Skip to main content

National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2018 Annual Report of the Division of Intramural Research

Genetic Disorders of Bone and Extracellular Matrix

Joan Marini
  • Joan C. Marini, MD, PhD, Chief, Section on Heritable Disorders of Bone and Extracellular Matrix
  • Chi-Wing Chow, PhD, Staff Scientist
  • Ying Liu, PhD, Biologist
  • Aileen M. Barnes, MS, Research Associate
  • Gali Guterman-Ram, PhD, Postdoctoral Fellow
  • Milena Jovanovic, PhD, Postdoctoral Fellow
  • Heeseog Kang, PhD, Postdoctoral Fellow
  • Smriti Aryal, PhD, Visiting Fellow
  • Marilyn Barragan, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Robert D. Carlson, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Jaelynn Lawrence, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • TzeYin Tang, BS, Postbaccalaureate Intramural Research Training Award Fellow
  • Ashita S. Vadlamudi, BS, Postbaccalaureate Intramural Research Training Award Fellow

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders known as osteogenesis imperfecta (OI). 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, in the seach for causes of recessive OI, we identified several key genes. Discoveries of defects in collagen modification have 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 knock-in murine model for OI with a classical collagen mutation as well as a murine model for recessive type IX OI and X-linked type XVIII OI, and we 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 III and IV OI, who form a longitudinal study group enrolled in age-appropriate clinical protocols for the treatment of their condition. We are also investigating melorheostosis, a very rare bone dysostosis, which is characterized by bone overgrowth in a radiographic pattern of "dripping candle wax." We recently identified mosaic mutations in the oncogene MAP2K1 as the cause of about half of cases of this benign condition. The causative mutations occur at a hot spot in the MAP2K1 negative regulatory domain and inhibit bone-morphometric protein 2 (BMP2)–induced bone differentiation. We are now developing animal models for studies of melorheostosis pathophysiology and treatment.

Mechanism of rare forms of osteogenesis imperfecta

Recessive null mutations in SERPINF1, which encodes pigment epithelium–derived factor (PEDF), cause OI type VI. PEDF is 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 a fish-scale pattern. However, we identified a patient with severe atypical type VI OI, whose osteoblasts displayed minimal secretion of PEDF, but whose SERPINF1 sequences were normal despite typical type VI OI bone histology. Surprisingly, exome sequencing on this proband and family members yielded a de novo mutation in IFITM5 (the gene encoding interferon-induced transmembrane protein 5, which is mutated in osteogenesis imperfecta type V) in one proband allele, causing a p.S40L substitution in the intracellular domain of BRIL, the encoded protein. The IFITM5 transcript and BRIL 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.

The endoplasmic reticulum (ER)–resident procollagen 3-hydroxylation complex is responsible for the 3-hydroxylation of type I collagen alpha1(I) chains. Deficiency of components of the collagen P3H (prolyl 3-hydroxylase) complex causes recessive OI. For type VIII OI, we investigated bone and osteoblasts with collaborators Nadja Fratzl-Zelman and Cathleen Raggio. Collagen has near-absent 3-hydroxylation from both bone and dermis, demonstrating that P3H1 is the unique enzyme responsible for collagen 3-hydroxylation. Bone histomorphometry revealed patches of increased osteoid, although the overall osteoid surface was normal. Quantitative backscattered electron imaging (qBEI) showed increased mineralization of cortical and trabecular bone, as in other OI types. However, the proportion of bone with low mineralization was higher in Type VIII bone than type VII, consistent with patchy osteoid only occurring in type VIII.

The third member of the complex, cyclophilin B (CyPB), encoded by PPIB, is an ER–resident peptidyl-prolyl cis-trans isomerase (PPIase). CyPB is the major PPIase catalyzing collagen folding. We characterized the first patient with deficiency in 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. Intracellular collagen folding occurs more slowly in CyPB-null cells, supporting the enzyme's role as 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 a further collagen PPIase. We found that CyPB supports collagen lysyl hydroxylase 1 (LH1) activity, demonstrating significantly reduced hydroxylation of the helical crosslinking residue K87, which directly affects both the extent and type of collagen intermolecular crosslinks in bone. However, CyPB deficiency results in increased hydroxylation at telopeptide crosslinking sites in tendon, with moderate increase in glycosylation.

Recessive type XIV OI is a moderately severe bone dysplasia caused by null mutations in TMEM38B, which encodes TRIC-B. TRIC-B forms a monovalent cation channel in the ER membrane that is thought to counterbalance inositol triphosphate receptor (IP3R)–mediated calcium release from the ER to the cytoplasm. We found that TRIC-B was undetectable in fibroblasts and osteoblasts of three independent probands. Together with our collaborators Yoshi Yamada and Joshua Zimmerberg, we showed that absence of TRIC-B results in reduced calcium flux from the ER and abnormal store-operated calcium entry, but that ER steady-state calcium is normal. As expected, the disturbed calcium flux causes ER stress along the PERK (protein kinase RNA–like endoplasmic reticulum kinase, an ER–resident transmembrane protein kinase that initiates both pro-apoptotic and pro-survival signaling pathways) pathway of the unfolded protein response (UPR) and elevated BiP, an ER chaperone protein and regulator of ER stress. Disruption of calcium dynamics also alters the expression and activity of several collagen-modifying enzymes and chaperones in the ER. As a result, lysyl hydroxylation of the collagen helix by LH1 is reduced by 30%. Procollagen chain assembly is also delayed, likely through sequestration of protein disulfide isomerase (PDI) by calreticulin. These data support a role for TRIC-B in calcium homeostasis and directly connect TMEM38B defects to the collagen-related paradigm of OI. Other ER pathways are likely also disrupted by abnormal calcium flux in these cells.

In collaboration with Vorasuk Shotelersuk and Cecilia Giunta, we identified a new OI causative gene on the X-chromosome. This is the first type of OI with X-linked inheritance, and it causes a moderate to severe bone dysplasia with pre- and postnatal fractures of ribs and long bone, bowing of long bones, low bone density, kyphoscoliosis and pectal deformities, and short stature. The affected individuals have missense mutations in MBTPS2, which encodes the protein S2P. S2P is a transmembrane protein in the Golgi and is a critical component of regulated membrane proteolysis (RIP). In RIP, regulatory proteins are transported from the ER membrane to the Golgi in times of cell stress or sterol depletion, where they are sequentially cleaved by S1P/S2P to release activated N-terminal fragments that enter the nucleus and activate gene transcription. Mutant S2P protein is stable but has impaired RIP functioning, with deficient cleavage of the ER–stress transducers OASIS, ATF6, and SREBP. Furthermore, hydroxylation of the collagen residue K87 is reduced by half in proband bone, consistent with reduced lysyl hydroxylase in proband osteoblasts. Reduced collagen crosslinks presumably undermine bone strength. The mutations in MBTPS2 demonstrate that RIP plays a fundamental role in bone development.

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 (procollagen proteinase C, also known BMP-1 or bone-morphometric protein). Probands with substitutions at any of the four cleavage site residues have a high-bone-mass form of OI, first reported by our lab in collaboration with Katarina Lindahl. The patients have elevated bone density DEXA Z-scores and, in bone histology, patchy unmineralized osteoid. The processing of the C-propeptide from collagen secreted by proband cells is delayed. Using bone mineralization density distribution (BMDD), we investigated mineralization to show that, in the alpha2(I) cleavage site mutation, the bone had a uniformly higher mineral density, while in the alpha1(I) mutation, the average mineral density was markedly heterogeneous, with areas of either very high or low bone density.

To investigate the role of the C-propeptide in bone mineralization and developmental progression, we developed 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 processing intermediate 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 exhibit extreme brittleness on mechanical testing, as well as reduced fracture load. BMDD measurement on femora from 2-, 6-, and 12-month-old mice show significantly increased mineralization compared with wild-type (WT), which continues to increase in HBM (high bone mass) mice even after WT mineralization plateaus at 6 months. PINP and TRAP, serum markers of bone remodeling, are significantly increased in HBM mice. Osteocyte density is reduced but lacunar area is increased. We are currently investigating osteoblasts, osteocytes, and osteoclast in HBM mice.

Insights from the Brtl mouse model for OI

The Brtl mouse model for OI, generated by our lab, is a knock-in mouse that contains a Gly349Cys substitution in the alpha1(I) collagen 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. In a treatment trial of the bisphosphonate alendronate in Brtl and wild-type (WT) littermates, 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 (see below).

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. Treatment with Scl-Ab caused no detrimental change in Brtl bone material properties. Nano-indentation studies indicating unchanged mineralization showed that the hyper-mineralization 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. Because Scl-AB is a short-acting drug, we recently investigated sequential Scl-AB/bisphosphonate treatment. The study showed that administration of a single dose of bisphosphonate after Scl-AB cessation preserved anabolic gains from the Scl-AB treatment. Alternatively, a single low dose of bisphosphonate concurrent with Scl-AB treatment facilitated the anabolic action of Scl-AB by increasing availability of trabecular surfaces for new bone formation.

Brtl mice also provided important information about the cytoskeletal organization in OI osteoblasts and their potential role in the phenotypic variability. Abnormal cytoskeletal organization was demonstrated 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 cofilin-1. The alterations affected osteoblast proliferation, collagen deposition, integrin, and TGF-beta signaling. The data open the possibility that cytoskeletal elements may be 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, however, 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 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 published the cardiopulmonary aspects of our natural history study on types III and IV OI. Longitudinal evaluations were completed in 23 children with type III OI and 23 children with type IV OI, who had pulmonary function tests every 1–2 years. Compared with size-matched children, our patients 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 the lung related to structurally abnormal collagen. The studies are important because pulmonary issues are the most prevalent cause of morbidity and mortality in OI. Affected individuals should seek anticipatory evaluation and treatment.

Currently, OI–specific growth curves are not available, despite the fact that short stature is one of the cardinal features of OI. We assembled longitudinal length, weight, head circumference, and body mass index (BMI) data on 100 children with type III and IV OI, with known mutations in type I collagen, to generate sex- and type-specific growth curves for OI. We examined effects of gender, OI type, and pathogenic variant, using multilevel modeling, and we constructed OI–specific centile curves, using a generalized additive model for location, scale, and shape (GAMLSS). The data show that gender and OI type, but not the collagen chain in which the causative mutation is located, have significant effects on height in OI. Boys are taller than girls, and type IV OI boys and girls are taller than type III. In both genders, length curves for types III and IV OI overlap and the type IV 95th centile curve overlaps the lower US Centers for Disease Control and Prevention (CDC) curves for the general population. A pubertal growth spurt is generally absent or blunted in types III/IV OI. The body-mass-index 50th and 95th centile curves are distinctly shifted above respective US CDC curves in both genders. Weight differs by OI type, but not by gender or mutant collagen chain. Interestingly, head circumference does not differ by gender, OI type, or collagen mutation. Imposition of OI height curves on standard CDC curves reveals an overlapping of type III and IV percentiles and the absence of a growth spurt in type III OI. Standard growth curves for OI will be of great value to primary caregivers and families and will provide a baseline for treatment trials.

Our randomized controlled trial of bisphosphonate in children with types III and IV OI was the first randomized bisphosphonate trial for OI in the United States. It examined direct skeletal and secondary gains reported in uncontrolled trials. For skeletal outcomes, we found increased BMD (bone mineral density) Z-scores and improved vertebral geometry. 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 claims for improvement in ambulation level, lower-extremity strength, or alleviation of pain, suggesting these were placebo effects in observational trials. Our current recommendation is for 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 decline in the quality of bone material, it is important to determine the lowest cumulative dose that will provide vertebral benefits. Preliminary analysis indicates that OI children obtain comparable benefits from lower and higher doses of the bisphosphonate pamidronate.

Melorheostosis: genetic and clinical delineation

Melorheostosis is a very rare sporadic bone dysostosis that is characterized by metabolically active bone in the appendicular skeleton, which leads to asymmetric bone overgrowth, seen radiographically as "dripping candle wax," functional impairment, and pain. Skin overlying the bone lesion often has a hyper-pigmented, vascular lesion. The etiology of melorheostosis had been uncertain; attempts to find germline mutations were unsuccessful, and we hypothesized somatic mutations. Our collaborative team (with investigators Tim Bhattacharyya, Richard Siegel, and Nadja Fratzl-Zelman) was the first to look directly at bone samples. Fifteen patients with melorheostosis consented to paired biopsies of both affected and contralateral unaffected bone. DNA extracted from each bone tissue was subjected to 100x whole exome sequencing (WES), and each patient's affected samples were compared with their own unaffected samples.

Using WES, we identified somatic mosaic MAP2K1 (which encodes mitogen-activated protein kinase kinase 1 and is also known as MEK1) mutations in the affected, but not in unaffected, bone of eight unrelated patients and in the skin overlying lesions, but not in blood. There was no evidence of an underlying germline mutation. In affected bone, the mutant allele frequency ranged from 3–34%. Given that melorheostosis is a progressive but not a metastatic condition, it was striking to identify causative mutations in an oncogene. The activating mutations (Q56P, K57E, and K57N) cluster tightly in the MAP2K1 negative-regulatory domain and would be expected to raise MAP2K1 activity. Identical mutations have been found in several malignancies of other tissues (lung, blood, melanoma, colon), while only three instances of conversion of melorheostosis to osteosarcoma have been reported. Affected bone displays a mosaic pattern of increased p-ERK1/2 in osteoblast immunohistochemistry. Osteoblasts cultured from affected bone constitute two populations with distinct p-ERK1/2 levels by flow cytometry, enhanced ERK1/2 activation, and elevated cell proliferation. However, the MAP2K1 mutations inhibit BMP2–mediated osteoblast mineralization and differentiation in vitro, underlying the markedly increased osteoid detected in affected bone histology. Our data show that the MAP2K1 oncogene is important in human bone formation and implicate MAP2K1 inhibition as a potential treatment avenue for melorheostosis.

We also reported distinguishing clinical characteristics of melorheostosis patients with MAP2K1 mutations (MAP2K1–positive melorheostosis). Eight patients with mutations in MAP2K1 in affected bone were compared with the seven MAP2K1–negative patients to identify distinguishing characteristics. Patients with MAP2K1–positive melorheostosis had a distinct phenotype with the classic "dripping candle-wax" appearance on radiographs, characteristic vascular lesions on skin overlying affected bone, and higher prevalence of extraosseous mineralization and joint involvement. Melorheostotic bone from both MAP2K1–positive and MAP2K1–negative patients showed two zones of distinct morphology: an outer segment of parallel layers of primary lamellar bone and a deeper zone of intensely remodeled highly porous osteonal-like bone. Affected bone from MAP2K1–positive patients showed excessive osteoid, increased numbers of osteoblasts and osteoclasts, and increased vascularity on histology, compared with paired unaffected bone, which was not seen in affected bone in most MAP2K1–negative patients. The identification of a distinct phenotype of patients with MAP2K1–positive melorheostosis demonstrates clinical and genetic heterogeneity among patients with the disease. Further studies are needed to better understand the underlying pathophysiology and associated skin findings.

Additional Funding

  • NICHD DIR Director's Award


  1. Lindert U, Cabral WA, Ausavarat S, Tongkobpetch S, Ludin K, Barnes AM, Yeetong P, Weis M, Krabichler B, Srichomthong C, Makareeva E, Janecke A, Kennerknect I, Leikin S, Rothlisberger B, Rohrbach M, Eyre DR, Suphapecetiporn K, Giunta C, Marini JC, Shotelersuk V. MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-osteogenesis imperfecta. Nat Commun 2016;7:11920.
  2. Cabral WA, Ishikawa M, Garten M, Makareeva EN, Sargent BM, Weis MA, Barnes AM, Webb EA, Shaw NJ, Ala-Kokko L, Lacbawan FL, Högler W, Leikin S, Blank PS, Zimmerberg J, Erye DR, Yamada Y, Marini JC. Absence of the ER cation channel TMEM38B/TRIC-B disrupts intracellular calcium homeostasis and dysregulates collagen synthesis in recessive osteogenesis imperfecta. PloS Genetics 2016;12(7):e1006156.
  3. Fratzl-Zelman N, Barnes AM, Weis MA, Carter E, Hefferan TE, Perino G, Chang W, Smith PA, Roschger P, Klaushofer K, Glorieux F, Eyre DR, Raggio C, Rauch F, Marini JC. Non-lethal type VIII osteogenesis imperfecta has elevated bone matrix mineralization. J Clin Endocrinol Metab 2016;101(9):3516-3525.
  4. Webb EA, Balasubramanian M, Fratzl-Zelamn N, Wayne WA, Titheradge H, Alsaedi A, Saraff V, Cole T, Stewart S, Crabtree N, Sargent BM, Gamsjaeger S, Paschalis EP, Rischger P, Klaushofer K, Marini JC, Shaw NJ, Högler W. Skeletal and bone material phenotype associated with osteogenesis imperfect due to mutations in TMEM38B suggests a novel patho-mechanism leading to increased bone fragility in humans. J Clin Endocrinol Metab 2017;102(6):2019-2028.
  5. Kang H, Jha S, Deng Z, Fratzl-Zelman N, Cabral WA, Ivovic A, Meylan F, Hanson EP, Lange E, Katz J, Roschger P, Klaushofer K, Cowen EW, Siegel RM, Marini JC, Bhattacharyya T. Somatic activating mutations in MAP2K1 cause melorheostosis. Nat Commun 2018;9(1):1390.


  • Patricia Becerra, PhD, Laboratory of Retinal Cell and Molecular Biology, NEI, Bethesda, MD
  • Timothy Bhattacharyya, MD, Clinical and Investigative Orthopedics Surgery Unit, NIAMS, Bethesda, MD
  • Anne De Paepe, MD, PhD, Universitair Ziekenhuis Gent, Ghent, Belgium
  • David Eyre, PhD, University of Washington, Seattle, WA
  • Antonella Forlino, PhD, Università degli Studi di Pavia, Pavia, Italy
  • Nadja Fratzl-Zelman, PhD, Ludwig Boltzmann-Institut für Osteologie, Hanusch Krankenhaus der WGKK und Unfallkrankenhaus Meidling, Vienna, Austria
  • Cecilia Giunta, PhD, Kinderspital Zürich, Zürich, Switzerland
  • Wolfgang Högler, MD, DSc, FRCPCH, Birmingham Children's Hospital NHS Foundation Trust, Birmingham, United Kingdom
  • Kenneth Kozloff, PhD, University of Michigan, Ann Arbor, MI
  • Sergey Leikin, PhD, Section on Physical Biochemistry, NICHD, Bethesda, MD
  • Katarina Lindahl, MD, Uppsala Universitet, Uppsala, Sweden
  • Scott Paul, MD, Rehabilitation Medicine, NIH Clinical Center, Bethesda, MD
  • Cathleen L. Raggio, MD, Weill Medical College of Cornell University, New York, NY
  • Frank Rauch, MD, Shriners Hospital for Children, Montreal, Canada
  • Vorasuk Shotelersuk, MD, FABMG, King Chulalongkorn Memorial Hospital, Bangkok, Thailand
  • Richard Siegel, MD, PhD, Autoimmunity Branch, NIAMS, Bethesda, MD
  • Yoshi Yamada, PhD, Molecular Biology Section, NIDCR, Bethesda, MD
  • Mitsuo Yamauchi, PhD, University of North Carolina, Chapel Hill, NC
  • Joshua Zimmerberg, MD, PhD, Section on Cellular and Membrane Biophysics, NICHD, Bethesda, MD


For more information, email

Top of Page