The Section conducts studies to elucidate the molecular and biochemical mechanisms of heritable disorders of connective tissue, specifically osteogenesis imperfecta (OI) and Ehlers- Danlos(EDS), and to apply this information to the treatment of these disorders. We generated a knock-in mouse model for the non-lethal form of OI and have designated this mouse Brittle (brtl). Brtl has a classic glycine substitution (G348C) in one of its collagen alpha1(I) chains and reproduces the molecular and biochemical features of OI. Its bone structure is disorganized and undermineralized. We have shown that this mutation reduces the temperature-dependent attractive forces responsible for collagen fibrillogenesis. We also investigated the variability of phenotype present in these mice and frequently seen in human OI. In mice with lethal and surviving outcomes, we demonstrated both equivalent expression of mutant collagen transcripts and tissue incorporation of mutant protein. We also engineered a ribozyme cleavage site into the Brtl mutant allele. We have been developing hammerhead ribozymes as a theraputic agent for OI and other dominant disorders. Cleavage of the mutant transcript by ribozyme can suppress expression of the mutant protein. We demonstrated allele-specific suppression of the mutant collagen transcript in fibroblasts to about 50% of its level in controls. For the murine ribozyme site, we have demonstrated in vitro specificity and efficiency. To create a delivery system for ribozymes, we have generated a transgenic mouse expressing a ribozyme construct specific for the Brtl ribozyme cleavage site. To determine the level of mutation expresssion that is compatible with functionally normal bone, we have identified two mosaic carriers who are the mothers of type II and IV OI affected children, respectively. We demonstrated the type IV carrier has a high percent of mutant cells in her leukocytes and fibroblasts. The carrier of the lethal mutation has been demonstrated to have 5-10% mutant cells in leukocytes and fibroblasts. We are initiating osteoblast studies on the carriers. Our studies of cultured OI osteoblasts are aimed at understanding the mechanism of OI as a bone disease. We have demonstrated that a greater proportion of electrophoretically abnormal collagen is detected both intracellularly and secreted into media by osteoblasts than by matched fibroblasts. Pulse labelling studies showed that overmodified chains appear more rapidly in osteoblasts than fibroblasts and have greater electrophoretic delay. In our studies of patients with EDS, we have identified a mutation in an alpha1(V) chain which causes haploinsufficiency and demonstrates that deficiency of normal type V collagen causes mild EDS. Affected individuals have a splicing defect which results in an out-of- frame transcript with a premature termination codon. Scanning electron microscopy demonstrates variation in dermal type I collagen fibril size and cauliflower fibrils. - osteogenesis imperfecta, ehlers-danlos syndrome, collagen, ribozymes - Human Subjects & Human Subjects: Interview, Questionaires, or Surveys Only
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