophysiology of related disorders of skeletal formation including idiopathic osteoporosis. The demonstration of specific type I collagen mutations (point mutations, deletions, insertions) has not shed light on the pathophysiology of OI, specifically with regard to two important questions; 1) the impact of specific mutations on normal skeletal formation and 2) factors which are responsible for the high degree of variable expressivity observed in human OI. The availability of a viable animal replica of OI will enormously facilitate the investigation of these important questions. The Osteogenesis Imperfecta (oim) mouse is the result of a spontaneous, nonlethal mutation first observed in the breeding stock of The Jackson Laboratory. This mutant appears to be biochemically and phenotypically similar to a well studied patient with severe OI type III. Like this OI patient, the phenotype of homozygous oim mice includes diminished growth, spontaneous fractures and progressive skeletal deformation, including scoliosis and osteoporosis. Biochemical study of homozygous oim dermal fibroblasts, skin, bone, tendon, heart, liver and kidney demonstrated a failure to synthesize alpha 2(I) chains. Also, we recently defined a putative G deletion at position 4060 of the proalpha 2 (I) gene that alters the last 49 amino acids of the COOH-propeptide. Therefore, only alpha1(I)3 homotrimer was found to accumulate in the skeletal matrix. alpha1(I)3 homotrimeric collagen is incorporated into a mineralized extracellular matrix, however, the resultant skeleton is osteopenic, deformed and smaller than in wild type mice. These data, combined with the radiographic and histochemical data, suggest that homotrimeric collagen does not provide a suitable template on which to form normal bone. We propose to 1) verify the putative mutation by sequencing the same region in a second homozygous oim mouse and in a heterozygous oim mouse, 2) maintain a mouse colony capable of generating sufficient animals, 3) determine the mutation's effect on skeletal formation, 4) culture calvarial osteoblasts to examine their ability to form a mineralizing, 3-dimensional extracellular matrix, 5) develop alpha2(I) minigenes utilizing different promoter: enhance elements and a full-length murine alpha2(I) CDNA, 6) develop transgenic osteoblast cultures and mice with these expression vectors, and 7) evaluate skeletal, dental and other tissues in transgenic mice for the effect of these constructs on the synthesis of alpha 1(I)3 homotrimer versus normal type I heterotrimer on bone formation in vitro and in vivo. Finally, we will attempt to rescue homozygous oim mice should provide a biochemical and phenotypic replica of human OI that will afford a unique opportunity to study the pathophysiology of type I collagen metabolism, and its relationship to skeletal formation.