Cell Senescence in Heterotopic Ossification.

The formation of bone outside the normal skeleton, or heterotopic ossification (HO), occurs through genetic and acquired mechanisms. Fibrodysplasia ossificans progressiva (FOP), the most devastating genetic condition of HO, is due to mutations in the ACVR1/ALK2 gene and is relentlessly progressive. Acquired HO is mostly precipitated by injury or orthopedic surgical procedures but can also be associated with certain conditions related to aging. Cellular senescence is a hallmark of aging and thought to be a tumor-suppressive mechanism with characteristic features such as irreversible growth arrest, apoptosis resistance, and an inflammatory senescence-associated secretory phenotype (SASP). Here, we review possible roles for cellular senescence in HO and how targeting senescent cells may provide new therapeutic approaches to both FOP and acquired forms of HO.

Successful preimplantation genetic testing for fibrodysplasia ossificans progressiva: a case report.

PURPOSE OF THE STUDY: Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant condition that leads to significant disability and morbidity, characterised by the formation of heterotopic hard tissues within connective tissues. The condition has an incidence of approximately one per two million people worldwide. There is no known single effective treatment available for FOP. We report the world's first case of a healthy infant born following in vitro fertilisation (IVF) and preimplantation genetic testing for monogenic disorder (PGT-M) using Karyomapping for FOP. CASE PRESENTATION: A 30-year-old Caucasian female with FOP presented with her partner seeking IVF with PGT-M to achieve a healthy pregnancy with an embryo unaffected by FOP. METHODS: The couple underwent IVF and PGT-M using Karyomapping as the testing method. A multi-disciplinary team approach was utilised in planning this case, considering the additional risks of oocyte retrieval, pregnancy and childbirth in women with FOP. MAIN FINDINGS: The oocyte retrieval was covered with a 5-day course of prednisolone to reduce the risk of a localised inflammatory reaction, which could result in subsequent heterotopic ossification. This was subsequently weaned down with reducing doses every two days. The patient underwent uncomplicated oocyte retrieval, yielding 12 mature oocytes. Following intracytoplasmic sperm injection (ICSI), ten zygotes having two pro-nuclei were cultured, and six underwent trophoectoderm biopsy and vitrification 5-6 days after retrieval. PGT-M via Karyomapping revealed four out of six (66.7%) of blastocysts were not carriers of the maternal high-risk FOP allele. In total, the patient had three separate embryo transfers. Pregnancy was achieved following the third frozen embryo transfer, which went to 37 weeks' gestation, and delivered by Caesarean section. The baby was born in excellent condition and is unaffected by FOP. CONCLUSION: IVF/ICSI and PGT-M using Karyomapping was successfully implemented to identify embryos carrying the high-risk FOP allele resulting in a healthy livebirth.

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating pathology where ectopic bone develops in areas of soft tissue. HO can develop as a consequence of traumatic insult or as a result of dysregulated osteogenic signaling, as in the case of the orphan disease fibrodysplasia ossificans progressiva (FOP). Traumatic HO (tHO) formation is mediated by the complex interplay of signaling between progenitor, inflammatory, and nerve cells, among others, making it a challenging process to understand. Research into the pathogenesis of genetically mediated HO (gHO) in FOP has established a pathway involving uninhibited activin-like kinase 2 receptor (ALK2) signaling that leads to downstream osteogenesis. Current methods of diagnosis and treatment lag behind pre-mature HO detection and progressive HO accumulation, resulting in irreversible decreases in range of motion and chronic pain for patients. As such, it is necessary to draw on advancements made in the study of tHO and gHO to better diagnose, comprehend, prevent, and treat both.

Hedgehog Signaling Controls Chondrogenesis and Ectopic Bone Formation via the Yap-Ihh Axis.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by abnormal bone formation due to ACVR1 gene mutations. The identification of the molecular mechanisms underlying the ectopic bone formation and expansion in FOP is critical for the effective treatment or prevention of HO. Here we find that Hh signaling activation is required for the aberrant ectopic bone formation in FOP. We show that the expression of Indian hedgehog (Ihh), a Hh ligand, as well as downstream Hh signaling, was increased in ectopic bone lesions in Acvr1R206H; ScxCre mice. Pharmacological treatment with an Ihh-neutralizing monoclonal antibody dramatically reduced chondrogenesis and ectopic bone formation. Moreover, we find that the activation of Yap in the FOP mouse model and the genetic deletion of Yap halted ectopic bone formation and decreased Ihh expression. Our mechanistic studies showed that Yap and Smad1 directly bind to the Ihh promoter and coordinate to induce chondrogenesis by promoting Ihh expression. Therefore, the Yap activation in FOP lesions promoted ectopic bone formation and expansion in both cell-autonomous and non-cell-autonomous manners. These results uncovered the crucial role of the Yap-Ihh axis in FOP pathogenesis, suggesting the inhibition of Ihh or Yap as a potential therapeutic strategy to prevent and reduce HO.

Immunologic Aspects in Fibrodysplasia Ossificans Progressiva.

BACKGROUND: Inflammation is a major driver of heterotopic ossification (HO), a condition of abnormal bone growth in a site that is not normally mineralized. PURPOSE OF REVIEW: This review will examine recent findings on the roles of inflammation and the immune system in fibrodysplasia ossificans progressiva (FOP). FOP is a genetic condition of aggressive and progressive HO formation. We also examine how inflammation may be a valuable target for the treatment of HO. Rationale/Recent findings: Multiple lines of evidence indicate a key role for the immune system in driving FOP pathogenesis. Critical cell types include macrophages, mast cells, and adaptive immune cells, working through hypoxia signaling pathways, stem cell differentiation signaling pathways, vascular regulatory pathways, and inflammatory cytokines. In addition, recent clinical reports suggest a potential role for immune modulators in the management of FOP. FUTURE PERSPECTIVES: The central role of inflammatory mediators in HO suggests that the immune system may be a common target for blocking HO in both FOP and non-genetic forms of HO. Future research focusing on the identification of novel inflammatory targets will help support the testing of potential therapies for FOP and other related conditions.

Matrix metalloproteinase-9 deficiency confers resilience in fibrodysplasia ossificans progressiva in a man and mice.

Single case studies of extraordinary disease resilience may provide therapeutic insight into conditions for which no definitive treatments exist. An otherwise healthy 35-year-old man (patient-R) with the canonical pathogenic ACVR1R206H variant and the classic congenital great toe malformation of fibrodysplasia ossificans progressiva (FOP) had extreme paucity of post-natal heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient post-natal inflammatory trigger for HO. A plasma biomarker survey revealed a reduction in total matrix metalloproteinase-9 (MMP-9) compared to healthy controls and individuals with quiescent FOP. Whole exome sequencing identified compound heterozygous variants in MMP-9 (c.59C > T, p.A20V and c.493G > A, p.D165N). Structural analysis of the D165N variant predicted both decreased MMP-9 secretion and activity that were confirmed by enzyme-linked immunosorbent assay and gelatin zymography. Further, human proinflammatory M1-like macrophages expressing either MMP-9 variant produced significantly less Activin A, an obligate ligand for HO in FOP, compared to wildtype controls. Importantly, MMP-9 inhibition by genetic, biologic, or pharmacologic means in multiple FOP mouse models abrogated trauma-induced HO, sequestered Activin A in the extracellular matrix (ECM), and induced regeneration of injured skeletal muscle. Our data suggest that MMP-9 is a druggable node linking inflammation to HO, orchestrates an existential role in the pathogenesis of FOP, and illustrates that a single patient's clinical phenotype can reveal critical molecular mechanisms of disease that unveil novel treatment strategies.

A healthy 35-year-old man (patient-R) with the classic fibrodysplasia ossificans progressiva (FOP) mutation and the congenital great toe malformation of FOP had extreme lack of heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient inflammatory trigger for HO. Blood tests revealed a reduction in the level of an inflammatory protein called matrix metalloproteinase-9 (MMP-9) compared to other individuals with FOP as well as healthy controls. DNA analysis in patient-R identified mutations in MMP-9, one of which predicted decreased activity of MMP-9 which was confirmed by further testing. Inflammatory cells (macrophages) expressing the MMP-9 mutations identified in patient-R produced significantly less Activin A, an obligate stimulus for HO in FOP. In order to determine if MMP-9 deficiency was a cause of HO prevention in FOP, we inhibited MMP-9 activity by genetic, biologic, or pharmacologic means in FOP mouse models and showed that MMP-9 inhibition prevented or dramatically decreased trauma-induced HO in FOP, locked-up Activin A in the extracellular matrix, and induced regeneration of injured skeletal muscle. Our data show that MMP-9 links inflammation to HO and illustrate that one patient's clinical picture can reveal critical molecular mechanisms of disease that unveil new treatment strategies.

iMSC-mediated delivery of ACVR2B-Fc fusion protein reduces heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by a gain-of-function mutation in ACVR1, which is a bone morphogenetic protein (BMP) type I receptor. Moreover, it causes progressive heterotopic ossification (HO) in connective tissues. Using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and mouse models, we elucidated the underlying mechanisms of FOP pathogenesis and identified a candidate drug for FOP. METHODS: In the current study, healthy mesenchymal stem/stromal cells derived from iPSCs (iMSCs) expressing ACVR2B-Fc (iMSCACVR2B-Fc), which is a neutralizing receptobody, were constructed. Furthermore, patient-derived iMSCs and FOP mouse model (ACVR1R206H, female) were used to confirm the inhibitory function of ACVR2B-Fc fusion protein secreted by iMSCACVR2B-Fc on BMP signaling pathways and HO development, respectively. RESULTS: We found that secreted ACVR2B-Fc attenuated BMP signaling initiated by Activin-A and BMP-9 in both iMSCs and FOP-iMSCs in vitro. Transplantation of ACVR2B-Fc-expressing iMSCs reduced primary HO in a transgenic mouse model of FOP. Notably, a local injection of ACVR2B-Fc-expressing iMSCs and not an intraperitoneal injection improved the treadmill performance, suggesting compound effects of ACVR2B-Fc and iMSCs. CONCLUSIONS: These results offer a new perspective for treating FOP through stem cell therapy.

The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A.

Mutations in activin-like kinase 2 (ALK2), e.g., ALK2-R206H, induce aberrant signaling to SMAD1/5/8, leading to Fibrodysplasia Ossificans Progressiva (FOP). In spite of extensive studies, the underlying mechanism is still unclear. Here, we quantified the homomeric and heteromeric interactions of ACVR2A, ACVR2B, ALK2-WT, and ALK2-R206H by combining IgG-mediated immobilization of one receptor with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of a co-expressed receptor. ACVR2B formed stable homomeric complexes that were enhanced by Activin A (ActA), while ACVR2A required ActA for homodimerization. ALK2-WT, but not ALK2-R206H, exhibited homomeric complexes unaffected by ActA. ACVR2B formed ActA-enhanced heterocomplexes with ALK2-R206H or ALK2-WT, while ACVR2A interacted mainly with ALK2-WT. The extent of the homomeric complex formation of ACVR2A or ACVR2B was reflected in their ability to induce the oligomerization of ALK2-R206H and ALK2-WT. Thus, ACVR2B, which forms dimers without ligand, induced ActA-independent ALK2-R206H clustering but required ActA for enhancing the oligomerization of the largely dimeric ALK2-WT. In contrast, ACVR2A, which undergoes homodimerization in response to ActA, required ActA to induce ALK2-R206H oligomerization. To investigate whether these interactions are translated into signaling, we studied signaling by the FOP-inducing hyperactive ALK2-R206H mutant, with ALK2-WT signaling as control. The activation of SMAD1/5/8 signaling in cells expressing ALK2-R206H alone or together with ACVR2A or ACVR2B was measured by blotting for pSMAD1/5/8 and by transcriptional activation assays using BRE-Luc reporter. In line with the biophysical studies, ACVR2B activated ALK2-R206H without ligand, while activation by ACVR2A was weaker and required ActA. We propose that the homodimerization of ACVR2B or ACVR2A dictates their ability to recruit ALK2-R206H into higher complexes, enabling the homomeric interactions of ALK2-R206H receptors and, subsequently, their activation.

Oxidative phosphorylation is a pivotal therapeutic target of fibrodysplasia ossificans progressiva.

Heterotopic ossification (HO) is a non-physiological bone formation where soft tissue progenitor cells differentiate into chondrogenic cells. In fibrodysplasia ossificans progressiva (FOP), a rare genetic disease characterized by progressive and systemic HO, the Activin A/mutated ACVR1/mTORC1 cascade induces HO in progenitors in muscle tissues. The relevant biological processes aberrantly regulated by activated mTORC1 remain unclear, however. RNA-sequencing analyses revealed the enrichment of genes involved in oxidative phosphorylation (OXPHOS) during Activin A-induced chondrogenesis of mesenchymal stem cells derived from FOP patient-specific induced pluripotent stem cells. Functional analyses showed a metabolic transition from glycolysis to OXPHOS during chondrogenesis, along with increased mitochondrial biogenesis. mTORC1 inhibition by rapamycin suppressed OXPHOS, whereas OXPHOS inhibitor IACS-010759 inhibited cartilage matrix formation in vitro, indicating that OXPHOS is principally involved in mTORC1-induced chondrogenesis. Furthermore, IACS-010759 inhibited the muscle injury-induced enrichment of fibro/adipogenic progenitor genes and HO in transgenic mice carrying the mutated human ACVR1. These data indicated that OXPHOS is a critical downstream mediator of mTORC1 signaling in chondrogenesis and therefore is a potential FOP therapeutic target.

Sex as a Critical Variable in Basic and Pre-Clinical Studies of Fibrodysplasia Ossificans Progressiva.

Heterotopic ossification (HO) is most dramatically manifested in the rare and severely debilitating disease, fibrodysplasia ossificans progressiva (FOP), in which heterotopic bone progressively accumulates in skeletal muscles and associated soft tissues. The great majority of FOP cases are caused by a single amino acid substitution in the type 1 bone morphogenetic protein (BMP) receptor ACVR1, a mutation that imparts responsiveness to activin A. Although it is well-established that biological sex is a critical variable in a range of physiological and disease processes, the impact of sex on HO in animal models of FOP has not been explored. We show that female FOP mice exhibit both significantly greater and more variable HO responses after muscle injury. Additionally, the incidence of spontaneous HO was significantly greater in female mice. This sex dimorphism is not dependent on gonadally derived sex hormones, and reciprocal cell transplantations indicate that apparent differences in osteogenic activity are intrinsic to the sex of the transplanted cells. By circumventing the absolute requirement for activin A using an agonist of mutant ACVR1, we show that the female-specific response to muscle injury or BMP2 implantation is dependent on activin A. These data identify sex as a critical variable in basic and pre-clinical studies of FOP.

The HIF-1α and mTOR Pathways Amplify Heterotopic Ossification.

Fibrodysplasia ossificans progressiva (FOP; MIM# 135100) is an ultra-rare congenital disorder caused by gain-of-function point mutations in the Activin receptor A type I (ACVR1, also known as ALK2) gene. FOP is characterized by episodic heterotopic ossification (HO) in skeletal muscles, tendons, ligaments, or other soft tissues that progressively causes irreversible loss of mobility. FOP mutations cause mild ligand-independent constitutive activation as well as ligand-dependent bone morphogenetic protein (BMP) pathway hypersensitivity of mutant ACVR1. BMP signaling is also a key pathway for mediating acquired HO. However, HO is a highly complex biological process involving multiple interacting signaling pathways. Among them, the hypoxia-inducible factor (HIF) and mechanistic target of rapamycin (mTOR) pathways are intimately involved in both genetic and acquired HO formation. HIF-1α inhibition or mTOR inhibition reduces HO formation in mouse models of FOP or acquired HO in part by de-amplifying the BMP pathway signaling. Here, we review the recent progress on the mechanisms of the HIF-1α and mTOR pathways in the amplification of HO lesions and discuss the future directions and strategies to translate the targeting of HIF-1α and the mTOR pathways into clinical interventions for FOP and other forms of HO.

Pay Attention to the Osteochondromas in Fibrodysplasia Ossificans Progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is an extremely rare disease characterized by malformation of the bilateral great toes and progressive heterotopic ossification. The clinical features of FOP occur due to dysfunction of the bone morphogenetic protein (BMP) signaling pathway induced by the mutant activin A type I receptor/activin-like kinase-2 (ACVR1/ALK2) which contributes to the clinical features in FOP. Dysregulation of the BMP signaling pathway causes the development of osteochondroma. Poor awareness of the association between FOP and osteochondromas always results in misdiagnosis and unnecessary invasive operation. CASE PRESENTATION: In this study, we present a case of classical FOP involving osteochondroma. An 18-year-old male adolescent, born with deformity of bilateral big toes, complained multiple masses on his back for 1 year. The mass initially emerged with a tough texture and did not cause pain. It was misdiagnosed as an osteochondroma. After two surgeries, the masses became hard and spread around the entire back region. Meanwhile, extensive heterotopic ossification was observed around the back, neck, hip, knee, ribs, and mandible during follow-up. Osteochondromas were observed around the bilateral knees. No abnormalities were observed in the laboratory blood test results. Whole exome sequencing revealed missense mutation of ACVR1/ALK2 (c.617G > A; p.R206H) in the patient and confirmed the diagnosis of FOP. CONCLUSION: In summary, classical FOP always behaves as a bilateral deformity of the big toes, as well as progressive ectopic ossification and osteochondromas in the distal femur and proximal tibia. An understanding of the association between osteochondromas and FOP aids in diagnosis and avoids unnecessary invasive management in patients.

A fibrodysplasia ossificans progressiva patient with a rare missense mutation in ACVR1 detected on 18F-FDG PET/CT.

Fibrodysplasia ossificans progressiva (FOP) is an exceedingly rare human genetic disorder characterized by the progressive and incapacitating formation of ectopic bone outside the skeleton. We report a case of FOP patient with mutations within the ACVR1 gene (c.982G>A; p.G328R). 18F-FDG positron emission tomography/computed tomography (PET/CT) was carried out for disease assessment. Previous studies have shown increased FDG uptake in regions of heterotopic ossification (HO) in FOP. However, in our study, the PET/CT features demonstrate that active ossificans exhibit increased 18F-FDG uptake, whereas end-stage ossifications do not. Collectively, 18F-FDG PET/CT emerges as a prospective approach to evaluate medication efficacy in the early stages, directing early intervention and pharmacological management of FOP before ossifications formation.

Navigating the Complex Landscape of Fibrodysplasia Ossificans Progressiva: From Current Paradigms to Therapeutic Frontiers.

Fibrodysplasia ossificans progressiva (FOP) is an enigmatic, ultra-rare genetic disorder characterized by progressive heterotopic ossification, wherein soft connective tissues undergo pathological transformation into bone structures. This incapacitating process severely limits patient mobility and poses formidable challenges for therapeutic intervention. Predominantly caused by missense mutations in the ACVR1 gene, this disorder has hitherto defied comprehensive mechanistic understanding and effective treatment paradigms. This write-up offers a comprehensive overview of the contemporary understanding of FOP's complex pathobiology, underscored by advances in molecular genetics and proteomic studies. We delve into targeted therapy, spanning genetic therapeutics, enzymatic and transcriptional modulation, stem cell therapies, and innovative immunotherapies. We also highlight the intricate complexities surrounding clinical trial design for ultra-rare disorders like FOP, addressing fundamental statistical limitations, ethical conundrums, and methodological advancements essential for the success of interventional studies. We advocate for the adoption of a multi-disciplinary approach that converges bench-to-bedside research, clinical expertise, and ethical considerations to tackle the challenges of ultra-rare diseases like FOP and comparable ultra-rare diseases. In essence, this manuscript serves a dual purpose: as a definitive scientific resource for ongoing and future FOP research and a call to action for innovative solutions to address methodological and ethical challenges that impede progress in the broader field of medical research into ultra-rare conditions.

AAV-Mediated Targeting of the Activin A-ACVR1R206H Signaling in Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by progressive disabling heterotopic ossification (HO) at extra-skeletal sites. Here, we developed adeno-associated virus (AAV)-based gene therapy that suppresses trauma-induced HO in FOP mice harboring a heterozygous allele of human ACVR1R206H (Acvr1R206H/+) while limiting the expression in non-skeletal organs such as the brain, heart, lung, liver, and kidney. AAV gene therapy carrying the combination of codon-optimized human ACVR1 (ACVR1opt) and artificial miRNAs targeting Activin A and its receptor ACVR1R206H ablated the aberrant activation of BMP-Smad1/5 signaling and the osteogenic differentiation of Acvr1R206H/+ skeletal progenitors. The local delivery of AAV gene therapy to HO-causing cells in the skeletal muscle resulted in a significant decrease in endochondral bone formation in Acvr1R206H/+ mice. These mice showed little to no expression in a major AAV-targeted organ, the liver, due to liver-abundant miR-122-mediated repression. Thus, AAV gene therapy is a promising therapeutic strategy to explore in suppressing HO in FOP.

Successful experience of tofacitinib treatment in patients with Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressive (FOP) is an ultra-rare genetic disorder that is caused by a mutation in the ACVR1 gene and provokes severe heterotopic ossification. Since flares of the disease are associated with inflammation, it is assumed that JAK inhibitors can control active FOP due to blocking multiple signaling pathways.

Reduced GS Domain Serine/Threonine Requirements of Fibrodysplasia Ossificans Progressiva Mutant Type I BMP Receptor ACVR1 in the Zebrafish.

Fibrodysplasia ossificans progressiva (FOP) is a rare human genetic condition characterized by altered skeletal development and extraskeletal bone formation. All cases of FOP are caused by mutations in the type I bone morphogenetic protein (BMP) receptor gene ACVR1 that result in overactivation of the BMP signaling pathway. Activation of the wild-type ACVR1 kinase requires assembly of a tetrameric type I and II BMP receptor complex followed by phosphorylation of the ACVR1 GS domain by type II BMP receptors. Previous studies showed that the FOP-mutant ACVR1-R206H required type II BMP receptors and presumptive glycine/serine-rich (GS) domain phosphorylation for overactive signaling. Structural modeling of the ACVR1-R206H mutant kinase domain supports the idea that FOP mutations alter the conformation of the GS domain, but it is unclear how this leads to overactive signaling. Here we show, using a developing zebrafish embryo BMP signaling assay, that the FOP-mutant receptors ACVR1-R206H and -G328R have reduced requirements for GS domain phosphorylatable sites to signal compared to wild-type ACVR1. Further, ligand-independent and ligand-dependent signaling through the FOP-mutant ACVR1 receptors have distinct GS domain phosphorylatable site requirements. ACVR1-G328R showed increased GS domain serine/threonine requirements for ligand-independent signaling compared to ACVR1-R206H, whereas it exhibited reduced serine/threonine requirements for ligand-dependent signaling. Remarkably, while ACVR1-R206H does not require the type I BMP receptor partner, Bmpr1, to signal, a ligand-dependent GS domain mutant of ACVR1-R206H could signal independently of Bmpr1 only when Bmp7 ligand was overexpressed. Of note, unlike human ACVR1-R206H, the zebrafish paralog Acvr1l-R203H does not show increased signaling activity. However, in domain-swapping studies, the human kinase domain, but not the human GS domain, was sufficient to confer overactive signaling to the Acvr1l-R203H receptor. Together these results reflect the importance of GS domain activation and kinase domain functions in regulating ACVR1 signaling and identify mechanisms of reduced regulatory constraints conferred by FOP mutations. © 2023 American Society for Bone and Mineral Research (ASBMR).

Most Fractures Treated Nonoperatively in Individuals With Fibrodysplasia Ossificans Progressiva Heal With a Paucity of Flareups, Heterotopic Ossification, and Loss of Mobility.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is an ultrarare genetic disorder with episodic and progressive heterotopic ossification. Tissue trauma is a major risk factor for flareups, heterotopic ossification (HO), and loss of mobility in patients with FOP. The International Clinical Council on FOP generally recommends avoiding surgery in patients with FOP unless the situation is life-threatening, because soft tissue injury can trigger an FOP flareup. Surprisingly little is known about flareups, HO formation, and loss of mobility after fractures of the normotopic (occurring in the normal place, distinct from heterotopic) skeleton when treated nonoperatively in patients with FOP. QUESTIONS/PURPOSES: (1) What proportion of fractures had radiographic evidence of union (defined as radiographic evidence of healing at 6 weeks) or nonunion (defined as the radiographic absence of a bridging callus at 3 years after the fracture)? (2) What proportion of patients had clinical symptoms of an FOP flareup because of the fracture (defined by increased pain or swelling at the fracture site within several days after closed immobilization)? (3) What proportion of patients with fractures had radiographic evidence of HO? (4) What proportion of patients lost movement after a fracture? METHODS: We retrospectively identified 36 patients with FOP from five continents who sustained 48 fractures of the normotopic skeleton from January 2001 to February 2021, who were treated nonoperatively, and who were followed for a minimum of 18 months after the fracture and for as long as 20 years, depending on when they sustained their fracture during the study period. Five patients (seven fractures) were excluded from the analysis to minimize cotreatment bias because these patients were enrolled in palovarotene clinical trials (NCT02190747 and NCT03312634) at the time of their fractures. Thus, we analyzed 31 patients (13 male, 18 female, median age 22 years, range 5 to 57 years) who sustained 41 fractures of the normotopic skeleton that were treated nonoperatively. Patients were analyzed at a median follow-up of 6 years (range 18 months to 20 years), and none was lost to follow-up. Clinical records for each patient were reviewed by the referring physician-author and the following data for each fracture were recorded: biological sex, ACVR1 gene pathogenic variant, age at the time of fracture, fracture mechanism, fracture location, initial treatment modality, prednisone use at the time of the fracture as indicated in the FOP Treatment Guidelines for flare prevention (2 mg/kg once daily for 4 days), patient-reported flareups (episodic inflammatory lesions of muscle and deep soft connective tissue characterized variably by swelling, escalating pain, stiffness, and immobility) after the fracture, follow-up radiographs of the fracture if available, HO formation (yes or no) as a result of the fracture determined at a minimum of 6 weeks after the fracture, and patient-reported loss of motion at least 6 months after and as long as 20 years after the fracture. Postfracture radiographs were available in 76% (31 of 41) of fractures in 25 patients and were independently reviewed by the referring physician-author and senior author for radiographic criteria of fracture healing and HO. RESULTS: Radiographic healing was noted in 97% (30 of 31) of fractures at 6 weeks after the incident fracture. Painless nonunion was noted in one patient who sustained a displaced patellar fracture and HO. In seven percent (three of 41) of fractures, patients reported increased pain or swelling at or near the fracture site within several days after fracture immobilization that likely indicated a site-specific FOP flareup. The same three patients reported a residual loss of motion 1 year after the fracture compared with their prefracture status. HO developed in 10% (three of 31) of the fractures for which follow-up radiographs were available. Patient-reported loss of motion occurred in 10% (four of 41) of fractures. Two of the four patients reported noticeable loss of motion and the other two patients reported that the joint was completely immobile (ankylosis). CONCLUSION: Most fractures treated nonoperatively in individuals with FOP healed with few flareups, little or no HO, and preservation of mobility, suggesting an uncoupling of fracture repair and HO, which are two inflammation-induced processes of endochondral ossification. These findings underscore the importance of considering nonoperative treatment for fractures in individuals with FOP. Physicians who treat fractures in patients with FOP should consult with a member of the International Clinical Council listed in the FOP Treatment Guidelines ( https://www.iccfop.org ). LEVEL OF EVIDENCE: Level IV, therapeutic study.

The serum levels of activin A and bone morphogenetic protein-4 and -6 in patients with fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is an ultrarare and disabling genetic disorder of connective tissue characterized by congenital malformation of the great toes, and progressive heterotopic ossification (HO) in soft connective tissues. A gain-of-function mutation of activin A receptor type I (ACVR1) enables ACVR1 to recognize activin A as an agonist with bone morphogenetic protein (BMP) signalling that leads to HO. Previous studies confirmed that activin A stimulates BMP signalling in vitro and drives HO in mouse models of FOP. However, the roles for BMP4 and BMP6 in FOP are supported only by correlative evidence in vitro. Thus, it remains unclear whether the circulating levels of activin A, BMP4 and BMP6 correlate with flare-ups in FOP patients. Hence, we investigated the protein levels of activin A, BMP4 and BMP6 in the serum of FOP patients. RESULTS: We recruited 16 untreated FOP patients and 16 age- and sex- matched healthy control subjects in this study. The 16 FOP patients were retrospectively divided into the flare-up group (n = 8) and remission group (n = 8) depending on whether they had flare-ups or worsening of any joint movement in the last 6 months. The serum activin A, BMP4 and BMP6 levels were detected by enzyme-linked immunosorbent assay. The serum activin A, BMP4 and BMP6 levels were slightly higher in FOP patients (median: 434.05 pg/mL, 459.48 pg/mL and 67.84 pg/mL) versus healthy control subjects (median: 364.14 pg/mL, 450.39 pg/mL and 55.36 pg/mL). However, there were no statistically significant differences between the two groups (p > 0.05 for all items), nor were there significant differences between the flare-up and remission groups of FOP (p > 0.05 for all items). Univariate and multivariate logistic regression analyses showed that age, sex, and serum activin A, BMP4 and BMP6 levels were not related to flare-up in FOP patients. CONCLUSIONS: There were no significant differences in the serum levels of activin A, BMP4 and BMP6 in FOP patients compared with healthy control subjects. Serum activin A, BMP4 and BMP6 proteins might not be the stimulators for FOP flare-up, and may not be biomarkers for FOP diagnosis.