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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2023 Annual Report of the Division of Intramural Research

Genetic Disorders of Bone and Extracellular Matrix

Joan Marini
  • Joan C. Marini, MD, PhD, Scientist Emeritus, Section on Heritable Disorders of Bone and Extracellular Matrix
  • Aileen M. Barnes, MS, Biologist
  • Milena Jovanovic, PhD, Postdoctoral Fellow
  • Elena F. Evans, BS, Postbaccalaureate Fellow

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders collectively 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 to apply this knowledge to patient treatment. We identified several key genes causing recessive and X-linked OI. Discoveries of defects in collagen modification generated a new paradigm for OI as a collagen-related disorder of matrix. We established that structural defects in collagen cause dominant OI, while deficiency of proteins that interact with collagen 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 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 one for X-linked type XVIII OI, and we are using these models to study disease pathogenesis, the skeletal matrix of OI, and the effects of pharmacological therapies. Our clinical studies involve children with the more prevalent types III and IV OI, as well as those with the rare recessive forms, 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 radiographic patterns of either ‘dripping candle wax’ or endosteal bone overgrowth. We recently identified mosaic mutations in the oncogene MAP2K1 as the cause of ‘dripping candle wax’ melorheostosis and somatic mutations in the gene SMAD3 as the cause of endosteal melorheostosis. In each gene, the causative mutations occur at a hot spot and result in gain of function. We are now developing animal models for studies of melorheostosis pathophysiology and treatment.

Mechanism of rare forms of osteogenesis imperfecta

Type XIV OI is a recessive OI form caused by null mutations in TMEM38B, which encodes the ER–membrane intracellular cation channel TRIC-B. Previously, we showed that absence of TMEM38B alters calcium flux in the ER of OI patient osteoblasts and fibroblasts, which further disrupts collagen synthesis and secretion. How the absence of TMEM38B affects osteoblast function is still poorly understood. We further investigated the role of TMEM38B in human osteoblast differentiation and mineralization [Reference 1]. TMEM38B–null osteoblasts showed altered expression of osteoblast marker genes and reduced mineralization. RNA-seq analysis revealed that cell-cell adhesion was one of the most downregulated pathways in TMEM38B–null osteoblasts, with further validation by real-time PCR and Western blot. Gap- and tight-junction proteins were also reduced by the absence of TRIC-B, both in patient osteoblasts and in calvarial osteoblasts of Tmem38b–null mice. Disrupted cell adhesion decreased mutant cell proliferation and cell-cycle progression. An important novel finding was that TMEM38B–null osteoblasts had elongated mitochondria with altered fusion and fission markers, MFN2 and DRP1. In addition, TMEM38B–null osteoblasts exhibited a significant increase in superoxide production in mitochondria, further supporting mitochondrial dysfunction. Together these results emphasize the novel role of TMEM38B/TRIC-B in osteoblast differentiation, affecting cell-cell proliferation, adhesion processes, gap- and tight-junction, cell-cycle, and mitochondrial function. We also collaborated with the Forlino laboratory on studies of the TMEM38B–null mouse that they generated. These studies showed that loss of TRIC-B function dysregulated SMAD signaling in bone. The impaired SMAD signaling detected in mutant mice was validated in OI patient osteoblasts, contributing to defects in osteoblast functioning [Reference 2].

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 lack serum PEDF and have 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 and whose bone had typical type VI histology, but whose SERPINF1 sequences were normal [Reference 3]. Exome sequencing of this proband and family members yielded a de novo mutation in IFITM5 (the gene encoding interferon-induced transmembrane protein 5, which is mutated in type V OI) in one proband allele, causing a p.S40L substitution in the intracellular domain of the encoded protein BRIL (an osteoblast-specific, mineralization-modifying, IFITM–like membrane 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 occur in connected cellular pathways that affect bone mineralization. We generated a murine model for atypical type VI OI. Investigations published to date have focused on bone material properties and on the lacunar canalicular network [Reference 4]. When Ifitm5/BRIL p.S42L murine bone is embedded with rhodamine, it is apparent that canicular density is lower than in wild-type (WT) mice at all ages studied, a feature that is also seen in normal bone with aging, where it is associated with impaired signal propagation in response to loading. Second harmonic generation was used to examine the organization of collagen in bone. Although the Ifitm5/BRIL p.S42L mouse does not have a defect in collagen structure, collagen is massively disorganized in their bone. Recent investigations of the Ifitm5/BRIL p.S42L mouse and its bone biology revealed that both heterozygous and homozygous Ifitm5/BRIL p.S42L mice are non-lethal and subject to spontaneous fractures, showing reduced bone strength and markedly increased brittleness on bone mechanics. Although Bril is not directly involved with type I collagen, the bones of the mice are hypermineralized on BMDD (bone-mineralization density distribution), similar to other collagen-based OI forms, with a tripling of CaHigh (highly mineralized bone matrix) in cortical bone. Like OI type aVI patients, young heterozygous mice have normal serum PEDF levels but elevated serum alkaline phosphatase. However, other aspects of bone histology did not recapitulate findings in the patients. Bone osteoid was decreased, as were the numbers of bone cells, both osteoblasts and osteoclasts, and bone formation rate, and the fish-scale lamellae were not detected under polarized light. Collagen secretion and cellular proliferation were increased in mutant osteoblasts, which is also atypical for OI. Second harmonic generation microscopy revealed disorganized matrix in male mice, similar to that detected among female mutants.

Type VI OI itself has been investigated in a knock-out murine model. We demonstrate that loss of PEDF delays osteoblast maturation as well as extracellular matrix (ECM) mineralization [Reference 5]. There was increased bone vascularization in KO mice, which correlated with elevated numbers of CD31(+)/Endomucin(+) endothelial cells, which are involved in coupling angiogenesis with osteogenesis. Global transcriptome analysis by RNA-seq of Serpinf1(−/−) mouse osteoblasts reveals that osteogenesis and angiogenesis are the biological processes most impacted by loss of PEDF. Intriguingly, TGF-β signaling is activated in type VI OI cells, and Serpinf1(−/−) osteoblasts are more sensitive to TGF-β stimulation than WT osteoblasts. These data suggest that the functional antagonism between PEDF and TGF-β pathways controls osteogenesis and bone vascularization and is implicated in type VI OI pathogenesis. The antagonism may be exploited for developing therapeutics for type VI OI.

The endoplasmic reticulum (ER)–resident procollagen 3-hydroxylation complex 3-hydroxylates type I collagen alpha1(I) chains. One member of the complex, cyclophilin B (CyPB), encoded by PPIB, is an ER–resident peptidyl-prolyl cis-trans isomerase (PPIase) and the major PPIase catalyzing collagen folding. Our group generated a Ppib knock-out (KO) mouse. We are studying the effect of absence of PPIB on the functioning of bone cells, both osteoblasts and osteoclasts, and the outcome for bone histomorphometry and correlating results with comparable studies in affected children.

In collaboration with Vorasuk Shotelersuk and Cecilia Giunta, we identified the first type of OI with X-linked inheritance. It causes 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. Affected individuals have missense mutations in MBTPS2, which encodes the protein S2P [Reference 3]. S2P is a transmembrane protein in the Golgi and is a critical component of regulated intramembrane 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, which enter the nucleus and activate gene transcription. Mutant S2P protein has impaired RIP cleavage of the transcription factors OASIS, ATF6, and SREBP. The mutations in MBTPS2 demonstrate that RIP plays a fundamental role in bone development. Recently, we have been investigating the differentiation of osteoblasts from two boys with type XVIII OI.

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 procollagen C-proteinase (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 a former collaboration with Katarina Lindahl [Lindahl et al., Hum Mutat 2011;32:598]. 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 development, we developed a knock-in murine model with a COL1A1 (the gene encoding pro-alpha1 type I collagen chain) cleavage-site mutation. Bone collagen fibrils showed a ‘barbed-wire’ appearance consistent with the presence of the processing intermediate pC-collagen, which 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 small, and their femora exhibit extreme brittleness on mechanical testing, as well as reduced fracture load. BMDD measurements on their femora show significantly higher mineralization than in WT mice, which continues to increase in the high bone-mass mice (HBM), even after mineralization plateaus at six months in the WT mice. PINP and TRAP, serum markers of bone remodeling, are significantly elevated in such HBM. Osteocyte density is reduced, but the lacunar area is increased.

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 reproduces type IV OI features. Brtl has provided important insights into the mechanism of OI and its treatment.

We 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 indicated unchanged mineralization, unlike the hyper-mineralization induced by bisphosphonates. In addition, Scl-AB was successfully anabolic in adult Brtl mice, and may thus 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 cessation of Scl-AB treatment preserved the anabolic gains from Scl-AB.

Brtl mice provided important information on the cytoskeletal organization in OI osteoblasts and their potential role in phenotypic variability. We observed abnormal cytoskeletal organization involving vimentin, stathmin (a microtubule-destabilizing phosphoprotein), and cofilin-1 (an actin-modulating protein) in lethal pups. Reduced vimentin (an intermediate filament) can lead to cytoskeletal collapse, and increased stathmin and cofilin-1 work in concert to disrupt cytoskeletal cellular functions. The alterations affected osteoblast proliferation, collagen deposition, integrin, and TGF-beta signaling. The data suggest that cytoskeletal elements present novel OI treatment targets.

We are also investigating an important unexplained feature of OI and other dominant skeletal disorders: the variability of expression, that is, very different phenotypic severity even in patients from the same family. We are studying variability both in human osteoblasts and in murine models. Osteoblasts from individuals with the same mutation but different severity of OI are being compared for differentiation and mineralization. More severe phenotypes are associated with greater reduction in mineralization. In addition, the Brtl(Cys)(G349C) mouse is being compared with a Brtl(Ser)(G349S) mouse with a different substitution at the same residue. Surprisingly, the serine substitution results in severely reduced collagen secretion and a bone volume only 13% of WT, leading to a lethal phenotype.

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

Pulmonary issues are the most prevalent cause of morbidity and mortality in OI. We previously 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 in pulmonary function over time, including in lung volumes and flow rates. The decline was worse in the 36 children with scoliosis but also occurred in 20 participants without scoliosis, who had declining function with restrictive disease, suggesting that pulmonary dysfunction of OI is attributable to a primary defect related to abnormal collagen in the lung. We have now published comprehensive pulmonary phenotyping results from a cohort of 37 individuals with OI evaluated at the NIH Clinical Center [Reference 6]. Lung function measurements, CT scans, and radiographic images from children and young adults with five different types of OI, predominantly the classical types III and IV OI, but also including the rare recessive types VI, VII, and XIV OI, were analyzed. We showed, for the first time, that arm span or ulnar lengths are comparable height surrogates for calculating pulmonary function testing (PFT) results in patients with OI. Most patients had restrictive lung disease even at this young age, accompanied by reduced gas exchange, pointing to parenchymal issues. In-depth analyses of CT scan images demonstrate a high prevalence of bronchial thickening at the level of small airways, which may be directly related to abnormal collagen or a secondary inflammatory response in OI. The functional impact of thickening of the walls of small bronchi is supported by reduced FEV25–75% (forced expiratory flow) air flow, which also measures small airways, in all patients with type III OI. In general, severity of pulmonary manifestations was more pronounced in patients with type III OI, which is consistent with overall severity of disease in patients with this disorder. We also found that decline in pulmonary function correlates with severity of scoliosis, supporting a role for extrinsic as well as intrinsic factors in OI lung disease.

Although short stature is a cardinal feature of OI, OI–specific growth curves were not previously available. We assembled longitudinal length, weight, head circumference, and body mass index (BMI) data on 100 children with types III and IV OI with known mutations in type I collagen, to generate sex- and type-specific growth curves for OI. The data show that gender and OI type, but not the specific mutant collagen chain, have significant effects on height in OI. A pubertal growth spurt is generally absent or blunted in types III/IV OI. The BMI 50th and 95th centile curves are distinctly shifted above respective CDC curves in both genders. Interestingly, head circumference does not differ by gender, OI type, or collagen mutation.

Our trial of bisphosphonate in children with types III and IV OI was the first randomized controlled bisphosphonate trial for OI in the United States. It examined direct skeletal and secondary gains reported in uncontrolled trials. We found increased BMD (bone mineral density) Z-scores and improved vertebral geometry. Vertebral BMD improvement tapered off after two years' treatment. Our treatment group did not experience fewer long-bone fractures, coinciding with equivocal improvement in fractures in other controlled trials. Our trial did not support claims for improved ambulation level, lower-extremity strength, or pain alleviation, suggesting these are placebo effects. Our recommendation is for treatment for two to three years, with subsequent follow-up of bone status. Our preliminary analysis of 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, indicates that OI children obtain comparable benefits from lower and higher doses of 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 sometimes has a hyperpigmented, vascular lesion. Because attempts to identify germline mutations causing melorheostosis were unsuccessful, we proposed that somatic mutations were causative. Our collaborative team (with Tim Bhattacharyya and Nadja Fratzl-Zelman) was the first to look directly at bone samples. Fifteen patients with melorheostosis had paired biopsies of both affected and contralateral unaffected bone. DNA from each patient’s two bone samples was subjected to whole-exome sequencing (WES), and sequences from each individual patient were compared. We identified two genes causing somatic mutations in melorheostotic lesions [Kang et al. Nat Commun 2018;9:1390; Kang et al. Exp Med 2020;217:e20191499]. Each gene was associated with one radiographic form of melorheostosis, and the bone lesions had distinct histology and mechanism along the TGFβ pathway.

Eight of the 15 patients had somatic mutations for MAP2K1 (dual-specificity mitogen-activated protein kinase 1), located in two adjacent residues of the negative regulatory domain and that would be expected to increase MEK1 (meiotic chromosome axis–associated kinase) activity. Erythematous skin lesions overlying the affected bone are often mosaic for the MAP2K1 mutations and have increased vascularity [Kang et al. Nat Commun 2018;9:1390]. Our data show that the MAP2K1 oncogene is important in human bone formation, and they implicate MAP2K1 inhibition as a potential treatment avenue for melorheostosis.

Four patients were determined to have causative somatic mutations in SMAD3, a component of the canonical TGFβ pathway [Kang et al. Exp Med 2020;217:e20191499]. SMAD3 phosphorylation was increased in affected bone, and downstream target genes of TGFβ signaling had elevated expression. The mutations were associated with an endosteal radiographic pattern. Cultured osteoblasts from affected bone exhibited reduced proliferation in vitro, increased osteoblast differentiation markers, and increased mineralization.

Melorheostotic bone from both MAP2K1–positive and SMAD3–positive patients showed two zones of distinct morphology [Fratzl-Zelman et al. J Bone Miner Res 2019;34:883-895]. In MAP2K1–positive melorheostosis, the inner osteonal zone is intensely remodeled and has increased osteoid. The zone is covered by an outer zone containing compact multi-layered lamellae. The remodeling zone has low bone mineralization and high porosity, reflecting high vascularity. The lamellar portion is less mineralized than the remodeling zone, indicating that the surgical hardness of this bone reflects its lamellar structure. We propose that the genetically induced deterioration of bone micro-architecture in the remodeling zone triggers a periosteal reaction.

Our current interests are to investigate communication between mutant and non-mutant cells in the affected bone, and to understand the mechanism of the SMAD3 mutation, using a recently generated murine model. Exploration of secreted cytokines and chemokines in the conditioned media of osteoblasts with a high variant allele frequency (VAF) of MAP2K1 mutations revealed factors that stimulate angiogenesis, inhibit osteoblast differentiation, and increase osteoclastogenesis and inflammation. Thus, it is likely that mutant cells recruit non-mutant cells into the bone overgrowth via these and other secreted factors.

Additional Funding

  • NICHD DIR Director’s Award


  1. Jovanovic M, Mitra A, Besio R, Contento BM, Wong KW, Derkyi A, To M, Forlino A, Dale RK, Marini JC. Absence of TRIC-B from Type XIV OI osteoblasts alters cell adhesion and mitochondrial function – A multi-omics study. Matrix Biol 2023 121:127–148.
  2. JBesio R, Contento BM, Garibaldi N, Filibian M, Sonntag S, Shmerling D, Tonelli F, Biggiogera M, Brini M, Salmaso A, Jovanovic M, Marini JC, Rossi A, Forlino A. CaMKII inhibition due to TRIC-B loss-of-function dysregulates SMAD signaling in osteogenesis imperfecta. Matrix Biol 2023 120:43–59.
  3. JJovanovic M, Guterman-Ram G, Marini JC. Osteogenesis Imperfecta: Mechanisms and signaling pathways connecting classical and rare OI types. Endocr Rev 2022 43(1):61–90.
  4. JHedjazi G, Guterman-Ram G, Blouin S, Schemenz V, Wagermaier W, Fratzl P, Hartmann M, Zwerina J, Fratzl-Zelman N, Marini JC. Alterations of bone material properties in growing Ifitm5/BRIL p.S42 knock-in mice, a new model for atypical type VI osteogenesis imperfecta. Bone 2022 162:116451.
  5. JKang H, Aryal Ac S, Barnes AM, Martin A, David V, Crawford S, Marini JC. Antagonism between PEDF and TGF-β contributes to type VI Osteogenesis Imperfecta bone and vascular pathogenesis. J Bone Miner Res 2022 37:925–937.
  6. JGochuico BR, Hossain M, Talvacchio S, Zuo MXG, Barton M, Dang Do AN, Marini JC. Intrinsic and extrinsic lung abnormalities in children and young adults with osteogenesis imperfecta affect pulmonary function. J Med Genet 2023 60:1067–1075.


  • Timothy Bhattacharyya, MD, Clinical and Investigative Orthopedics Surgery Unit, NIAMS, Bethesda, MD
  • Susan E. Crawford, DO, North Shore University Research Institute, University of Chicago Pritzker School of Medicine, Chicago, IL
  • 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
  • Kenneth Kozloff, PhD, University of Michigan, Ann Arbor, MI
  • Sergey Leikin, PhD, Section on Physical Biochemistry, NICHD, Bethesda, MD
  • Cathleen L. Raggio, MD, Weill Medical College of Cornell University, New York, NY
  • Vorasuk Shotelersuk, MD, FABMG, King Chulalongkorn Memorial Hospital, Bangkok, Thailand


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