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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
  • Angela Blissett, PhD, Postdoctoral Fellow
  • Adi Reich, PhD, Postdoctoral Fellow
  • Francina Abengowe, BS, Postbaccalaureate Fellow
  • Antonella Forlino, PhD, Guest Scientist

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 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. Thus, deficiency in CRTAP (collagen-associated protein) causes type VII OI (#610862 in the OMIM database), while deficiency in P3H1 causes type VIII OI (OMIM #610915). The 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 α1(I) chains. Null mutations in LEPRE1, the gene that encodes P3H1, or CRTAP cause a severe to lethal bone dysplasia whose phenotype overlaps with the severe/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/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 that encodes the proteins has a null defect, demonstrating mutual protection of these two components in the 3-hydroxylation complex. Also, in LEPRE1-null cells, the secretion of CRTAP into conditioned media is double the normal level. Secretion of CRTAP is important to OI pathology. We collaborated to study a proband with total deficiency of CRTAP. Importantly, collagen deposition into matrix is reduced in long-term culture of proband cells, and 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 then normal. More recently, we investigated siblings with non-lethal OI who have the first reported LEPRE1 mutation that eliminates the KDEL retrieval signal at the 3′-end of P3H1. P3H1 has the only KDEL signal for ER retention in the 3-hydroxylation complex. Even when other functional domains remain intact, loss of the KDEL sequence is sufficient to cause severe bone dysplasia. When P3H1 is absent from the cell, the collagen helix is overmodified, suggesting that the chaperone function of the complex is functionally inadequate, even while the Pro986 site in COL1A1 is 85% modified.

We identified a defect in the cyclophilin B (CyPB) gene PPIB, causing recessive OI in two siblings with moderately severe OI, a defect that alters the start codon; the siblings are homozygous for a point mutation in the start codon. The CyPB protein is undetectable by Western blot with three antibodies to different epitopes and by immunofluorescence microscopy. In the absence of CyPB, reduction of the 3-hydroxylation complex is reduced to half of normal. Thus, CyPB does provide some stabilization to the complex. Even though 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 unique 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, suggesting redundancy for this crucial isomerization in cells.

Investigations of the mechanism of type XI OI, caused by absence of the peptidylprolyl isomerase FKBP65 have been a new focus of our studies. FKBP65 is a collagen-binding ligand, and its deficiency causes progressive deforming OI, with or without congenital contractures. We demonstrated that type I collagen folding kinetics are normal in the absence of FKBP65, but that collagen cross-linking and deposition of collagen into matrix are strongly reduced, resulting in a matrix with sparse and disorganized collagen fibrils. The reduced collagen deposition is caused by near absence of collagen telopeptide hydroxylation in the proband-secreted collagen.

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 terminations codons. Homozygotes for the mutation die within months of birth, while compound heterozygotes can survive into their teen years 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 450kb conserved region. Calculations based on these data estimate that the mutation first occurred about 600 years ago, prior to the Atlantic slave trade, consistent with our hypothesis (2). 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 into the role of the C-propeptide date back over two decades. 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 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 is 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 collaboration with Adele Boskey, we found that 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 (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. Modeling of the collagen fibril 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. The 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. In order to study the developmental progression of a C-propeptide cleavage site mutation, we are developing a knock-in 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 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 littermates, focusing on the femur, which cannot be examined directly in pediatric trials. 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. Collaborative studies with Ken Kozloff, using an ulnar loading model, demonstrated that Brtl mouse long bone is more susceptible to microdamage. Brtl bone incurred increased microdamage during both normal cage activity and ulnar loading. The susceptibility may partially account for the decline in material quality when OI bone is treated with bisphosphonates, which inhibit the osteoclast activity necessary for microdamage repair. Recently, we also collaborated with 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 Brtl mice and increased bone mass and load to fracture. In a short-term treatment with Scl-Ab there was no change in Brtl bone material properties (3).

A third therapeutic trial involving Brtl was an in utero cell transplantation study using normal mesenchymal stem cells (MSC) tagged with GFP. 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 biomechanical properties of Brtl femora at two months were significantly improved. The results coincide with our previous data on human mosaic carriers who have less than 30% mutant bone cells and a very mild phenotype.

Brtl also provided important information about the cellular function in OI. Comparison of lethal and surviving Brtl pups found that ER stress was significantly increased in the lethal pups. Gadd153 (CHOP) expression was increased 2–3 fold in bone cells of lethal pups and was present in Western blots (CHOP is a component of an apoptosis pathway). Conversely, αβ-crystalline, a small heat shock protein that protects against apoptosis, is elevated in osteoblasts of surviving Brtl cells. Collaborative studies demonstrated impairment in the differentiation of Brtl MSCs to osteoblasts, including reduced expression of early and late osteoblastic markers. Conversely, Brtl MSCs generated more and larger adipocytes than wild-type (WT) MSCs. Other studies of surviving Brtl mice demonstrated the importance of osteoclasts to the OI phenotype. Osteoclast surface in Brtl is increased and is uncoupled from 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 we are currently exploring other osteoclast-stimulating pathways. Co-culture experiments with Brtl and WT MSC's 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 important targets 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 a German collaborator (5). 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 of pulmonary function, including lung volumes and flow rates, over time. 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. This suggests that the pulmonary dysfunction of OI is due to primary defect in lung related to structurally abnormal collagen. Clinically, these studies are of direct importance because pulmonary aspects of OI are the most prevalent cause of morbidity and mortality in OI, and affected individuals now have a scientifically based reason to 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 decreased long-bone fractures, coinciding with 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 biophysical mapping of the melting domains, which is being conducted by the Leikin laboratory; the lab deduced two flexible regions important for collagen fibril assembly and ligand binding. The Database also provided crucial material for 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 nine 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 features correlated in the previous analysis.


  • Barnes AM, Cabral WA, Weis WA, Makareeva E, Mertz E, Leikin S, Eyre D, Trujillo C, Marini JC. Absence of FKBP10 in recessive type XI OI leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix. Hum Mutat 2012;33(11):1589-1598.
  • Cabral WA, Barnes AM, Adeyemo A, Cushing K, Chitayat D, Porter FD, Panny SR, Majid F, Rebbeck TR, Tishkoff SA, Bailey-Wilson JE, Brody LC, Rotimi CN, Marini JC. A founder mutation in LEPRE1 carried by 1.5% of West Africans and 0.4% of Americans causes lethal recessive osteogenesis imperfecta. Genet Med 2012;14(5):543-551.
  • Sinder BP, Eddy MM, Caird MS, Marini JC, Kozloff KM. Sclerostin antibody improves skeletal parameters in a Brtl/+ mouse model of osteogenesis imperfecta. J Bone Miner Res 2012;E-pub ahead of print.
  • Gioia R, Panaroni C, Besio R, Palladini G, Merlini G, Giansanti V, Scovassi AI, Villani S, Villa I, Villa A, Vezzoni P, Tenni R, Rossi A, Marini JC, Forlino A. Impaired osteogenesis in a murine model of dominant osteogenesis imperfecta (OI), a new target for OI pharmacological therapy. Stem Cells 2012;30:1465-1476.
  • Thiele F, Cohrs CM, Flor A, Lisse TS, Przemeck GHK, Horsch M, Schrewe A, Gialus-Durner V, Ivandic B, Katus H, Wurst W, Reisenberg C, Chaney H, Fuchs H, Hans W, Beckers J, Marini JC, Hrabe de Angelis M. Cardiopulmonary dysfunction in the osteogenesis imperfect mouse model aga2 and human patients are caused by bone-independent mechanisms. Hum Mol Genet 2012;21(16):3535-3545.


  • 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, 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
  • 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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