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Genetic Disorders of Bone and Extracellular Matrix

Joan C. Marini, MD, PhD
  • Joan C. Marini, MD, PhD, Chief, Bone and Extracellular Matrix Branch
  • Aileen M. Barnes, MS, Research Associate
  • Wayne A. Cabral, AB, Chemist
  • Helen Rajpar, PhD, Senior Fellow
  • Antonella Forlino, PhD, Guest Scientist
  • Angela Blissett, PhD, Postdoctoral Fellow
  • Adi Reich, PhD, Postdoctoral Fellow
  • Laura Felley, BS, Postbaccalaureate Fellow
  • Francina Abengowe, BS, Postbaccalaureate Fellow
  • Laurie Colston, MSN, FNP-BC, Nurse Practitioner

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 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 have established that structural defects in collagen cause dominant OI while defects in the components of a complex in the endoplasmic reticulum that modifies collagen 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 and are using the model 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.

Recessive osteogenesis imperfecta mechanism

Deficiency of components of the collagen prolyl 3-hydroxylation complex causes recessive OI. Deficiency in the component CRTAP causes type VII OI (#610862 in the OMIM database), while deficiency in P3H1 causes type VIII OI (OMIM #610915). This ER–resident 3-hydroxylation complex, of which cyclophilin B is the third member, is responsible for the 3-hydroxylation of the Pro986 residue of the alpha1(I) chains. Null mutations in LEPRE1, the gene that encodes P3H1, or in CRTAP cause a severe-to-lethal bone dysplasia whose phenotype overlaps with the severe-to-lethal dominant forms of OI in terms of crumpled and undertubulated long bones, disorganized bone matrix, and extreme bone fragility. However, the two recessive forms share other characteristics that are distinct from those of dominant OI; affected individuals have white or light sclerae, small-to-normal rather than enlarged head circumferences, and rhizomelia of long bones. Biochemically, the type I collagen produced by affected children lacks Pro986 hydroxylation, but, surprisingly, exhibits overmodification of the helical region of collagen chains by the P4H1 and LOH modification system, indicating that folding of the helix is delayed. We found that both CRTAP and P3H1 are absent from cells when either gene has a null defect, demonstrating mutual protection of these two components in the 3-hydroxylation complex. Stable transfection of CRTAP expression constructs into CRTAP-null cells restores P3H1, as well as CRTAP, in these cells. In addition, in LEPRE1-null cells, the secretion of CRTAP into conditioned media is twice the normal level.

Recently, we identified a defect in PPIB, which alters the start codon of cyclophilin B (CyPB) and causes causing recessive OI. Two siblings have moderately severe recessive OI with white sclerae and normal dentition but without rhizomelia. They are homozygous for a point mutation in the CyPB start codon, and each parent and the unaffected sibling are carriers. In the probands, PPIB transcript levels are reduced to half of normal. The CyPB protein is undetectable by Western blot with three antibodies to different epitopes and by immunofluorescence microscopy. The protein is undetectable after cells are treated with proteosomal inhibitors, indicating that it is not rapidly degraded after synthesis, and is also undetectable in guanidinium extracts, indicated that the protein did not elude detection in Westerns because it had formed insoluble aggregates. The absence of CyPB demonstrates moderate reduction of the 3-hydroxylation complex, whose level is half of normal. Thus, CyPB does provide some stabilization to the complex, although not at the mutual-protection level that CRTAP and P3H1 provide. Although reduced, the level of the P3H1/CRTAP complex is sufficient to fully 3-hydroxylate the Pro986 residue. Lastly, CyPB is a well-known peptidyl-prolyl cis-trans isomerase, which was thought to be the only isomerase catalyzing the rate-limiting step in collagen folding. Surprisingly, collagen helical modification and alpha-chain gel migration are normal in proband cells despite the absence of CyPB. These data implied redundancy for this crucial isomerization in cells.

We collaborated on investigations of a case of non-lethal type VII OI. The proband is a 7-year-old boy who is the product of consanguinous mating. He has the height of an average 2.5 year old boy, DXA z-score of -3SD, and radiographic features of severe deforming OI. At the molecular level, he is homozygous for a frame-shifting inserting/deletion in exon 1 of CRTAP, which leads to absence of both CRTAP and P3H1 protein from proband cells. Proband type I collagen is overmodified with an increased Tm (melting temperature). Importantly, collagen deposition into matrix is reduced in long-term culture; the minimal deposited matrix is disorganized. A matrix-chase experiment confirmed deposition of about 10% of the normal amount of matrix by proband cells, with normal turnover of the deposited collagen, showing that this is a defect of deposition. In vivo, proband collagen dermal fibrils have increased diameter, with irregular edges and more variability in size than normal. Thus, the role of CRTAP in matrix may be an important component of OI pathology.

West African LEPRE1 allele for type VIII OI

Our initial studies of probands with type VIII OI identified a recurring mutation, IVS5+1G→T, in African Americans and West African immigrants to the United States. The mutation was subsequently identified in other individuals of African origin, but not in any other racial group. The mutation results in five alternative spliced forms of the transcript, each leading to premature termination codons. Homozygotes for the mutation die within months of birth, while compound heterozygotes can survive into their teens but with extremely severe bone disease. We hypothesize that this is a West African founder mutation, which was transported to the Caribbean and the Americas with the Atlantic slave trade. To investigate the incidence of mutation carriers and the molecular anthropology of the disease, we used a custom SNP (single nucleotide polymorphism) assay to screen contemporary African Americans from Maryland, Pennsylvania, and Washington, DC as well as contemporary Africans. In the three African American cohorts, the incidence of carriers was 1/200–300 individuals, typical of a rare metabolic disorder and predicting a frequency of homozygous type VIII OI among African Americans of about 1/200,000 births. For West African studies, we collaborated with Charles Rotimi, who shared over 1,200 DNA samples from contemporary Ghanaians and Nigerians. To our surprise, we found that the carrier frequency for this lethal bone dysplasia in West Africa is over 1.5%. This particularly high incidence of carriers predicts that the rate of recessive OI in West Africa will equal the rate of dominant OI. To determine whether the mutation is pan-African rather than limited to West Africa, we collaborated with Sarah Tishkoff and Timothy Rebbeck to screen samples from Cameroon, Chad, CAR, and Senegal—countries surrounding Nigeria and Ghana. No carriers were found in these samples. Haplotype analysis of African American pedigrees and West African triads on the 4.2 MB region surrounding the LEPRE1 gene yielded a 450 kb conserved region. Calculations based on these data estimate that the mutation first occurred about 600 years ago, prior to the Atlantic slave trade, which is consistent with our hypothesis. We are interested in determining the forces responsible for maintaining the elevated carrier frequency of this lethal mutation in contemporary West Africans.

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. Investigations on the role of the C-propeptide began two decades ago. 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; cleavage is enhanced by the C-proteinase enhancer protein (PCPE-1). Probands with substitutions at three of the four cleavage site residues have been identified; we are studying two of those probands. One child is the patient of Katarina Lindahl, our Swedish collaborator, and the other is an NIH patient. Surprisingly, the two children have elevated bone density DEXA z-scores, although their fracture history, radiographs, and bone histomorphometry are typical of mild OI. The only abnormality in collagen biochemistry in these children is delayed cleavage of the C-propeptide. This led us to investigate matrix mineralization of these children. In a collaboration with Adele Boskey, FTIR (Fourier transform infrared spectroscopy) demonstrated a higher mineral/matrix ratio in both the trabecular and cortical bone of each patient than in either age-matched normal or classical OI controls, as well as more marked maturation of collagen cross-links, although crystallinity and mineral composition were typical of classical OI. We extended the investigation of mineralization with BMDD (bone mineral density distribution) and BEI (back-scattered electron imaging) to show that, in the alpha2(I) cleavage site mutation, the bone had a uniformly higher mineral density, while in the alpha1(I) mutation (in the COL1A1 gene), the average mineral density was typical of classical OI but markedly more heterogeneous, with areas of very high and low bone density. Modeling of the collagen fibril has indicated that the C-terminal end is located on the surface of the fibril. If the C-propeptide is not cleaved, it will be located on the surface of the fibril where it apparently generates an initiating locus for increased mineralization. These studies showed that mutations in the C-propeptide cleavage site cause a distinctive form of OI and revealed important basic information about the propeptide's role in bone mineralization. To study the developmental progression of a C-propeptide cleavage site mutation, we are developing a KI murine model with a COL1A1 mutation.

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 alpgha1(I) chain. Brtl was modeled on a type IV OI child and accurately reproduces most features of type IV OI. Brtl has provided important insights into both potential OI treatments and the mechanism of OI. First, we conducted a controlled treatment trial of the bisphosphonate alendronate in Brtl and wild-type littermates, focusing on the effect of bisphosphonate on the OI femur, which cannot be examined directly in children in treatment trials. We found that bone density and bone volume improved with treatment, with a dramatic increase in trabecular number. The femora also achieved increased loading before fracture. However, during the timeframe in which Brtl BMD and femur loading improved, important detrimental side effects appeared. Retention of mineralized cartilage and decline in the strength of bone material likely account for the paradoxical increase in bone fractures seen after prolonged bisphosphonate treatment. There was also a toxic effect on osteoblast morphology—previously cuboidal cells became flat lining cells. These results reinforce the conclusion of the pediatric trial to limit the duration of bisphosphonate treatment. In preparation for site-specific deposition studies to understand the decreased material quality associated with bisphosphonate deposition, collaborative studies with Ken Kozloff validated the fluorescent bisphosphonate analog FRFP as an accurate biomarker of bisphosphonate deposition and retention (Kozloff et al., J Bone Min Res 2010;25:1748).

A second therapeutic trial involving Brtl was an in utero cell-transplantation study using normal mesenchymal stem cells (MSC) tagged with GFP; the trial clearly demonstrated uptake of normal bone-forming cells into Brtl bone. Surprisingly, despite the low-level of cell uptake into bone, the day-one lethality outcome for 30% of Brtl mice was almost entirely rescued and the biomechanics of Brtl femora at two months improved significantly. These results agree with our previous data on human mosaic carriers who have less than 30% mutant bone cells and a very mild phenotype.

Brtl has also provided important information about the cellular function in OI. Comparison of lethal and surviving Brtl pups found that ER stress was significantly elevated in the lethal pups. Gadd153 (CHOP) expression increased 2- to 3-fold in bone cells of lethal pups and was present in Western blots; CHOP is a component of an apoptosis pathway. Conversely, alpha-beta-crystallin, a small heat shock protein that protects against apoptosis, is elevated in osteoblasts of surviving Brtl cells. Other studies of surviving Brtl mice have shown the importance of osteoclasts to the OI phenotype. Osteoclast surface in Brtl is increased and is uncoupled from the osteoblast surface, resulting in a decline in bone formation rate (BFR). Surprisingly, the number is TRAP+ cells is elevated in Brtl marrow cultures, suggesting elevated numbers of precursors. We demonstrated that this increase is independent of the RANKL/OPG signaling pathway and are currently exploring other osteoclast-stimulating pathways. Co-culture experiments with Brtl and wild-type (WT) MSC's and osteoclast precursors yielded elevated ocsteoclast numbers from WT or Brtl precursors grown with Brtl MSCs, but not with WT MSCs, indicating that an osteoblast product is necessary and sufficient for elevated osteoclast numbers and could provide important targets for treatment of OI. Finally, a collaborative study with Joseph Wallace and Mark Banaszak Holl demonstrated that the nanoscale morphology of type I collagen fibrils is altered in Brtl bone (Wallace et al., J Structural Biol 2010;173:146). Altered fibril structure may be detected by osteoblasts as part of OI pathology; additional studies may link the morphological observations to nanoscale mechanical integrity.

Bisphosphonate treatment of children with types III and IV OI

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 of the bisphosphonate pamidronate examined both direct skeletal gains and secondary gains reported in uncontrolled trials. For the primary skeletal outcomes, we found increased BMD z-scores, and improved vertebral area and compressions. We were the first to note, however, that improvement in vertebral BMD tapered off after one to two years of treatment, a conclusion later extended by histomorphometry from the Shriner's Hospital Montreal trial, which showed that almost all bone histology gains occurred in the first 2–3 years of treatment. We also found that the treatment group did not experience a decrease in long-bone fractures, which again coincided with the lack of fracture improvement or equivocal improvement in fractures in other controlled trials. The BEMB controlled trial did not support the secondary gains claimed in observational trials; treated children did not experience significant improvement in ambulation level, or lower-extremity strength, or alleviation of pain, suggesting that previously reported gains in these parameters were a placebo effect. Our current treatment protocol recommends treatment for 2–3 years, with subsequent follow-up of bone status. Furthermore, we are now engaged in a dose comparison trial, in which the dose from our first trial is compared with a lower dose, achieved by increasing the cycle interval but giving 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, on bone healing, bone modeling, and 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. We are also focusing on the important secondary features of OI, including hearing loss, pulmonary function, and cardiac abnormalities, to determine their incidence and progression in our study population.

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 twice 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 alpha1(I) chain on the collagen monomer and the overlapping of the regularly spaced clusters of lethal mutations along the alpha2(I) chain with the proteoglycan binding sites on the collagen fibril. The modeling for alpha2(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 biophysical mapping of the melting domains, which is being conducted by the Leikin laboratory, which deduced two flexible regions important for collagen fibril assembly and ligand binding. The Database also provided crucial material 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 has been in an active assembly phase over the past year and now contains over 1,570 mutations from 9 international laboratories, including 1,253 glycine substitutions and 326 exon splicing defects. An upcoming analysis of this database will add examination of the effects of interchain salt bridges and re-nucleation residues C-terminal to the substituted glycine to the features correlated in the previous analysis.


  • Barnes AM, Carter EM, Cabral WA, Weis M, Chang W, Makareeva E, Leikin S, Rotimi CN, Eyre DR, Raggio CL, Marini JC. Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding. N Engl J Med 2010;362:521-528.
  • Chang W, Barnes AM, Cabral WA, Bodurtha JN, Marini JC. Prolyl 3-hydroxylase 1 and CRTAP are mutually stabilizing in the endoplasmic reticulum collagen prolyl 3-hydroxylation complex. Hum Mol Genet 2009;19:223-234.
  • Forlino, A, Cabral, W.A., Barnes, A.M., and Marini, J.C. New perspectives on osteogenesis imperfecta. Invited Review. Nat Rev Endocrinol 2011;7:540-557.
  • Lindahl K, Barnes AM, Fratzl-Zelman J, Whyte M, Hefferan TE, Makareeva E, Yaszemski MJ, Rubin C-J, Kindmark A, Roschger P, Klaushofer K, McAlister WH, Mumm S, Leikin S, Kessler E, Boskey AL, Ljunggren O, Marini JC. COL1-C-propeptide cleavage site mutations cause high bone mass osteogenesis imperfecta. Hum Mutat 2011;32:598-609.
  • Valli M, Barnes A, Gallanti A, Cabral W, Viglio S, Weis M, Makareeva E, Eyre D, Leikin S, Antoniazzi F, Marini J, Mottes M. Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix. Clin Genet 2011;[Epub ahead of print].


  • Mark M. Banaszak Holl, PhD, University of Michigan, Ann Arbor, MI
  • Adele Boskey, PhD, Weill Medical College of Cornell University, New York City
  • David Eyre, PhD, University of Washington, Seattle, WA
  • Steven Goldstein, PhD, University of Michigan, Ann Arbor, MI
  • Kenneth Kozloff, PhD, University of Michigan, Ann Arbor, MI
  • Sergey Leikin, PhD, Section on Physical Biochemistry, NICHD, Bethesda, MD
  • Katarina Lindahl, MD, Uppsala University, 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
  • Timothy Rebbeck, PhD, University of Pennsylvania, Philadelphia, PA
  • Charles N. Rotimi, PhD, NIH Center for Research on Genomics and Global Health, NHGRI, Bethesda, MD
  • James San Antonio, PhD, Jefferson University, Philadelphia, PA
  • Sarah Tishkoff, PhD, University of Pennsylvania, Philadelphia, PA
  • Joseph Wallace, PhD, University of Michigan, Ann Arbor, MI
  • The OI Mutation Consortium, NICHD, Bethesda, MD


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