Heritable Disorders of Connective Tissue

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

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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. 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. 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. 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. 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 have conducted a follow-up examination to our 2007 Mutation Consortium study of genotype-phenotype relationships in classical OI. This revealed that lethality for COL1A1 mutations is associated with their location in regions important for collagen interaction with other protein, while in COL1A2, mutations resulting in splice-site variants are predominantly lethal. New modulators for phenotypic variability should be sought using murine disease models. 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 treatment group experienced improvement in vertebral parameters, including BMD z-scores, central vertebral height and vertebral area, which tapered 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. 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. We also derived BMI curves for OI, demonstrating distinct shifts above the CDC curves. Lung disease is the major cause of illness and death in individuals with osteogenesis imperfecta (OI), a collagen-related bone fragility disorder. Previously, external factors such as scoliosis and chest wall deformities were considered responsible for abnormal lung function in OI. Prior data from the NICHD OI Research Program showed that pulmonary function declined with age even in children who did not have scoliosis, suggesting an intrinsic component for OI lung disease. In this investigation, we studied 37 children and young adults with several types of OI, 29 of whom have structural mutations in type I collagen. Type I collagen is a component of lung parenchyma as well as the major matrix protein of bone. Study participants underwent lung function tests, X-Rays and CTs of lung tissue. Most patients had restrictive lung disease and decreased gas exchange, independent of scoliosis. Analysis of chest scans and breathing tests showed thickening of the walls of small airways and lung tissue abnormalities. We conclude that OI also causes intrinsic lung disease that negatively impacts Quality of Life (QOL) in OI. Further study of intrinsic lung abnormalities may lead to improved treatment options and improve QOL.