Heritable Disorders Of Connective Tissue

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). 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 Branch has generated a knock-in murine model for OI with a classical collagen mutation, and we are using this model to study disease pathogenesis and the skeletal matrix of OI, the effects of pharmacological therapies, and approaches to gene therapy.We are also continuing our clinical studies of children with types III and IV OI, who form a longitudinal study group enrolled in age-appropriate clinical protocols for treatment of their condition. [unreadable] [unreadable] The Brtl mouse model for OI continues to be investigated to understand the pathological and cellular mechanism of OI. We have investigated the cellular contribution to Brtl bone properties.Brtl cortical and trabecular bone are reduced before and after puberty, with BV/TV decreased 40-45%. Brtl osteoblast surface is comparable to wild-type, and Brt and wild-type marrow generate equivalent numbers of osteoblast precursors at both ages. Interestingly, the osteoclast surface is increased[unreadable] in Brtl at both ages (36-45%), as are the number of TRAP positive cells (57-47%). After puberty, Brtl osteoblast matrix production decreases to half of wild-type values. In contrast, osteoclasts remain significantly elevated compared to wild-type. The combination of osteoblast and osteoclast changes[unreadable] results in a decline in BFR from normal to half of wild-type values at 6 months. We investigated whether the RANKL/OPG signaling pathway is responsible for the uncoupled cell numbers.Both immunohistochemnistry and real-time RT-PCR show increased RANK, RANKL and OPG levels in Brtl, but a[unreadable] normal RANKL/OPG ratio. Surprisingly, we found that TRAP+ precursors are markedly elevated in Brtl marrow cultures and form more osteoclasts,suggesting that osteoclast increases arise from increased numbers of precursors. This exciting data suggest that OI pathology is due to acombination of deficient bone formation by osteoblasts and increased numbers of osteoclasts,triggered independently by abnormal bone matrix.[unreadable] [unreadable] We have also completed a collaborative study which shows that the Brtl mouse develops an early and rapidly progressive osteoarthritis. The knee joint OA was detected by micro-MRI and micro-CT and confirmed by histology and immunohistochemistry. Brtl mice show thinning of subchondral bone by 2[unreadable] months of age; by 12 months Brtl has severe OA with joint space narrowing.Safranin O staining showed reduced cartilage proteoglycans compared to wild type mice of the same age. We demonstrated that this cartilage degeneration was not due abnormal spacial expression of collagen by immunohistochemistry. These studies are important for the common condition, osteoarthristis, as well as for the rare dysplasia OI. They demonstrate that abnormalities in subchondral bone can cause OA in mice with[unreadable] initially normal cartilage.[unreadable] [unreadable] To better understand the relationship of genotype and phenotype in human OI, the BEMB led and international consortium of connective tissue laboratories to assemble and analyze a mutation database containing over 830 mutations. Genotype-phenotype modeling revealed different functional relationships for each chain of type I collagen. Lethal mutations in alpha 1 (I) coincide with the Major Ligand Binding Regions. Lethal regions in alpha 2(I) continue to support the Regional Model first proposed by the BEMB, with lethal mutations in regularly-spaced clusters along the chain that coincide with proteoglycan binding regions. This model correctly predicts clinical outcome in 86% of alpha 2(I) mutations. [unreadable] [unreadable] We have participated in several collaborations which have extended our understanding of genotype-phenotype relationships. Our long and continuing collaboration with the laboratory of Sergey Leikin at NICHD has yielded insight into structural heterogeneity in the the type I collagen triple helix and its role in osteogenesis imperfecta. Using differential scanning calorimetry and circular dichroism, changes in collagen melting temperature were measured for 41 different glycine substitutions. There was no correlation of melting temperature with substituting residue. Instead, the variations in Tm defined regions along the triple helix which were further confirmed with an activation energy map. We deduced two large, flexible regions important for collagen fibril assembly and ligand binding, one of which aligned with a lethal region for glycine substitutions in the alpha[unreadable] 1(I) chain.[unreadable] [unreadable] The mapping of collagen functional domains was also extended in terms of cell and matrix interaction domains on the collagen fibril. This effort utilized a database of hundreds of type I collagen ligand binding sites and mutations on a 2D model of the fibril. This fibril based model supports important roles for the interaction with integrins in a cell interaction domain, as well as a matrix interaction domain important for fibril connections with proteoglycans. Complementary to the larger mapping enterprise,was our collaboration with investigators mapping the osteonectinbinding sites on type I collagen, using OI mutations to validate the importance of a major and secondary binding site.[unreadable] [unreadable] The BEMB 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 (increased bone density and decreased fractures) 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. However, the increment 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 children with OI treated with pamidronate. Hence the changes previously reported appear to have been a placebo effect in uncontrolled trials. We are now recommending that treatment of children with types III and IV OI with pamidronate be limited to one to two (or at most three) years, with subsequent follow-up of bone status. Furthermore, we are currently engaged in a dose comparison trial, using the dose from our first trial and a lower dose.[unreadable] [unreadable] We have also done a clinical radiographic investigation which parallels our studies of recessive OI. We have demonstrated that popcorn calcifications occur in recessive OI; now we delineate its incidence and progression in dominant OI. We retrospectively examined serial lower limb radiographs of 45 children with known dominant mutations in type I collagen. The mean age of onset of popcorn calcifications was 7 years, with a range of 4-14 years. Those children with popcorn always had this finding in their distal femora, and most also had it in proximal tibiae. Unilateral popcorn calcifications were shown to contribute to leg length discrepancy, but not to the severe linear growth deficiency of OI. The type I collagen mutations associated with popcorn calcification occur equally in both COL1A1 and COL1A2 and have no preferential location along the chains.