David L. Glaser

A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification. We mapped FOP to chromosome 2q23-24 by linkage analysis and identified an identical heterozygous mutation (617G → A; R206H) in the glycine-serine (GS) activation domain of ACVR1, a BMP type I receptor, in all affected individuals examined. Protein modeling predicts destabilization of the GS domain, consistent with constitutive activation of ACVR1 as the underlying cause of the ectopic chondrogenesis, osteogenesis and joint fusions seen in FOP.NOTE: In the version of this article initially published, several contributing authors were listed collectively under the name The International FOP Research Consortium. In order to facilitate the electronic citation of author contributions, the authors have chosen to delete the Consortium name and replace it with the names of the individual consortium authors in alphabetical order. This error has been corrected in the HTML and PDF versions of the article.

Classic and atypical fibrodysplasia ossificans progressiva (FOP) phenotypes are caused by mutations in the bone morphogenetic protein (BMP) type I receptor ACVR1

Fibrodysplasia ossificans progressiva (FOP) is an autosomal dominant human disorder of bone formation that causes developmental skeletal defects and extensive debilitating bone formation within soft connective tissues (heterotopic ossification) during childhood. All patients with classic clinical features of FOP (great toe malformations and progressive heterotopic ossification) have previously been found to carry the same heterozygous mutation (c.617G>A; p.R206H) in the glycine and serine residue (GS) activation domain of activin A type I receptor/activin‐like kinase 2 (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor. Among patients with FOP‐like heterotopic ossification and/or toe malformations, we identified patients with clinical features unusual for FOP. These atypical FOP patients form two classes: FOP‐plus (classic defining features of FOP plus one or more atypical features) and FOP variants (major variations in one or both of the two classic defining features of FOP). All patients examined have heterozygous ACVR1 missense mutations in conserved amino acids. While the recurrent c.617G>A; p.R206H mutation was found in all cases of classic FOP and most cases of FOP‐plus, novel ACVR1 mutations occur in the FOP variants and two cases of FOP‐plus. Protein structure homology modeling predicts that each of the amino acid substitutions activates the ACVR1 protein to enhance receptor signaling. We observed genotype‐phenotype correlation between some ACVR1 mutations and the age of onset of heterotopic ossification or on embryonic skeletal development. Hum Mutat 0, 1–12, 2008.

Identification of progenitor cells that contribute to heterotopic skeletogenesis.

BACKGROUND Individuals who have fibrodysplasia ossificans progressiva develop an ectopic skeleton because of genetic dysregulation of bone morphogenetic protein (BMP) signaling in the presence of inflammatory triggers. The identity of progenitor cells that contribute to various stages of BMP-induced heterotopic ossification relevant to fibrodysplasia ossificans progressiva and related disorders is unknown. An understanding of the cellular basis of heterotopic ossification will aid in the development of targeted, cell-specific therapies for the treatment and prevention of heterotopic ossification. METHODS We used Cre/loxP lineage tracing methods in the mouse to identify cell lineages that contribute to all stages of heterotopic ossification. Specific cell populations were permanently labeled by crossing lineage-specific Cre mice with the Cre-dependent reporter mice R26R and R26R-EYFP. Two mouse models were used to induce heterotopic ossification: (1) intramuscular injection of BMP2/Matrigel and (2) cardiotoxin-induced skeletal muscle injury in transgenic mice that misexpress BMP4 at the neuromuscular junction. The contribution of labeled cells to fibroproliferative lesions, cartilage, and bone was evaluated histologically by light and fluorescence microscopy. The cell types evaluated as possible progenitors included skeletal muscle stem cells (MyoD-Cre), endothelium and endothelial precursors (Tie2-Cre), and vascular smooth muscle (Smooth Muscle Myosin Heavy Chain-Cre [SMMHC-Cre]). RESULTS Vascular smooth muscle cells did not contribute to any stage of heterotopic ossification in either mouse model. Despite the osteogenic response of cultured skeletal myoblasts to BMPs, skeletal muscle precursors in vivo contributed minimally to heterotopic ossification (<5%), and this contribution was not increased by cardiotoxin injection, which induces muscle regeneration and mobilizes muscle stem cells. In contrast, cells that expressed the vascular endothelial marker Tie2/Tek at some time in their developmental history contributed robustly to the fibroproliferative, chondrogenic, and osteogenic stages of the evolving heterotopic endochondral anlagen. Importantly, endothelial markers were expressed by cells at all stages of heterotopic ossification. Finally, muscle injury and associated inflammation were sufficient to trigger fibrodysplasia ossificans progressiva-like heterotopic ossification in a setting of chronically stimulated BMP activity. CONCLUSIONS Tie2-expressing progenitor cells, which are endothelial precursors, respond to an inflammatory trigger, differentiate through an endochondral pathway, contribute to every stage of the heterotopic endochondral anlagen, and form heterotopic bone in response to overactive BMP signaling in animal models of fibrodysplasia ossificans progressiva. Thus, the ectopic skeleton is not only supplied by a rich vasculature, but appears to be constructed in part by cells of vascular origin. Further, these data strongly suggest that dysregulation of the BMP signaling pathway and an inflammatory microenvironment are both required for the formation of fibrodysplasia ossificans progressiva-like lesions.

Heparan Sulfate Proteoglycans (HSPGs) Modulate BMP2 Osteogenic Bioactivity in C2C12 Cells

Cell surface heparan sulfate proteoglycans (HSPGs) have been implicated in bone morphogenetic protein (BMP)-mediated morphogenesis by regulating BMP activity and gradient formation. However, the direct role of HSPGs in BMP signaling is poorly understood. Here we show that HSPGs directly regulate BMP2-mediated transdifferentiation of C2C12 myoblasts into osteoblasts. HSPGs sequester BMP2 at the cell surface and mediate BMP2 internalization. Depletion of cell surface HSPGs by heparinase III treatment or decreased glycosaminoglycan chain sulfation with sodium chlorate enhances BMP2 morpho-genetic bioactivity. The addition of exogenous heparin, a widely used anticoagulant, reduced BMP2 signaling. Our results suggest that cell surface HSPGs mediate BMP2 internalization and modulate BMP2 osteogenic activity.

In Vivo Somatic Cell Gene Transfer of an Engineered Noggin Mutein Prevents BMP4-Induced Heterotopic Ossification

BACKGROUND The formation of the skeleton requires inductive signals that are balanced with their antagonists in a highly regulated negative feedback system. Inappropriate or excessive expression of BMPs (bone morphogenetic proteins) or their antagonists results in genetic disorders affecting the skeleton, such as fibrodysplasia ossificans progressiva. BMP signaling mediated through binding to its receptors is a critical step in the induction of abnormal ossification. Therefore, we hypothesized that engineering more effective inhibitors of this BMP-signaling process may lead to the development of therapies for such conditions. METHODS BMP4-induced heterotopic ossification was used as a model for testing the ability of the BMP antagonist Noggin to block de novo bone formation, either by local or systemic delivery. Since Noggin naturally acts locally, a Noggin mutein, hNOGDeltaB2, was engineered and was shown to circulate systemically, and its ability to block heterotopic ossification was tested in a mouse model with use of adenovirus-mediated somatic cell gene transfer. RESULTS A mouse model of BMP4-induced heterotopic ossification was developed. Local delivery of wild-type NOG inhibited heterotopic ossification, but systemic administration was ineffective. In contrast, systemic delivery of the adenovirus encoding hNOGDeltaB2 resulted in systemic levels that persisted for more than two weeks and were sufficient to block BMP4-induced heterotopic ossification. CONCLUSIONS BMP4-induced heterotopic ossification can be prevented in vivo either by local delivery of wild-type Noggin or after somatic cell gene transfer of a Noggin mutein, hNOGDeltaB2. Furthermore, the data in the present study provide proof of concept that a naturally occurring factor can be engineered for systemic delivery toward a desirable pharmacological outcome. CLINICAL RELEVANCE Blocking bone formation is clinically relevant to disorders of heterotopic ossification in humans, such as fibrodysplasia ossificans progressiva. Furthermore, development of BMP antagonists as therapeutic agents may provide modalities for the treatment of other pathologic conditions that arise from aberrant expression of BMPs and/or from a lack of their antagonists.

Osteoporosis: Definition and Clinical Presentation

Osteoporosis is a skeletal condition characterized by decreased density (mass/volume) of normally mineralized bone. The reduced bone density leads to decreased mechanical strength, thus making the skeleton more likely to fracture. Postmenopausal osteoporosis (Type I) and age‐related osteoporosis (Type II) are the most common primary forms of bone loss seen in clinical practice. Secondary causes of osteoporosis include hypercortisolism, hyperthyroidism, hyperparathyroidism, alcohol abuse, and immobilization. In the development of osteoporosis, there is often a long latent period before the appearance of the main clinical manifestation, pathologic fractures. The earliest symptom of osteoporosis is often an episode of acute back pain caused by a pathologic vertebral compression fracture, or an episode of groin or thigh pain caused by a pathologic hip fracture. In the diagnostic process, the extent and severity of bone loss are evaluated and secondary forms of bone loss are excluded. A careful diagnostic work‐up that includes clinical history, physical examination, laboratory evaluation, bone densitometry, and radiographic imaging will allow the clinician to determine the cause of osteoporosis and to institute medical interventions that will stabilize and even reverse this frequently preventable condition.

Early Diagnosis of Fibrodysplasia Ossificans Progressiva

BACKGROUND. Fibrodysplasia ossificans progressiva is a rare and disabling genetic condition characterized by congenital malformation of the great toes and by progressive heterotopic ossification in specific anatomic patterns. Most patients with fibrodysplasia ossificans progressiva are misdiagnosed early in life before the appearance of heterotopic ossification and undergo diagnostic procedures that can cause lifelong disability. Recently, the genetic cause of fibrodysplasia ossificans progressiva was identified, and definitive genetic testing for fibrodysplasia ossificans progressiva is now available before the appearance of heterotopic ossification. METHODS. We recently evaluated 7 children for diagnosis of fibrodysplasia ossificans progressiva before the onset of heterotopic ossification. A medical history, physical examination, and skeletal survey were obtained on all of the patients, as well as clinical genetic testing for the canonical fibrodysplasia ossificans progressiva mutation. RESULTS. All 7 of the children (4 girls and 3 boys; ages 3 months to 6 years) had congenital malformations of the great toes, but none had radiographic evidence of heterotopic ossification at the time of evaluation. Five of the 7 children had soft tissue lesions of the neck and back, suggestive of early fibrodysplasia ossificans progressiva flare-ups, 3 of whom had undergone invasive diagnostic procedures that exacerbated their condition. Two children had no history or signs of soft tissue swelling or flare-ups. DNA sequence analysis found that all 7 of the children had the recurrent fibrodysplasia ossificans progressiva missense mutation, a single nucleotide substitution (c.617G>A) at codon 206 in the glycine-serine activation domain of activin receptor IA, a bone morphogenetic protein type 1 receptor. CONCLUSION. Clinical suspicion of fibrodysplasia ossificans progressiva early in life on the basis of malformed great toes can lead to early clinical diagnosis, confirmatory diagnostic genetic testing, and the avoidance of additional harmful diagnostic and treatment procedures. This is the first report of genetic confirmation of fibrodysplasia ossificans progressiva before the appearance of heterotopic ossification. Pediatricians should be aware of the early diagnostic features of fibrodysplasia ossificans progressiva, even before the appearance of heterotopic ossification. This awareness should prompt early genetic consultation and testing and the institution of assiduous precautions to prevent iatrogenic harm.

Hematopoietic Stem-Cell Contribution to Ectopic Skeletogenesis

BACKGROUND Fibrodysplasia ossificans progressiva is a rare genetic disorder of ectopic skeletogenesis associated with dysregulation of bone morphogenetic protein (BMP) signaling. Hematopoietic cells have been implicated in the ectopic skeletogenesis of fibrodysplasia ossificans progressiva, and their replacement has been postulated as a possible cure. However, the definitive contribution of hematopoietic cells to the pathogenesis of ectopic skeletogenesis remains obscure. METHODS We employed both careful clinical observation and in vivo murine transplantation studies to more precisely determine the contribution of hematopoietic cells to ectopic skeletogenesis. We identified a patient with fibrodysplasia ossificans progressiva who had undergone bone marrow transplantation for the treatment of intercurrent aplastic anemia twenty-five years earlier and investigated whether the clinical course of the fibrodysplasia ossificans progressiva had been influenced by bone marrow replacement or immunosuppression, or both. In complementary studies, we transplanted hematopoietic stem cells from constitutively expressing LacZ transgenic mice to identify the contribution of hematopoietic cells to BMP4-induced heterotopic ossification, a histopathologic model of fibrodysplasia ossificans progressiva. RESULTS We found that replacement of hematopoietic cells was not sufficient to prevent ectopic skeletogenesis in the patient with fibrodysplasia ossificans progressiva but pharmacologic suppression of the apparently normal donor immune system following transplantation in the new host modulated the activity of the fibrodysplasia ossificans progressiva and diminished the expression of skeletal ectopia. In complementary murine transplantation studies, we found that cells of hematopoietic origin contributed to the early inflammatory and late marrow-repopulating stages of BMP4-induced heterotopic ossification but were not represented in the fibroproliferative, chondrogenic, or osteogenic stages of heterotopic ossification. Interestingly, both recombinant human BMP4 induction in an animal model and the dysregulated BMP signaling pathway in a patient with fibrodysplasia ossificans progressiva were sufficient to recruit at least two populations of cells, one of hematopoietic origin and at least one of non-hematopoietic origin, that contribute to the formation of an ectopic skeleton. CONCLUSIONS Taken together, these findings demonstrate that bone marrow transplantation did not cure fibrodysplasia ossificans progressiva in the patient in this study, most likely because the hematopoietic cell population is not the site, or at least not the dominant site, of the intrinsic dysregulation of the BMP signaling pathway in fibrodysplasia ossificans progressiva. However, following transplantation of bone marrow from a presumably normal donor, immunosuppression of the immune system appeared to ameliorate activation of ectopic skeletogenesis in a genetically susceptible host. Thus, cells of hematopoietic origin may contribute to the formation of an ectopic skeleton, although they are not sufficient to initiate the process alone.

Fibrodysplasia ossificans progressiva

Abstract Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder of connective tissue. The condition is characterized by congenital malformation of the great toes and by progressive heterotopic ossification of the tendons, ligaments, fasciae, and striated muscles. Fibrodysplasia ossificans progressiva occurs sporadically and is transmitted as a dominant trait with variable expression and complete penetrance. Reproductive fitness is low. There are fewer than 150 known patients with the disorder in the United States. A point prevalence of one affected patient in every 2 million of population has been observed. There is no sexual, racial, or ethnic predilection. The disease presents in early life; its course is unavoidably progressive. Most patients are confined to a wheelchair by the third decade of life and often succumb to pulmonary complications in the 5th/6th decade of life. At present there is no effective prevention or treatment. The recent discovery of overproduction of bone morphogenetic protein-4 in lesional cells and lymphocytic cells of affected patients provides a clue to both the underlying pathophysiology and potential therapy. The FOP gene has recently been mapped to human chromosome 4 q 27–31.

Circulating Osteogenic Precursor Cells in Heterotopic Bone Formation

Cells with osteogenic potential can be found in a variety of tissues. Here we show that circulating osteogenic precursor (COP) cells, a bone marrow‐derived type I collagen+/CD45+ subpopulation of mononuclear adherent cells, are present in early preosseous fibroproliferative lesions in patients with fibrodysplasia ossificans progressiva (FOP) and nucleate heterotopic ossification (HO) in a murine in vivo implantation assay. Blood samples from patients with FOP with active episodes of HO contain significantly higher numbers of clonally derived COP cell colonies than patients with stable disease or unaffected individuals. The highest level of COP cells was found in a patient just before the clinical onset of an HO exacerbation. Our studies show that even COP cells derived from an unaffected individual can contribute to HO in genetically susceptible host tissue. The possibility that circulating, hematopoietic‐derived cells with osteogenic potential can seed inflammatory sites has tremendous implications and, to our knowledge, represents the first example of their involvement in clinical HO. Thus, bone formation is not limited to cells of the mesenchymal lineage, and circulating cells of hematopoietic origin can also serve as osteogenic precursors at remote sites of tissue inflammation. STEM CELLS 2009;27:2209–2219