Heritable Disorders of Connective Tissue

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications, trials & patents

Abstract

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorder osteogenesis imperfecta (OI). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. Our Section has generated a knock-in murine model for OI with a COL1A1 collagen mutation, the Brtl mouse. Recently, we collaborated to investigate the material properties of Brtl cortical and endosteal bone using acoustic transmission microscopy. Collagen orientation in Brtl endosteal bone had a strong reduction in periodically alternating collagen orientation compared to WT. Also, sound velocity was significantly increased in Brtl endosteal bone, demonstrating that the predominant effect of the Brtl mutation is on endosteal bone. Brtl is also being used as the model for testing an anabolic therapy for OI, anti-sclerostin antibody (SclAB), which works by stimulating bone formation along the canonical wnt pathway. SclAB was demonstrated to be effective in increasing cortical bone formation in both young and adult Brtl mice. Brtl femora increased cortical bone formation and mechanical strength, without exacerbating the underlying brittleness of OI bone material. These data suggest SclAB treatment does not impair material properties. Although SclAB is a short-acting drug, a single dose of bisphosphonate will preserve the gains to trabecular bone mass following cessation of antibody. Concurrent administration of low dose bisphosphonate with anti-sclerostin antibody revealed synergistic effects on trabecular mass and vertebral stiffness. Cortical gains in mass and stiffness occurred through SclAB alone. Thus, minimal antiresorptive treatment may be able to amplify the effects of SclAB, without incurring the detrimental effects of BP on bone material quality. Recently, our collaborative studies have demonstrated that treatment of Brtl mice with SclAB does not change cranial shape in ways that would be significant for impingement of neural foramina. We identified a novel high bone density form of OI caused by mutations in the C-proteinase cleavage site. The Asp-Ala dipeptide between the telopeptide and the C-propeptide of each chain is cleaved by C-proteinase/BMP1 to release mature collagen. Children with substitutions at these residues present with fractures and a high DEXA z-score. Interestingly, despite the high DEXA, radiographs and histology are similar to type I OI and point to matrix deficiency. Pericellular processing of procollagen C-propeptide is delayed. FTIR and BSEM revealed bone mineral content higher than other forms of OI. These data not only reveal a novel form of OI but also provide new fundamental information on roles of procollagen processing and the mechanism of tissue mineralization. We have now generated a mouse model for HBM OI, to investigate the role of type I procollagen C-propeptide cleavage in the mechanism of increased bone mineralization, both at the matrix and intracellular levels. We have also studied amino acid substitutions in the procollagen C-propeptide itself. These mutations are intriguing because they occur in a region of the promolecule that is not incorporated into matrix. On immumofluorescence microscopy, procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to normal localization at the ER membrane. Pericellular processing of the mutant C-propeptide was defective, as were in vitro cleavage assays with purified BMP1. Our data suggest that the 3D structure of the C-proteinase cleavage site is altered by these mutations. In bench studies aimed at understanding the basis of the phenotypic variability of patients with the identical OI-causing mutation, we collaborated on investigations of cellular cytoskeleton in Brtl lethal and surviving mice. Components of intermediate filaments, microtubules and actin filaments were all shown to be abnormal only in tissues from lethal mice. This data was extended to cells from patients with lethal and non-lethal mutations caused by identical glycine substitutions. They point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment. We also collaborated on studies in which Brtl cells were exposed to drugs to counter ER-stress. General protein secretion, as well as specific type I collagen secretion, were improved in these cells. We are continuing our clinical studies of children with types III and IV OI. The SHDBEM undertook the first randomized controlled trial of bisphosphonate in children with types III and IV OI. The aim was to test both the primary skeletal gains and secondary gains (improved functional level and muscle strength and decreased pain) reported in observational trials. The treatment group experienced improvement in vertebral parameters, including BMD z-scores, central vertebral height and vertebral area. The increase in vertebral BMD in the treatment group tapered off after one to two years of treatment. There was no significant change in ambulation level, lower-extremity strength or pain in treated children, which appear to be placebo effects in uncontrolled trials. We recommend that pamidronate treatment of children with types III/IV OI be limited to three years, with subsequent follow-up of bone status. We are currently engaged in a dose comparison trial and focusing on the variability of response to treatment. These differences may be related to important individual variation in ability to synthesize new bone or to remodel bone. Short stature is one of the cardinal features of OI. The SHDBEM has established OI-specific longitudinal growth curves for children with types III and IV OI based on data from 100 children with structural mutations in type I collagen. These curves show that the height trajectory of OI children is influenced by both type and gender, but only OI type affected the weight curves. Interestingly, head circumference did not differ by gender or type, and length, weight were not influenced by the chain mutated. We also derived BMI curves for OI, demonstrating distinct shifts above the CDC curves. A collaborative clinical study focused on the effect of intramedullary rods with a high diameter on OI long bones. Patients showed diaphyseal atrophy that was significant by the 2nd year post-rodding, with secondary effects on muscle strength, gait and scoliosis. Replacement of the large intramedullary rod with an appropriately sized smaller diameter rod resulted in significant diaphyseal recovery. This study should lead surgeons to smaller diameter rods for osteotomies and to a technique for reversal of atrophy where a large diameter rod was previously used.