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Genetic disorders of bone and extracellular matrix

Joan C. Marini, MD, PhD, Head, Section on Connective Tissue Disorders Armando Flor, MD, Clinical Associate Aarthi Ashok, PhD, Postdoctoral Fellow Thomas Uveges, PhD, Postdoctoral Fellow Antonella Forlino, PhD, Contractor Anne Letocha, MSN, CRNP, Senior Research Assistant Wayne A. Cabral, AB, Chemist Aileen M. Barnes, MS, Research Associate Sarah Milgrom, BS, Postbaccalaureate Fellow

In a unique 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 primary collagen defects cause skeletal fragility and other significant connective tissue symptoms and then to apply the knowledge gained from our studies to the treatment of children with these conditions. An understanding of the interactions of mutant collagen molecules with the normal collagenous and noncollagenous components of extracellular matrix will also enhance our understanding of normal bone function and may yield insights applicable to the more common forms of osteoporosis. We recently focused on the development of a nonlethal animal model for OI with a classical collagen mutation. This nonlethal knockin Brtl mouse (Brtl), with a glycine substitution mutation in the alpha1(I) chain, is an excellent model for pharmacological treatment trials, for developing gene therapy suitable for dominant disorders, and for investigations of the skeletal matrix of OI. Our clinical studies involve children with types III and IV OI who are enrolled in age-appropriate clinical protocols for treatment and who form a longitudinal study group. The OI/EDS region of the alpha1(I) collagen chain Cabral, Letocha, Marini; in collaboration with Leikin A distinct subset of osteogenesis imperfecta patients are those with OI/EDS. In addition to the skeletal fragility of OI, they have characteristics of EDS, including severe laxity of large and small joints and early-onset scoliosis. In seven children with OI/EDS, we delineated mutations in the first 90 residues of the helical region of the alpha1(I) chain. We determined that these collagen mutations cause abnormal N-propeptide processing, incorporation of pN-collagen into matrix, and decreased diameter of dermal fibrils. These data provide a mechanism for the children’s EDS symptoms while the helical changes per se are responsible for bone fragility. Thus, the mechanism of the subject children’s EDS is shared with patients with EDS VIIA and B attributable to the absence of the N-proteinase cleavage site from the alpha1(I) or alpha2(I) chain, respectively. We identified seven mutations by direct sequencing of RT-PCR amplification products of alpha1(I) mRNA. In contrast to normal procollagen and collagen, which have an identical melting peak, the thermal stabilities of each of the mutant collagens differed from those of the corresponding procollagens. Our observations also stand in contrast to collagen mutations beyond the first 90 amino acids; their differential scanning calorimetry tracings usually show both normal and lower stability peaks, but the melting curves of procollagen and collagen are identical for each mutation. In vitro cleavage with N-proteinase processed only 25 percent of proband pro-alpha1(I) chains for exon 7 mutations and 65 to 85 percent of pro-alpha1(I) chains for exon 8-11 mutations. The pericelluar processing of the seven mutants was also delayed. The pN-collagen is incorporated into matrix deposited by cultured fibroblasts, with pN-alpha1(I) collagen prominently present in the newly incorporated and immaturely cross-linked fractions. Electron microscopy of dermal fibrils of six patients revealed that fibril diameters of all six were significantly smaller than those of matched controls, as seen in EDS VII. The above assays defined a folding region of alpha1(I) in which mutations cause a distinct OI/ED phenotype by altering the triple helical structure and secondary structure of the N-proteinase cleavage site. The retention of N-propeptide in a substantial proportion of collagen chains limits fibril diameter. The abnormal fibrils may cause laxity of joints and paraspinal ligaments either directly by reduced resistance to shearing forces or indirectly by altering interactions between collagen and other matrix components in the overlap zone of the D-periods. Type I collagen C-propeptide mutations Marini, Barnes, Ashok Mutations in the C-propeptide of type I collagen have been found in a small number of patients with OI. The phenotype of these patients ranges from lethal to moderately severe. The mutations are of special interest because they are located in a region that is cleaved from procollagen before collagen fibril assembly. Therefore, the mutations per se are not expected to be present in collagen fibrils in tissues. The implication is that the pathophysiological mechanism of these mutations must differ from that associated with mutations in the helical region of the alpha chains; the latter mutations are incorporated into matrix and exert a dominant negative effect. In the collagen of children with types III and IV OI, we identified four novel C-propeptide mutations at conserved residues. All mutations delayed incorporation of alpha1 chains into heterotrimers, with delay ranging from two- to six-fold the chain incorporation time of normal control collagen. A pericellular processing assay suggests a delay in C-propeptide removal from secreted collagens containing these mutations. Mutant collagens are incorporated into fibroblast matrix in culture and form mature cross-links. Using immunofluorescence assays, we investigated the intracellular interaction of the mutant procollagen molecules with ER chaperones in OI fibroblasts by comparing the behavior of normal control molecules with that of molecules with a C-terminal helical mutation. Our results demonstrate a clear correlation between the presence/type of mutation and the subcellular localization pattern of procollagen. Normal procollagen and procollagens with mutations in the carboxyl end of the helical domain display a distinct reticular pattern of ER localization while the procollagens with C-propeptide mutations exhibit a diffuse ER localization. The diffuse pattern of procollagen staining displays an almost complete overlap with the immunofluorescence pattern of the ER chaperones, Hsp-47, and protein disulfide isomerase (PDI) and a minimal overlap with the ER-membrane–associated chaperone calnexin. In contrast, the reticular pattern of procollagen fluorescence observed in normal and helical mutation–bearing fibroblasts shows poor overlap with both Hsp-47 and PDI, but highly significant overlap with calnexin. Thus, procollagens with mutations near the carboxyl end of the helix and C-propeptide mutations display different intracellular behavior. The location of the mutation along the procollagen chains directs the nature of ER chaperone interactions. Alendronate treatment of Brtl mouse Marini, Uveges; in collaboration with Goldstein, Gronowicz Bisphosphonate drugs are widely administered to children with OI, but the drugs’ effects on OI bone containing abnormal type I collagen have not been directly examined. The Brtl mouse model for type IV OI has a glycine substitution (G349C) knocked into one COL1A1 allele. We treated Brtl and wild-type offspring of Brtl x CD-1 matings from two to 14 weeks of age with either alendronate (0.219 mg/kg/wk, gift of Merck) or saline placebo. Brtl mouse weight and femor length were significantly smaller than in wild-type mice and were unchanged by alendronate. Whole-bone density of femurs and lumbar vertebrae, measured by using a Lunar Piximus, were significantly increased in both treated Brtl and wild-type mice; treated Brtl samples attained the average untreated wild-type bone mineral density (BMD). Micro CT data suggest that the differences in Brtl are attributable to increases in bone volume rather than to mineralization. Distal femoral bone volume per total volume doubled with treatment in both Brtl and wild type due to increased trabecular number. Diaphyseal cortical thickness increased by periosteal bone deposition in Brtl and wild-type femurs. In treated Brtl, the overall geometry of femurs reshaped to a more rounded structure, similar to that in the untreated wild-type mice. We tested the mechanical properties of femurs in four-point bending. Alendronate treatment increased femoral stiffness and decreased pre-yield displacement in wild-type mice, indicating that the treatment is not benign for normal bone. Stiffness, pre-yield displacement, and yield load were unchanged in treated Brtl femora. In both genotypes, we observed an increase in the ultimate load at which femurs fracture. Unfortunately, alendronate has a negative impact on several aspects of bone quality. First, treatment reduces the predicted material strength and modulus of Brtl and wild-type bone. Second, the brittleness (post-yield displacement) of treated Brtl femurs did not improve; post-yield displacement decreased further in treated Brtl as compared with untreated wild-type mice. After the yield point is reached, Brtl femurs fracture with similar additional load and deformation as if untreated. Third, the metaphyses of treated Brtl femurs exhibit increased remnants of mineralized cartilage. The matrix discontinuities caused by the presence of mineralized cartilage in the bone may increase the risk of fracture initiation and account for some of the observed increase in BMD. Fourth, we observed a detrimental effect on bone cells. After 12 weeks of alendronate treatment, bone formation rates (BFR/BS), mineral apposition rate (MAR), and mineralized surface (MS/BS) decline to less than 25 percent of pretreatment values in Brtl and wild-type mice. At that time, the percent of osteoblast surface is significantly lower in both genotypes while the percent of osteoclast surface remains stable. In addition, the morphology of the Brtl osteoblasts changes from the plump cuboidal osteoblasts seen in untreated femurs to an intermediate morphology, evidence of a toxic effect on the cells. Our interpretation is that alendronate treatment of Brtl improves bone geometry and increases loading before fracture but decreases predicted bone material quality and alters osteoblast surface and morphology. The data suggest that limited treatment duration may be optimal for obtaining improved bone geometry and minimizing the detrimental effect of extended treatment on bone quality. Pamidronate treatment of children with types III and IV OI Letocha, Marini; in collaboration with Gerber, Paul Uncontrolled trials of bisphosphonates in OI children report increased vertebral bone density and height, improved strength and functional level, and decreased fractures and bone pain. We undertook a randomized controlled trial of pamidronate in children with Types III and IV OI. The first study year was controlled; children in the treatment group received pamidronate (10 mg/m^2/day for three days every three months); children in both treatment and control groups underwent quarterly rehabilitation and physical therapy assessments, including measurements of function, strength, and pain. Children in the treatment group received pamidronate for an additional six to 21 months. All patients underwent L1-L4 DEXA, spine qCT, spine radiographs, and musculoskeletal and functional testing. In the controlled phase, treated patients experienced a significant increase in vertebral BMD z-score as compared with the controls. They also had significant increases in L1-L4 mid-vertebral height and total vertebral area as compared with the controls. The treatment group did not experience decreased long bone fractures. In the extended treatment phase, DEXA z-scores and vertebral heights and areas did not increase significantly beyond the 12-month value. In the context of maximized physical rehabilitation, we did not see an additional functional effect from bisphosphonate treatment. In contrast to reports from uncontrolled trials, we found no significant changes in ambulation level, lower extremity strength, or pain in OI children treated with pamidronate. We assessed motor skills related to ambulation with the 10-point Brief Assessment of Motor Function (BAMF). At initiation, the BAMF of the treatment group was 6.1±1.8 versus 6.7±1.9 at 12 months. At initiation, the BAMF of the control group was 6.6±2 versus 7.02±1.31 at 12 months. Manual muscle testing was assessed as the sum (total points 110) of abdominal, straight leg raise, hip abduction, extension, and flexion, and quadriceps strength. Lower extremity muscle strength did not change. There was no significant decrease in pain on a four-point scale. Some patients reported increased endurance or decreased back pain, but most reported no perceptible changes. The previously reported changes in these measures appear to have been placebo effects in the uncontrolled trials. Interestingly, the response within the treatment group showed considerable variety. Some children had a robust response in all measurements; others had increased bone density, but not increased area or height. Changes in DEXA z-scores ranged from less than 1 SD to more than 3 SD. The variability of response has not been previously reported and is presumably related to differences in bone matrix caused by the underlying collagen mutations. Cabral WA, Marini JC. High proportion of mutant osteoblasts is compatible with normal skeletal function in mosaic carriers of osteogenesis imperfecta. Am J Hum Genet 2004;74:752-760. Chernoff E, Letocha AD, Marini JC. Osteogenesis imperfecta. In: Allanson J, Cassidy S, eds. Clinical Management of Common Genetic Syndromes. New York: Wiley & Sons, 2004, in press. Kozloff KM, Carden A, Berwitz C, Forlino A, Uveges TE, Morris MD, Marini JC, Goldstein SA. Brittle IV mouse model for osteogenesis imperfecta IV demonstrates post pubertal adaptations to improve whole bone strength. J Bone Min Res 2004;19:614-622. Kuznetsova NV, Forlino A, Cabral WA, Marini JC, Leikin S. Structure, stability and interactions of type I collagen with Gly 349 Cys substitution in alpha1(I) chain in a murine osteogenesis imperfecta model. Matrix Biol 2004;23:101-112. Walker LC, Overstreet MA, Willing MC, Marini JC, Cabral WA, Pals G, Bristow J, Atsawasuwan P, Yamauchi M, Yeowell HN. Heterogeneous basis of the type VIB form of Ehlers-Danlos syndrome (EDS VIB) that is unrelated to decreased collagen lysyl hydroxylation. Am J Med Genet 2004;131A:155-162. COLLABORATORS Antonella Forlino, PhD, University of Pavia, Italy Lynn Gerber, MD, Rehabilitation Medicine, NIH Clinical Center, Bethesda, MD Steven Goldstein, PhD, University of Michigan, Ann Arbor, MI Gloria Gronowicz, PhD, University of Connecticut Health Center, Farmington, CT Sergey Leikin, PhD, Unit on Molecular Forces and Structure, NICHD, Bethesda, MD Scott Paul, MD, Rehabilitation Medicine, NIH Clinical Center, Bethesda, MD For further information, contact mailto:marinij@mail.nih.gov