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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 Annual Report of the Division of Intramural Research

Genetic Disorders of Bone and Extracellular Matrix

Joan Marini
  • Joan C. Marini, MD, PhD, Chief, Bone and Extracellular Matrix Branch
  • Aileen M. Barnes, MS, Research Associate
  • Wayne A. Cabral, PhD, Chemist
  • Simone M. Smith, PhD, Research Fellow
  • Heeseog Kang, PhD, Postdoctoral Fellow
  • Adi Reich, PhD, Postdoctoral Fellow
  • Vihang V. Nakhate, BS, Postbaccalaureate Fellow
  • Brandi M. Owens, BA, Postbaccalaureate Fellow

In an integrated program of laboratory and clinical investigation, the Bone and Extracellular Matrix Branch (BEMB) studies 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 to apply this knowledge to the treatment of children with these conditions. Recently, we identified the long-sought cause of recessive OI. Discoveries of defects in collagen modification generated a new paradigm for collagen-related disorders of matrix. We established that structural defects in collagen cause dominant OI while deficiency of proteins that interact with OI for folding, post-translational modification, or processing cause recessive OI. Our challenge now is to understand the cellular and biochemical mechanisms of recessive OI. We also generated a knockin murine model for OI with a classical collagen mutation as well as a murine model for recessive type IX OI and are using these models to study disease pathogenesis and the skeletal matrix of OI, the effects of pharmacological therapies, and approaches to gene therapy. Our clinical studies involve children with types II and IV OI, who form a longitudinal study group enrolled in age-appropriate clinical protocols for the treatment of their condition.

Mechanism of rare forms of osteogenesis imperfecta (OI)

The endoplasmic reticulum (ER)–resident 3-hydroxylation complex is responsible for the 3-hydroxylation of type I collagen α1(I) chains. Deficiency of components of the collagen P3H (prolyl 3-hydroxylase) complex causes recessive OI. Thus, deficiency in CRTAP (collagen-associated protein) causes type VII OI, while deficiency in P3H1 causes type VIII OI. Types VII and VIII OI are severe to lethal bone dysplasias that are indistinguishable clinically because these two components are mutually protective in the complex. They share characteristics that are distinct from dominant OI; affected individuals have white or light sclerae, small/normal rather than enlarged head circumferences, and rhizomelia of long bones. We are now investigating the subcellular localization of CRTAP. The protein has a transmembrane sequence at the amino end. Its tethering to the ER membrane could be important for the coordination of collagen chain folding and modification.

The third member of the complex, Cyclophilin B (CyPB), encoded by PPIB, is an ER–resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions both independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Previously, the BEMB characterized the first patient with deficiency of PPIB, which causes recessively inherited Type IX OI. Our group generated a Ppib knock-out (KO) mouse model that recapitulates the Type IX OI phenotype, including growth deficiency and brittle bones (Reference 2). Collagen from KO mouse tissue and cells is nearly lacking 3-hydroxylation. We investigated the role of CyPB in collagen biosynthesis and found that intracellular collagen folding occurs more slowly in CyPB-null cells, supporting its role in the rate-limiting step of folding. However, treatment of KO cells with the cyclophilin inhibitor cyclosporin A caused further delay in folding, providing support for the existence of another collagen PPIase. We also extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Analysis of bone and osteoblast type I collagen revealed site-specific alterations in helical lysine hydroxylation, in particular significantly reduced hydroxylation of the helical crosslinking residue K87. The alteration directly affects both the extent and type of collagen intermolecular crosslinks that form in bone tissue. Our studies demonstrated novel consequences of the effect of CyPB on collagen hydroxylation, glycosylation, crosslinking, and fibrillogenesis, showing that CyPB not only functions to regulate collagen folding and 3-hydroxylation in the ER but also indirectly regulates bone development and mechanical properties. A collaboration with Ehud Cohen brought an additional important novel finding for CyPB that extended its foldase role to presenilin 1 in Alzheimer's disease. Reduced quantities of active properly folded presenilin were observed in brains of CyPB KO mice, supporting the emerging mechanism that Alzheimer's disease can arise from altered presenilin folding and function. Thus, ER chaperones may be targets for the development of therapies for neurodegenerative disorders.

OI type V is caused by a recurrent dominant mutation (c.-14C→T) in IFITM5, which encodes BRIL, a bone-restricted interferon-induced transmembrane protein–like protein most strongly expressed in osteoblasts, which plays a role in mineralization. Patients with type V OI have distinctive clinical manifestations with overactive bone mineralization and mesh-like lamellation on bone histology. We identified eight patients with the recurrent type V OI mutation and used cultured osteoblasts from these patients to study the mechanism of this type of OI at the bone cell level. We demonstrated that the mutant Bril transcripts and protein were stable and expressed at levels comparable to control. Both early and late markers of osteoblast differentiation are elevated in type V OI osteoblasts, including the osteoblast differentiation factor Runx2, alkaline phosphatase, bone sialoprotein, osteopontin, and osteocalcin. Mineralization by osteoblasts, assayed by alizarin red staining, was also elevated in type V OI osteoblasts. However, this occurs despite the seemingly paradoxical reduction in transcripts for type I collagen in mid to late differentiation, which leads to a concomitant reduction in cross-linked collagen in matrix and an altered appearance of fibrils deposited in culture, which have a patchy rather than a network appearance. These studies demonstrated that a gain-of-function mechanism underlies type V OI and establish its collagen-related defect.

Recessive null mutations in SERPINF1, which encodes pigment epithelium–derived factor (PEDF), cause OI type VI. PEDF is already well-known as a potent anti-angiogenic factor. Type VI OI patients have no serum PEDF, elevated alkaline phosphatase (ALPL) as children, and bone histology with broad unmineralized osteoid and fish-scale pattern. At first, types V and VI OI appear to be unconnected, caused by different genes, with distinct phenotypes and histology. However, we identified a patient with severe OI whose phenotype is most like type VI OI. Her osteoblasts displayed minimal secretion of PEDF, but her serum PEDF was normal. SERPINF1 sequences were normal despite bone histomorphometry typical of type VI OI, and elevated childhood serum ALPL. To identify the mutation, exome sequencing on the proband, parents, and an unaffected sibling surprisingly yielded a de novo mutation in IFITM5 in one allele of the proband, causing a p.S40L substitution in the BRIL intracellular domain. IFITM5 transcript and BRIL protein level were normal in proband fibroblasts and osteoblasts. SERPINF1 expression and PEDF secretion were reduced in proband osteoblasts. In contrast, osteoblasts from a typical case of type V OI have elevated SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, the data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and their roles in bone mineralization (Reference 1). We are extending this investigation using the murine model for type VI OI. Our current studies focus on delineating the pathway connecting type V and VI OI in osteoblasts and the relationship of the angiogenic effect of PEDF to its effect on osteoblasts and bone tissue.

Studies of a very rare defect in TAPT1, which encodes the highly conserved transmembrane anterior posterior transformation 1 protein, have been conducted in collaboration with the Belgian laboratory studying OI, led by Paul Coucke and Anne De Paepe. Defects in this gene cause a complex lethal osteochondrodysplasia that overlaps with the findings in a ciliopathy and skeletal dysplasia. Two large pedigrees with several pregnancies that were lethal in the perinatal period as a result of skeletal undermineralization and fractures, as well as neurological abnormalities such as ventriculomegaly and hypoplastic cerebellum and abnormalities of lungs and kidney, were investigated. A homogygous acceptor splice site variant in TAPT1 in one family, and a homozygous missense change in a highly conserved region of TAPT1 in the second family, were identified. Both mutations affect the second extracellular/luminal loop of this transmembrane protein. In human fibroblasts, TAPT1 was shown to localize to the centrosome and ciliary basal body region in control cells. In cells from family 1, the primary cilium did not form, while in the missense mutation the structure of the primary cilium was disturbed. Proband cells also have disturbed collagen biochemistry. Collagen folds more slowly in these cells and the trafficking of secreted proteins through the Golgi was disturbed, with the Golgi more disperse and distended. Knockdown of tapt1b in zebrafish induced severe craniofacial cartilage manifestations and delayed ossification, consistent with the human phenotype (Reference 5).

C-propeptide cleavage site mutations increase bone mineralization.

Type I procollagen is processed to mature collagen by the removal of both N- and C-terminal propeptides. The C-propeptide is cleaved at the Ala-Asp peptide bond between the telopeptide and the C-propeptide of each chain by C-proteinase/BMP-1. Probands with substitutions at the four cleavage site residues have been identified. Our investigations of two of those probands has identified a High Bone Mass form of OI. These individuals have elevated bone density DEXA z-scores and patchy unmineralized osteoid on bone histology. The processing of the C-propeptide from collagen secreted by proband cells is delayed. In collaboration with Adele Boskey, we found that Fourier transform infrared spectroscopy (FTIR) demonstrated a higher mineral/matrix ratio in both trabecular and cortical bone of each patient than in either age-matched normal or classical OI controls, as well as marked maturation of collagen cross-links. We extended the investigation of mineralization with BMDD (bone mineral density distribution) and BEI (backscattered electron imaging) to show that, in the α2(I) cleavage site mutation, the bone had a uniformly higher mineral density, while in the α1(I) mutation, the average mineral density was typical of classical OI but markedly more heterogeneous, with areas of very high and low bone density.

To investigate the role of the C-propeptide in bone mineralization and developmental progression, we developing a knock-in murine model with a COL1A1 cleavage site mutation. Bone collagen fibrils showed a "barbed-wire" appearance consistent with the presence of the pC-collagen that was detected in extracts of bone from mutant mice, and with impaired collagen processing in vitro. Impaired C-propeptide processing affects skeletal size and biomechanics. The mice are smaller than wild-type litter mates. Their femora have extreme brittleness on mechanical testing, as well as reduced fracture load. We are currently investigating bone mineralization in these mice.

Insight from the Brtl mouse model for OI

The Brtl mouse model for OI, generated by the BEMB, is a knock-in mouse that contains a Gly349Cys substitution in the α1(I) chain. Brtl was modeled on a type IV OI child and accurately reproduces features of type IV OI. Brtl has provided important insights into both potential OI treatments and the mechanism of OI. First, we conducted a treatment trial of the bisphosphonate alendronate in Brtl and wild-type (WT) littermates. We found that bone density, bone volume, and trabecular number improved with treatment, as did load-to-fracture. However, detrimental side effects such as retained mineralized cartilage, reduced material properties, and altered osteoblast morphology occurred with treatment. The results reinforce the conclusion of the pediatric trial to limit the duration of bisphosphonate treatment. Recently, we also collaborated with Kenneth Kozloff's group to investigate a potential anabolic therapy, sclerostin antibody (Scl-Ab), which stimulates osteoblasts via the canonical wnt pathway. Scl-Ab stimulated bone formation in young Brtl mice and increased bone mass and load-to-fracture. In treatment with Scl-Ab, there was no detrimental change in Brtl bone material properties. Nano-indentation studies indicating unchanged mineralization showed that the hypermineralization of bisphosphonate treatment did not occur. In addition, Scl-AB was successfully anabolic in adult Brtl mice, and may be a therapy for adult patients who have fewer treatment options.

A third therapeutic trial involving Brtl approached allele-specific silencing of the col1a1 mutation, undertaken in collaboration with Antonella Forlino. Specific small interfering RNA (siRNA) were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction in mutant transcripts by about half was associated with a 40% decrease in mutant collagen chain. Further testing of siRNA delivered by lentivirus might allow treatment of OI patients by autologous transplantation.

Brtl also provided important information about the cytoskeletal organization in OI osteoblasts and their potential role in the phenotypic variability of OI. Abnormal cytoskeletal organization was demonstrated to occur only in lethal pups. Comparison of lethal and surviving Brtl pups' skin/bone and bone/skin hybrid networks highlighted three proteins involved in cytoskeletal organization: vimentin, stathmin, and coffin-1. The alterations were shown to affect osteoblast proliferation, collagen deposition, integrin, and TGF-beta signaling. Furthermore, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not non-lethal, OI patients with an identical glycine substitution. The data open the possibility of cytoskeletal elements as novel treatment targets for OI.

Two basic insights have emerged from Brtl studies. The hyper-mineralization of OI bone was previously thought to be a passive process. Altered levels for osteocyte transcripts involved in bone mineralization, such as Dmp1 and Sost1, demonstrated the presence of an actively directed component. Second, the osteoclast is important to the OI phenotype, with elevated numbers and TRAP (tartrate-resistant acid phosphatase) staining of osteoclasts and precursors. Co-culture experiments with Brtl and wild-type (WT) mesenchymal stem cells (MSCs) and osteoclast precursors yielded elevated osteoclast numbers from WT or Brtl precursors grown with Brtl MSCs, but not with WT MSCs. The results indicate that an osteoblast product is necessary and sufficient for elevated osteoclast numbers and could provide an important target for treatment of OI.

Natural history and bisphosphonate treatment of children with types III and IV OI

We recently brought the cardiopulmonary aspects of our natural history study on types III and IV OI to publication in collaboration with translational murine studies of our collaborator, Martin Hrabe de Angelis. The longitudinal evaluations were completed in 23 children with type III OI and 23 children with type IV OI, who had serial pulmonary function tests every 1–2 years. Comparison of their results with size-matched children showed a significant decline over time in pulmonary function, including lung volumes and flow rates. The decline was worse in the 36 children with scoliosis (average curve 25 degrees) but also occurred in 20 participants without scoliosis, who had declining function with restrictive disease, suggesting that the pulmonary dysfunction of OI is attributable to a primary defect in lung related to structurally abnormal collagen. The studies are important because pulmonary issues are the most prevalent cause of morbidity and mortality in OI, and affected individuals should now seek anticipatory evaluation and treatment.

Our randomized controlled trial of bisphosphonate in children with types III and IV OI was the first randomized trial in the United States and one of four worldwide. Our trial examined both direct skeletal and secondary gains reported in uncontrolled trials. For skeletal outcomes, we found increased BMD Z-scores and improved vertebral area and compressions. We noted that vertebral BMD improvement tapered off after two years treatment. Our treatment group did not experience fewer long-bone fractures, coinciding with the lack of improvement or equivocal improvement in fractures in other controlled trials. The BEMB controlled trial did not support the secondary gains claimed in observational trials, including improvement in ambulation level, lower-extremity strength, or alleviation of pain, suggesting these were placebo effects in observational trials. Our current recommendation is treatment for 2–3 years, with subsequent follow-up of bone status. We are now engaged in a dose-comparison trial, comparing the dose from our first trial with a lower dose, achieved by increasing the cycle interval at the same dose/kg/cycle. Given the decade-long half-life and side effects of bisphosphonate on normal as well as dysplastic bone, including osteonecrosis of the jaw (ONJ), bone healing, bone modeling, and decline in the quality of the bone material, it is important to determine the lowest cumulative dose that will provide vertebral benefits. Preliminary analysis indicates that OI children obtain benefits from lower pamidronate doses that are comparable to the benefits from higher doses.

OI Mutation Consortium

The BEMB assembled and leads an international consortium of connective tissue laboratories for the compilation and analysis of a database of mutations in type I collagen that cause OI. The Consortium Database assembled double the previously available number of collagen mutations, and when the first analysis of the database was published in 2007, it listed over 830 mutations, including 682 glycine substitutions and 150 splice-site defects. Genotype-phenotype modeling revealed distinct functions for each alpha chain of type I collagen, including the occurrence of exclusively lethal mutations in the Major Ligand Binding Regions (MLBR) of the α1(I) chain on the collagen monomer and the overlapping of the regularly spaced clusters of lethal mutations along the α2(I) chain with the proteoglycan binding sites on the collagen fibril. The modeling for α2(I) supports the Regional Model for mutation that was first proposed by the BEMB over 15 years ago and now correctly predicts 86% of clinical outcomes. The Consortium Database has provided the basis for our collaborators James San Antonio and Joseph Orgel to model functional domains in terms of the cell and matrix interactions of the collagen fibril. The Consortium Database now contains over 1,570 mutations from nine international laboratories. An upcoming analysis of this database will also examine the effects of interchain salt bridges and re-nucleation residues C-terminal to the substituted glycine.

Additional Funding

  • NICHD Director's Award


  1. Farber CR, Reich A, Barnes AM, Becerra P, Rauch F, Cabral WA, Bae A, Quinlan A, Glorieux FH, Clemens TL, Marini JC. A novel IFITM5 mutation in severe atypical osteogenesis imperfect type VI impairs osteoblast production of PEDF. J Bone Miner Res 2014; 29:1402-1411.
  2. Cabral WA, Perdivara I, Weis MA, Terajima M, Blissett AR, Chang W, Makareeva EN, Mertz E, Leikin S, Tomer KB, Eyre DR, Yamauchi M, Marini JC. Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta. PLoS Genetics 2014; 10(6):e1004465.
  3. Reich A, Bae AS, Barnes AM, Cabral WA, Hinek A, Stimee J, Hill SC, Chitayat D, Marini JC. Type V primary osteoblasts display increased mineralization despite decreased COL1A1 expression. Metabolism 2015; 100(2):E325-332.
  4. Forlino A, Marini JC. Osteogenesis Imperfecta: new forms reveal novel gene functions in bone development and unexpected protein-protein interactions. Lancet, Invited Seminar. Online 2015; in press.
  5. Symoens S, Barnes AM, Gistelinck C, Malfait F, Guillemyn B, Steyaert W, Syx D, D'hondt S, Biervliet M, DeBacker J, Witten EP, Leikin S, Makareeva E, Gillessen-Kaesbach G, Huysseune A, Vleminskx K, Willaert A, DePaepe A, Marini JC, Coucke PJ. Genetic defects in TAPT1 disrupt ciliogenesis and cause a complex lethal Osteochondrodysplasia. Am J Hum Genet 2015; 97:521-534.


  • Patricia Becerra, PhD, Laboratory of Retinal Cell and Molecular Biology, NEI, Bethesda, MD
  • Adele Boskey, PhD, Weill Medical College of Cornell University, New York City
  • Ehud Cohen, PhD, The Hebrew University of Jerusalem, Jerusalem, Israel
  • Paul Coucke, PhD, Ghent University Hospital, Ghent, Belgium
  • Anne De Paepe, MD, PhD, Ghent University Hospital, Ghent, Belgium
  • David Eyre, PhD, University of Washington, Seattle, WA
  • Martin Hrabe de Angelis, PhD, Institute of Experimental Genetics, Helmholtz Zentrum München, Munich, Germany
  • Charles R. Farber, PhD, University of Virginia, Charlottesville, VA
  • Antonella Forlino, PhD, Università degli Studi di Pavia, Pavia, Italy
  • Kenneth Kozloff, PhD, University of Michigan, Ann Arbor, MI
  • Sergey Leikin, PhD, Section on Physical Biochemistry, NICHD, Bethesda, MD
  • Katarina Lindahl, MD, Uppsala University, Uppsala, Sweden
  • Joseph Orgel, PhD, Illinois Institute of Technology, Chicago, IL
  • Philip Osdoby, PhD, Washington University, St. Louis, MO
  • Scott Paul, MD, Rehabilitation Medicine, NIH Clinical Center, Bethesda, MD
  • James San Antonio, PhD, Jefferson University, Philadelphia, PA
  • Joseph Wallace, PhD, University of Michigan, Ann Arbor, MI
  • Bernd Wollnik, MD, Zentrum für Molekulare Medizin Köln, Uniklinik Köln, Cologne, Germany
  • Brant Weinstein, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
  • Mitsuo Yamauchi, PhD, University of North Carolina, Chapel Hill, NC
  • The OI Mutation Consortium, NICHD, Bethesda, MD


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