Fibrodysplasia ossificans progressiva: mechanisms and models of skeletal metamorphosis

Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of connective tissue metamorphosis. It is characterized by malformation of the great (big) toes during embryonic skeletal development and by progressive heterotopic endochondral ossification (HEO) postnatally, which leads to the formation of a second skeleton of heterotopic bone. Individuals with these classic clinical features of FOP have the identical heterozygous activating mutation (c.617G>A; R206H) in the gene encoding ACVR1 (also known as ALK2), a bone morphogenetic protein (BMP) type I receptor. Disease activity caused by this ACVR1 mutation also depends on altered cell and tissue physiology that can be best understood in the context of a high-fidelity animal model. Recently, we developed such a knock-in mouse model for FOP (Acvr1^R206H/+) that recapitulates the human disease, and provides a valuable new tool for testing and developing effective therapies. The FOP knock-in mouse and other models in Drosophila, zebrafish, chickens and mice provide an arsenal of tools for understanding BMP signaling and addressing outstanding questions of disease mechanisms that are relevant not only to FOP but also to a wide variety of disorders associated with regenerative medicine and tissue metamorphosis.     

ACVR1 gene mutations in four Turkish patients diagnosed as fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized with congenital malformations of the great toes and progressive heterotopic ossifications in the skeletal muscles and soft tissue. FOP has been associated with a specific point mutation on the ACVR1 (Activin A receptor type I) gene. Four sporadic cases clinically diagnosed as FOP have been included in this study for mutational analysis. In three patients, heterozygote c.617G > A; p.R206H mutation was detected by both DNA sequence analyses and by HphI restrictive enzyme digestion. In the fourth patient, a heterozygote c.774G > T; p.R258S mutation in exon 5 was detected by DNA sequence analysis.     

进行性骨化性纤维増殖不良症临床及基因突变分析

目的：研究一例具有超经典型临床特征的FOP患者,并对其ACVR1/ALK2基因进行分析。方法：根据患者的大踇趾畸形和进行性异位骨化等表现进行临床诊断,确诊为FOP。经患者及家属同意,采集患者、父母外周血,提取DNA,通过PCR扩增并直接测序测定ACVR1基因全部外显子序列,以此来确定突变位点。结果：患者具有超经典型FOP的临床表现：先天性大踇趾畸形,先天性双手拇指、食指远端关节僵直和进行性异位骨化,父母无FOP的相关临床表现。基因测序分析示该患者在ACVR1第七外显子发现存在c.1067G〉A（p.G356D）杂合错义突变,而其父母无此杂合突变。结论：该患者在ACVR1的c.1067G〉A（p.G356D）发生杂合错义突变,这有助于我们更好地理解认识中国FOP患者的临床表现和发病机制。     

进行性骨化性纤维增殖不良症临床及基因突变分析

目的:研究一例具有超经典型临床特征的FOP患者,并对其ACVR1/ALK2基因进行分析。方法:根据患者的大踇趾畸形和进行性异位骨化等表现进行临床诊断,确诊为FOP。经患者及家属同意,采集患者、父母外周血,提取DNA,通过PCR扩增并直接测序测定ACVR1基因全部外显子序列,以此来确定突变位点。结果:患者具有超经典型FOP的临床表现:先天性大踇趾畸形,先天性双手拇指、食指远端关节僵直和进行性异位骨化,父母无FOP的相关临床表现。基因测序分析示该患者在ACVR1第七外显子发现存在c.1067G>A(p.G356D)杂合错义突变,而其父母无此杂合突变。结论:该患者在ACVR1的c.1067G>A(p.G356D)发生杂合错义突变,这有助于我们更好地理解认识中国FOP患者的临床表现和发病机制。     

Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.     

Fibrodysplasia ossificans progressiva, a heritable disorder of severe heterotopic ossification, maps to human chromosome 4q27-31

Fibrodysplasia ossificans progressiva (FOP) is a severely disabling, autosomal-dominant disorder of connective tissue and is characterized by postnatal progressive heterotopic ossification of muscle, tendon, ligament, and fascia and by congenital malformation of the great toes. To identify the chromosomal location of the FOP gene, we conducted a genomewide linkage analysis, using four affected families with a total of 14 informative meioses. Male-to-male transmission of the FOP phenotype excluded X-linked inheritance. Highly polymorphic microsatellite markers covering all human autosomes were amplified by use of PCR. The FOP phenotype is linked to markers located in the 4q27-31 region (LOD score 3.10 at recombination fraction 0). Crossover events localize the putative FOP gene within a 36-cM interval bordered proximally by D4S1625 and distally by D4S2417. This interval contains at least one gene involved in the bone morphogenetic protein-signaling pathway.     

Fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva is a rare autosomal dominant genetic disorder and the most disabling condition of heterotopic ossification in humans. Congenital malformation of the great toes in terms of hypoplasia or aplasia and fibular deviation is noted in almost all patients, and a hypoplasia of the thumbs in about half of the patients. Mutations in the ACVR1 gene were identified as a genetic cause of FOP. We summarize the clinical and molecular data of 25 patients from our cohort as well as of 47 patients with known mutations, and the clinical information described in the literature so far. We discuss these summarized data in terms of genotype-phenotype correlation.     

Localization of the gene for fibrodysplasia ossificans progressiva (FOP) to chromosome 17q21-22

Fibrodysplasia ossificans progressiva (FOP) is a very rare disease characterized by congenital malformation of the great toes and progressive heterotopic ossification of muscles. To identify the chromosomal localization of the FOP gene, we conducted a genomewide linkage analysis using seven affected families. The FOP phenotype is linked to markers located in the 17q21-22 region (LOD score of 3.41 at the recombination fraction theta = 0). Crossover events localize the putative FOP gene within a 12cM interval, bordered proximally by D17S809 and distally by D17S1838. Noggin (NOG) gene, located in 17q22, is an excellent candidate gene for FOP.     

Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2: Inhibiting the Receptor That Is Overactive in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.     

Progressive osseous heteroplasia: an uncommon cause of ossification of soft tissues

Whereas the deposition of calcium within soft tissues is not infrequent, the development of highly structured, mineralized tissue histologically identifiable as true bone is uncommon and can cause a variety of clinical features. This article reports the clinical and radiological features in a patient with progressive osseous heteroplasia (POH), a recently identified disorder characterized by heterotopic ossification. The patient, a female, was 20 years of age at presentation. In addition to abnormal ossifications, she had short metacarpals at the fourth and fifth rays and short metatarsals at the second rays. Her parents were unaffected. Until the results of Rosenfeld and Kaplan in 1995 reporting POH in two boys, typical features had only been reported in females (n=8). POH is usually sporadic; however, familial associations and atypical phenotypes have been reported.     

Molecular Consequences of the ACVR1R206H Mutation of Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP), a rare genetic and catastrophic disorder characterized by progressive heterotopic ossification, is caused by a point mutation, c.617G>A; p.R206H, in the activin A receptor type 1 (ACVR1) gene, one of the bone morphogenetic protein type I receptors (BMPR-Is). Although altered BMP signaling has been suggested to explain the pathogenesis, the molecular consequences of this mutation are still elusive. Here we studied the impact of ACVR1 R206H mutation on BMP signaling and its downstream signaling cascades in murine myogenic C2C12 cells and HEK 293 cells. We found that ACVR1 was the most abundant of the BMPR-Is expressed in mesenchymal cells but its contribution to osteogenic BMP signal transduction was minor. The R206H mutant caused weak activation of the BMP signaling pathway, unlike the Q207D mutant, a strong and constitutively active form. The R206H mutant showed a decreased binding affinity for FKBP1A/FKBP12, a known safeguard molecule against the leakage of transforming growth factor (TGF)-β or BMP signaling. The decreased binding affinity of FKBP1A to the mutant R206H ACVR1 resulted in leaky activation of the BMP signal, and moreover, it decreased steady-state R206H ACVR1 protein levels. Interestingly, while WT ACVR1 and FKBP1A were broadly distributed in plasma membrane and cytoplasm without BMP-2 stimulation and then localized in plasma membrane on BMP-2 stimulation, R206H ACVR1 and FKBP1A were mainly distributed in plasma membrane regardless of BMP-2 stimulation. The impaired binding to FKBP1A and an altered subcellular distribution by R206H ACVR1 mutation may result in mild activation of osteogenic BMP-signaling in extraskeletal sites such as muscle, which eventually lead to delayed and progressive ectopic bone formation in FOP patients.     

The effect of Activin-A on periodontal ligament fibroblasts-mediated osteoclast formation in healthy donors and in patients with fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation.     

A new mutation of the noggin gene in a French Fibrodysplasia ossificans progressiva (FOP) family

A new mutation of the Noggin gene in a French Fybrodysplasia ossificans progressiva (FOP) family: Fibrodysplasia ossificans progressiva (FOP) is a very rare disease characterized by congenital malformation of the great toes and progressive heterotopic ossification of the muscles. We previously located a FOP gene in the 17q21-22 region and described several mutations of the noggin (NOG) gene (located in 17q22) in four FOP patients, including the G91C mutation which is transmitted dominantly in a Spanish FOP family. We describe in the present study a new mutation of the NOG gene in a French FOP family. This new mutation is a guanine to adenine change at nucleotide 283 (283G --> A) of the NOG gene, and is transmitted in the family (in the heterozygote form) by the affected mother to her two affected children. At the peptide level this mutation (A95T) substitutes an Alanine residue by a Threonine at position 95 of the Noggin protein. The Alanine mutated residue is located just adjacent to the myristoylation site of the protein, where all the mutations we described until now are located.     

Cellular and morphological aspects of fibrodysplasia ossificans progressiva: Lessons of formation,repair, and bone bioengineering

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disease that causes bone formation within the muscles, tendons, ligaments and connective tissues. There is no cure for this disorder and only treatment of the symptoms is available. The purpose of this study was to review the literature and describe the clinical, cellular and molecular aspects of FOP. The material used for the study was obtained by reviewing scientific articles published in various literature-indexed databases. In view of its rarity and of the lack of insightful information and the unpredictability of its course, FOP is a challenging disorder for professionals who are confronted by it. However, this rare disease raises a great deal of interest because understanding the mechanism of mature bone formation can encourage research lines related to bone regeneration and the prevention of heterotopic ossification.     

Cellular and morphological aspects of fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disease that causes bone formation within the muscles, tendons, ligaments and connective tissues. There is no cure for this disorder and only treatment of the symptoms is available. The purpose of this study was to review the literature and describe the clinical, cellular and molecular aspects of FOP. The material used for the study was obtained by reviewing scientific articles published in various literature-indexed databases. In view of its rarity and of the lack of insightful information and the unpredictability of its course, FOP is a challenging disorder for professionals who are confronted by it. However, this rare disease raises a great deal of interest because understanding the mechanism of mature bone formation can encourage research lines related to bone regeneration and the prevention of heterotopic ossification.     

Radiation therapy in treatment of fibrodysplasia ossificans progressiva: a case report and review of the literature

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare genetic disorder with diffuse extra-skeletal bone formation. The genetic mutation responsible for FOP has recently been discovered and is connected with excessive activation of bone morphogenetic protein receptor. This disease usually begins with typical ossification pattern in early childhood, causing increasing disability and making patients totally disabled by the age of 30. Ectopic ossification develops spontaneously and can be triggered by any trauma and even intramuscular injections. The symptoms of FOP are often misdiagnosed as cancer, causing unnecessary biopsies, which can precipitate further progressive heterotopic ossification. There is no effective treatment for this severe condition. Radiotherapy can be helpful in impeding ossification, although the strict evidence for that is lacking. There are only two reports in the literature referring to the use of radiotherapy in treatment of FOP. Herein, we present a 35-year-old patient successfully treated with small doses of fractionated radiotherapy in several courses. This case indicates that radiotherapy can be useful in treating patients with FOP.     

Loss-of-function of ACVR1 in osteoblasts increases bone mass and activates canonical Wnt signaling through suppression of Wnt inhibitors SOST and DKK1

BMPs (Bone morphogenetic proteins) such as BMP2 and BMP7 have been used about one decade as bone anabolic agents in orthopaedics. The BMP receptor ACVR1, which is a key receptor of BMP7, is expressed in bone. The pathological role of ACVR1 in humans has been reported: a point mutation in ACVR1 can cause fibrodysplasia ossificans progressiva (FOP) in which ectopic ossification occurs in skeletal muscles and deep connective tissues. The physiological function of ACVR1 in bone, however, is totally unknown. The purpose of this study is to investigate the endogenous role of ACVR1 in osteoblasts, one of the most dominant cell-types in bone. We generated Acvr1-null mice in an osteoblast-specific manner using an inducible Cre-loxP system. Surprisingly, we found that bone mass was increased in the Acvr1-null mice. Interestingly, canonical Wnt signaling was increased and expression levels of Wnt inhibitors Sost and Dkk1 were both suppressed in the null bones during the developmental stages. In addition, we confirmed that expression levels of both Sost and Dkk1 were upregulated by BMP7 dose-dependently in vitro. These results suggest that the Acvr1-deficiency can increase bone mass by activating Wnt signaling in which both Sost and Dkk1 expression levels are diminished. This study leads to a new concept of the BMP7-ACVR1-SOST/DKK1 axis in osteoblasts, in which BMP7 signaling through ACVR1 can reduce Wnt signaling via SOST/DKK1 and then inhibits osteogenesis. Although this concept is beyond the current known function of BMP7, it can explain the varied outcomes of BMP7 treatment. We believe BMP signaling can exhibit multifaceted effects by context and cell type.     

Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists

Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-like kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans.     

A novel mutation of ALK2, L196P, found in the most benign case of fibrodysplasia ossificans progressiva activates BMP-specific intracellular signaling equivalent to a typical mutation, R206H

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic ossification in muscle tissues. Constitutively activated mutants of a bone morphogenetic protein (BMP) receptor, ALK2, have been identified in patients with FOP. Recently, a novel ALK2 mutation, L196P, was found in the most benign case of FOP reported thus far. In the present study, we examined the biological activities of ALK2(L196P) in vitro. Over-expression of ALK2(L196P) induced BMP-specific activities, including the suppression of myogenesis, the induction of alkaline phosphatase activity, increased BMP-specific luciferase reporter activity, and increased phosphorylation of Smad1/5 but not Erk1/2 or p38. The activities of ALK2(L196P) were higher than those of ALK2(G356D), another mutant ALK2 allele found in patients with FOP and were equivalent to those of ALK2(R206H), a typical mutation found in patients with FOP. ALK2(L196P) was equally or more resistant to inhibitors in comparison to ALK2(R206H). These findings suggest that ALK2(L196P) is an activated BMP receptor equivalent to ALK2(R206H) and that ALK2(L196P) activity may be suppressed in vivo by a novel molecular mechanism in patients with this mutation.