A Novel Gain-of-Function Mutation in BMPR2 in a Patient with Variant Fibrodysplasia Ossificans Progressiva

A Novel Gain-of-Function Mutation in BMPR2 in a Patient with Variant Fibrodysplasia Ossificans Progressiva | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Novel Gain-of-Function Mutation in BMPR2 in a Patient with Variant Fibrodysplasia Ossificans Progressiva Yonghwan Kim, Tae-Joon Cho, Myung-Jin Kim, Eunyoung Jung, Dayeon Kim, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8453923/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract Heterotopic ossification (HO), a pathological process in which bone forms in soft tissues is rare and debilitating without effective treatment. Gain-of-function mutations in ACVR1 cause fibrodysplasia ossificans progressiva (FOP). Here we report a novel, ultrarare gain of function mutation in BMPR2 (c.1126G > A, p.E376K) that causes a systemic HO simulating FOP. The pathological features associated with BMPR2 E376K appear reminiscent of classic FOP, yet manifest a number of distinct hallmarks, including lack of stereotypic malformation of the big toes. BMPR2 E376K appears to function as a neomorph, displaying an exaggerated response to Activin A stimulation by selectively interacting with ACVR1. These findings are consistent with the central role of Activin A mediated ACVR1 signaling in FOP. Taken together, our data illustrates the complex molecular features underlying the pathophysiology of HO and highlight the importance of BMPR2 as a nexus for ACVR1 and Activin A interaction. Moreover, our findings provide a theoretical framework for developing novel therapeutic options for HO. Health sciences/Pathogenesis Biological sciences/Physiology/Bone Gain-of-function BMPR2 Heterotopic ossification Fibrodysplasia Ossificans Progressiva Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Heterotopic ossification (HO) is a pathological process characterized by the formation of bone tissue in non-skeletal regions, such as muscles, tendons, or other soft tissues 1 – 3 . While HO can occur following traumatic injuries, burns, or surgical interventions, it is also associated with rare genetic disorders 4 . HO represents a significant clinical challenge, often leading to pain, restricted joint movement, and in severe cases, complete immobilization. Despite its debilitating effects, the underlying mechanisms that govern this abnormal bone formation remain incompletely understood, particularly in the context of genetic predispositions. One of the most extensively studied diseases characterized by hereditary HO is fibrodysplasia ossificans progressiva (FOP, MIM #135100). FOP is an ultra-rare and devastating genetic disorder driven by gain-of-function mutations in the activin A type I receptor ( ACVR1) gene 1 , 5 – 7 . Approximately 97% of affected individuals have classic FOP defined as characteristic malformations of the great toes, progressive HO and a recurrent ACVR1 (R206H) mutation 7 – 9 . The remaining ~ 3% have variant FOP, which is defined by either atypical or normal great toes, progressive HO, and is driven by other gain-of-function mutations in ACVR1 7 . To date, all individuals with classic or variant FOP have gain-of-function mutations in ACVR1 gene 10 . These mutations are known to dysregulate bone morphogenetic protein (BMP) signaling 11 – 14 . ACVR1 functions as a BMP type I receptor, forming a heterotetrameric complex with type II receptors upon ligand binding. This complex phosphorylates receptor-regulated SMAD proteins (SMAD1/5/9), which translocate to the nucleus to regulate the expression of target genes involved in endochondral bone formation 15 – 17 . The R206H mutation in ACVR1 leads to a gain-of-function alteration, resulting in activation of the receptor. Consequently, the mutant receptor exhibits increased basal activity and heightened sensitivity to BMP ligands, leading to excessive phosphorylation of SMAD1/5/8, upregulation of osteogenic target genes and aberrant BMP pathway signaling. One of the pivotal mediators of heterotopic bone formation in FOP is Activin A, a member of the TGF-β superfamily. Under normal physiological conditions, Activin A acts as an antagonist to BMP signaling through the formation of non-signaling complex (NSC) with ACVR1 receptor 18 , 19 . It was proposed that Activin A dampens BMP signaling by forming a complex with ACVR1 and a type II receptor, both of which are required for initiation of BMP pathway signaling 19 . However, in FOP patients harboring the ACVR1 R206H mutation, Activin A paradoxically activates the mutant receptor, amplifying BMP signaling pathways 20 – 22 . While the precise molecular mechanisms underlying Activin A-mediated activation of BMP signaling remain largely unresolved, significant progress has been made using cell and mouse models 11 , 20 , 21 . Studies have demonstrated that inhibition of Activin A with a neutralizing antibody blocks the formation of new heterotopic bone lesions, halts the growth and even reduces the size of pre-existing actively growing lesions, underscoring its essential role in driving HO 23,24 . Furthermore, the depletion or deletion of ACVR2A and ACVR2B markedly diminishes BMP signaling, highlighting their critical role in the neofunction of ACVR1 R206H 18,19,25 . Inflammation is another central driver of HO in FOP. Localized inflammatory episodes, whether triggered by trauma, infection, or spontaneous flare-ups, create a pro-inflammatory microenvironment enriched with cytokines, growth factors, and immune cells 26 , 27 . Notably, Activin A is an indispensable ligand for the initiation of HO in FOP, and activation of ACVR1 R206H by Activin A is required to drive the pathological ossification process 22 . Fibro/adipogenic progenitors (FAPs) play a critical role in the pathophysiology of FOP by driving enforced differentiation into chondrocytes, a pivotal step in endochondral HO 11,28,29 . In FOP, mutations in the ACVR1 gene lead to aberrant activation of BMP pathway signaling, particularly in response to Activin A, thereby skewing FAPs differentiation toward osteogenic and chondrogenic lineages. This enforced differentiation underpins the endochondral HO characteristic of FOP and validates the pathogenic role of the causative genetic alteration. Animal models, such as the Acvr1 R206H knock-in mouse line, have demonstrated that FAPs contribute significantly to HO through their osteochondrogenic potential, offering insights into disease mechanisms and potential therapeutic targets 11 . Understanding the interplay between FAPs differentiation and the pathological microenvironment in FOP is crucial for elucidating the molecular basis of HO and for developing effective treatments to mitigate disease progression 30 . Bone morphogenetic protein receptor type 2 (BMPR2) is a critical player in the BMP signaling pathway, which regulates a range of cellular processes, including growth, differentiation, and apoptosis 31 . Mutations in the BMPR2 gene have been extensively studied in pulmonary arterial hypertension (PAH), a severe vascular disorder characterized by progressive narrowing of pulmonary arteries, ultimately leading to right heart failure. Most cases of PAH linked to BMPR2 mutations involve loss-of-function variants, which disrupt normal BMP signaling 32 , 33 . Consequently, endothelial cell dysfunction, excessive proliferation of smooth muscle cells, and resistance to apoptosis drive vascular remodeling central to PAH pathogenesis. However, to dates, no known human disorders are due to the gain-of-function mutation of BMPR2 gene. In this study, we present a unique case of a patient with a phenotypic variant of FOP, characterized by progressive manifestation of systemic HO but normal great toes, who lacks mutations in the commonly implicated ACVR1 and GNAS genes 1 . Through whole exome sequencing, we identified a novel gain-of-function mutation in the BMPR2 gene, which encodes a type II receptor integral to the BMP signaling pathway and functionally validated the pathogenesis of the causative mutation using multiple cell types including patient-derived dermal fibroblasts. This is the first report of a BMPR2 gain-of-function mutation in variant FOP, providing critical new insights into genetic diversity underlying this rare disorder. While the ACVR1 R206H mutation remains the most prevalent genetic driver of FOP, these findings highlight the need to broaden the genetic landscape of the disease, as other mutations within the BMP signaling pathway may also shape its pathophysiology. These findings expand the mechanistic understanding of FOP at the molecular level, providing new avenues for research into the interplay of Activin A, inflammation, and BMP pathway signaling in disease pathogenesis. RESULTS Identification of a gain-of-function BMPR2 mutation in a patient presented with FOP phenotype A 16-year-old boy presented with flare-ups of the left pectoral region after treatment for dental caries. The patient was a product of a full-term pregnancy and normal vaginal delivery to a healthy Korean couple. Birth weight was 2.98 kg. No perinatal problems were encountered. Motor and cognitive development were normal. The patient recovered uneventfully from laparotomy for pyloric stenosis in childhood. Subcutaneous migrating nodules were noted over the scalp at age 2 and over the posterior neck at age 4. Flare-ups and stiffness of the neck and back developed at age 6 and progressed to the limbs. At age 19, he developed severe dizziness. Magnetic resonance imaging of the brain revealed multifocal gadolinium-enhanced tumors involving the suprasellar area, septum pellucidum, and medulla oblongata that were diagnosed as mixed germ cell tumors because the serum beta-hCG level was moderately elevated. Following radiation therapy, the tumors disappeared, and the patient has remained in complete remission for 3 years but developed diabetes insipidus. Physical examination revealed a Cumulative Analogue Joint Involvement Scale (CAJIS) score of 20/30 34 . The neck, back, both shoulders, elbows, hips, and the left knee were functionally ankylosed. The feet and toes appeared normal. At age 22, the patient’s height was 145 cm (z < -4) and weight was 57 kg. The neck, back, both shoulders, hips, and the left knee were ankylosed. Both elbows maintained only 10 to 30 degrees of flexion-extension motion, and the right knee maintained 80 degrees of motion. Radiographic examination revealed extensive HO in the back, periscapular, peripelvic regions and thighs (Fig. 1 a). Ankylosis of the posterior column of the cervical vertebrae was noted. Unlike in patients with classic FOP, the individual had normal great toes (Fig. 1 a). The patient had progressive HO in characteristic anatomic patterns with normal great toes and was diagnosed as having a clinical variant of FOP. The clinical manifestations were fully consistent with variant FOP based on the normal great toes 7 and progressive HO in characteristic anatomic patterns 7 (Fig. 1 a). However, full sequencing of the coding region of ACVR1 (MIM #102576) of the proband showed two silent sequence variations, both of which were previously reported in the normal population: c.270C > T (NCBI dbSNP rs2227861) and c.690G > A (rs1146031). The patient exhibited no clinical manifestations consistent with Progressive Osseous Heteroplasia (POH), and no mutations were identified in the GNAS gene, which is recognized as the causative gene for POH 1 . In order to find causative mutations for the disorder, we performed whole exome sequencing on the genomic DNA from the proband and identified a heterozygous mutation in BMPR2 (MIM #600799), c.1126G > A (p.E376K). Sanger sequencing and restriction enzyme digestion (data not shown) confirmed the sequence variation in the proband and absence in the genome of the parents and the sibling (Fig. 1 b, c). The mutation of the BMPR2 E376K variant is located in the kinase domain 33 (Fig. 1 d) where the sequence is highly conserved among species (Fig. 1 e). The BMPR2 E376K sequence variation was not found in the general populations of the 100 genome, ExAC, dbSNP, or Exome Sequencing Project databases. Hyper-activation of BMP signaling in patient-derived dermal fibroblasts To gain insight into the molecular basis underlying disease phenotype, we examined the molecular consequences of the BMPR2 E376K variant by performing immunoblot analysis on lysates prepared from dermal fibroblasts obtained from the patient’s skin and a normal control (BJ cells). As shown in Fig. 1 f (left panel), lysates from the patient-derived cells showed robust phosphorylation of SMAD1/5/9, and resultant increased expression of ID1 and ID3 35–40 , which were not observed in the lysates prepared from the normal control cells (Fig. 1 f). Notably, we also observed phosphorylation of SMAD2 in the patient-derived cells, although SMAD2 phosphorylation is not typically associated with BMP pathway signaling. In addition, when cultured in differentiation media, patient-derived fibroblasts expressed bone-associated proteins 41 , including osteocalcin (OCN), alkaline phosphatase (ALP), and RUNX2, even in the absence of exogenous BMP ligands (Fig. 1 f, right panel). These results strongly suggest that BMPR2 E376K induces hyper-activation of BMP pathway signaling potentially due to the gain-of-function mutation. At the cellular level, the patient-derived cells were positive for the alkaline phosphatase (ALP) staining and ALP activity 11 while normal control cells barely expressed ALP (Fig. 1 g). Similarly, alizarin red S staining revealed significant calcium accumulation 42 in the patient-derived cell cultures after 21 days (Fig. 1 h). Taken together, these findings provide compelling evidence that BMPR2 E376K drives hyper-activation of BMP pathway, promoting osteogenic differentiation. Functional validation of pathogenicity of the BMPR2 variant Functional validation of pathogenicity of the potential causative gene mutation is critical for establishing its role in disease pathogenesis. As DNA sequence analysis showed heterozygous BMPR2 mutation resulting in the FOP like phenotype, we assumed that the mutation is autosomal dominant. We therefore hypothesized that deletion of the BMPR2 E376K allele would normalize the increased BMP signaling. To test this, we deleted the BMPR2 E376K allele using CRISPR-Cas9 in the patient-derived cells. Simultaneously, we reverted the c.1126G > A variant back to the wild-type (WT) sequence using CRISPR-Cas9 knock-in methods. Multiple single clones were isolated and the sequence of the BMPR2 gene in individual clones was determined by a MiSeq system (Fig. 2 a and Fig. S1 ). From the gene editing experiments, we successfully obtained allele specific knock-out (KO) and knock-in (KI) patient-derived fibroblast clones. These modified clones exhibited a loss of SMAD1/5/9 phosphorylation (Fig. 2 b), as well as a significant reduction in ALP staining, and calcium deposition (Fig. 2 c, d), strongly demonstrating that BMPR2 E376K is a gain-of-function mutation causing BMP pathway activation. To further validate the functional dominance of BMPR2 E376K , we assessed whether heterotopic expression of the mutant allele could recapitulate the molecular and cellular changes observed in patient-derived cells. To test this, HEK293T cells were transfected with an empty vector, BMPR2 WT , or BMPR2 E376K (Fig. 2 e, left panel). Only cells expressing BMPR2 E376K led to hyperphosphorylation of SMAD1/5/9 and expression of ID1 and ID3. In addition, to test if the enhanced BMP signaling due to the expression of BMPR2 E376K results in BMP responsive gene expression, we established a transcriptional reporter assay system in which luciferase expression is controlled by BMP-responsive elements 43 . Consistent with the gain-of-function hypothesis, cells expressing BMPR2 E376K showed significantly elevated luciferase activity (Fig. 2 f, left panel). In the same experimental setting, similar results were obtained when we expressed the classic ACVR1 R206H mutant in HEK293T cells (Fig. 2 e, f, right panels). Collectively, these in vitro experiments highlight the critical gain-of-function properties of the BMPR2 E376K variant and its role in hyperactivating BMP signaling. Enhanced chondrogenic differentiation of MSCs expressing BMPR2 variant HO in FOP lesions involves chondrogenic differentiation from fibroadipogenic (FAP) progenitor cells due to enhanced BMP signaling 41 , 44 . As BMPR2 E376K stimulates BMP pathway signaling in the absence of BMP ligands, we hypothesized that BMPR2 E376K might stimulate mesenchymal cells to become chondrocytes. As a test of this concept, we first generated induced pluripotent stem cells from the normal control dermal fibroblasts (BJ cells), the patient-derived cells and the restored KI patient-derived cells (#203) (Fig. S2). Then these iPSCs were partially differentiated into MSCs (Fig. S3). Consistent with previous findings, Western blot analysis showed that only the MSC from the patient-derived cells were positive for BMP signaling, indicated by the robust phosphorylation of SMAD1/5/9 and increased expression of ID1 and ID3 (Fig. 3 a). At the cellular level, only the MSC from the patient derived cells were positive for alcian blue staining (Fig. 3 b) and alizarin S staining (Fig. 3 c), which was not the case of the MSC from normal control cells and knock-in patient-derived cells. These findings demonstrated that expression of BMPR2 E376K results in enforced MSC differentiation to chondrocytes, which later undergo ossification during the endochondral ossification. We additionally confirmed that the expression of osteogenic genes, including ALP and OCN, was significantly increased compared to the normal control and knock-in patient-derived cells (Fig. 3 d), indicating that BMPR2 E376K expression leads to the activation of BMP signaling. To rule out the possibility of additional unidentified genetic alteration responsible for enhanced BMP pathway signaling in the patient-derived cells, we individually expressed an empty vector, BMPR2 WT , or BMPR2 E376K in the mouse MSC cell line C3H10T1/2 45 . As shown in Fig. 4 a, we found that temporal expression of BMPR2 E376K led to phosphorylation of SMAD1/5/9 and expression of its downstream effectors including ID1, ID3, and SOX9. In addition, C3H10T1/2 cells expressing BMPR2 E376K exhibited a chondrocytic phenotype, based on the Alcian blue staining 46 (Fig. 4 b). Indeed, later in culture, expression of BMPR2 E376K showed enhanced expression of COL2A1, COL10A1, and Aggrecan, all of which are highly expressed in osteoblasts 47 (Fig. 4 c). Taken together, these findings suggest that BMPR2 E376K is a gain-of-function mutation and is likely causative for the enhanced BMP signaling and HO phenotype in our patient. Mechanistic insights into the pathogenicity of the BMPR2 variant BMP signaling is typically activated in the presence of BMP ligands, which leads to engagement of type I and type II receptors 48 . Given that BMPR2 E376K showed hyper-activation of BMP signaling, we hypothesized that the mutant receptor might engage with the type I receptor ACVR1 WT even in the absence of BMP ligands. To test this, we transiently co-expressed V5-tagged ACVR1 WT with either wild-type (WT) or mutant (E376K) HA-tagged BMPR2 in HEK293T cells. Surprisingly, we found that ACVR1 WT co-immunoprecipitated with BMPR2 E376K , whereas BMPR2 WT did not associate with ACVR1 WT (Fig. 5 a). These results suggest that the BMPR2 E376K variant promotes ligand-independent oligomerization with ACVR1 WT , providing a molecular explanation for its ability to activate BMP signaling in a dysregulated manner. Additionally, combined expression of ACVR1 R206H and BMPR2 E376K showed additive effects of SMAD1/5/9 phosphorylation (Fig. 5 b), indicating that BMPR2 E376K activates BMP signaling through a distinct molecular mechanism compared to ACVR1 R206H -mediated BMP activation. Interestingly, we observed enhanced SMAD2 phosphorylation in the patient-derived cells (Fig. 1 f). Consistently, as shown in the Fig. 6 c, HEK293T cells expressing BMPR2 E376K , showed robust SMAD2 phosphorylation, which was not detected in cells expressing the ACVR1 R206H variant or in cells expressing BMPR2 WT (Fig. 5 c). By performing real-time quantitative PCR, we further confirmed that the expression of downstream genes 49 – 51 of SMAD2 was increased (Fig. S4), implying that SMAD2 phosphorylation is because of the presence of BMPR2 E376K . To identify the type I receptor potentially interacting with BMPR2 E376K and driving SMAD2 phosphorylation 52 , we individually depleted seven type I receptors in patient-derived cells. Notably, depletion of TGFβR1 completely abrogated SMAD2 phosphorylation in patient-derived cells (Fig. 5 d) as well as in other cells stably expressing BMPR2 E376K (Fig. S5a, b). These findings implicate TGFβR1 as the key type I receptor mediating SMAD2 activation in the context of BMPR2 E376K . To examine the role of SMAD2 activation in cells expressing BMPR2 E376K , we used mouse myogenic C2C12 cells 53 , 54 . Through lentiviral transduction, we established C2C12 cell lines which stably express empty vector, human BMPR2 WT , or BMPR2 E376K . As expected, SMAD1/5/9 phosphorylation and its downstream target ID 35 were detected only in the cells expressing BMPR2 E376K (Fig. 5 e). Consistently, cells expressing BMPR2 E376K were positive for ALP staining and activity indicating osteogenic differentiation (Fig. S6a, b). Addition of BMP2 or BMP4 further increased ALP expression in both BMPR2 WT and BMPR2 E376K backgrounds (Fig. S6a, b). Importantly, we found that treatment with dorsomorphin, a potent inhibitor of BMP pathway signaling 55 (Fig. 5 f), demonstrating that the BMP signaling plays a central role in BMPR2 E376K -mediated effects. Interestingly, we observed that inhibition of SMAD2-mediated TGF-β signaling using SB431542, a potent inhibitor TGF-β pathway signaling 56 , also reduced the ALP staining, albeit to a lesser extent (Fig. 5 g), suggesting a possibility that BMPR2 E376K -dependent SMAD2 signaling might be partially involved in the processes of accelerated chondro-osseous differentiation. Together, these results indicate a complex interplay between BMP and TGF-β signaling pathways in the development of HO driven by BMPR2 E376K . BMPR2 E376K exhibits enhanced responsiveness to Activin A Activin A has been demonstrated as a culprit ligand for the HO phenotype in the context of ACVR1 R206H variant, although Activin A functions as an antagonist of signaling mediated by wildtype ACVR1 23,24 . It was proposed that Activin A induces clustering or dimerization of ACVR1 R206H , which enhances autophosphorylation of ACVR1 R206H25 . On the other hand, when wild-type ACVR1 and its associated type II receptors are engaged by Activin A, it results in the formation of non-signaling complexes that sequester ACVR1 and the type II receptors, rendering them unavailable for BMP signaling 19 . Given that the BMPR2 E376K variant drives enhanced BMP signaling and an FOP-like phenotype, we hypothesized that Activin A might similarly turn on BMP signaling in cells expressing BMPR2 E376K . Remarkably, similar to ACVR1 R206H 20,21 (Fig. 6 a), Activin A treatment significantly increased SMAD1/5/9 phosphorylation in cells expressing BMPR2 E376K , but not in cells transfected with an empty vector or BMPR2 WT (Fig. 6 b). These findings suggest that Activin A plays a pivotal role in driving the HO phenotype in our patient with the BMPR2 E376K variant, mirroring its role in patients with the ACVR1 R206H variant. Consistently, neutralization of Activin A or BMP ligands using ACVR2A-Fc, ACVR2B-Fc, or follistatin, each of which effectively sequesters Activin A or BMPs, substantially reduced SMAD1/5/9 phosphorylation in BMPR2 E376K -expressing cells (Fig. 6 c). These results further support the neofunction of BMPR2 E376K as a driver of enhanced responsiveness to Activin A and underscore its critical role in the pathogenesis of HO. The discovery of BMPR2 E376K , and its ability to enable wildtype ACVR1 to respond to Activin A, similar to ACVR1 R206H , provides an additional tool to investigate the molecular mechanisms by which these FOP-causing variants convert their complexes with Activin A and partner type II receptors into signaling complexes. DISCUSSION To date, ACVR1 is the only known gene responsible for FOP 43 , 57 . In this study, we report that a novel gain-of-function mutation in the BMPR2 gene causes non-classic FOP. We showed that the BMPR2 E376K is a neofunction variant much like the FOP-causing variant of ACVR1 R206H and may not only be neoresponsive to Activin A in association with wildtype ACVR1 but also be implicated in SMAD2/3 phosphorylation. This discovery introduces a previously unreported mechanism of HO involving a type II receptor in the BMP signaling pathway. While FOP has historically been linked to gain-of-function mutations in the ACVR1 gene, most notably the ACVR1 R206H variant present in over 97% of cases, our findings expand the spectrum of genetic contributors to FOP-like HO phenotypes and highlight potential distinctions and overlaps in molecular mechanisms. Unlike classic FOP cases, the patient in our study presented with a variant phenotype characterized by the absence of the typical great toe malformation. Cellular and biochemical analysis demonstrated Activin-A responsive activation of BMP signaling in patient-derived cells expressing BMPR2 E376K . Functional assays validated the pathogenicity of this mutation, showing its significant role in dysregulated signaling. Specifically, BMPR2 E376K enhanced mesenchymal stem cell (MSC) differentiation into chondrocytes, a precursor to endochondral ossification. These findings provide a mechanistic explanation for HO observed in the patient and shed light on the broader implications of dysregulated BMP signaling. In addition to its canonical BMP pathway effects, BMPR2 E376K exhibited novel properties, including the activation of SMAD2 phosphorylation and downstream gene expression. Further investigation revealed that TGFβR1 is implicated in the SMAD2 signaling. This noncanonical signaling was found to contribute to accelerated chondro-osseous differentiation, aligning with prior evidence implicating TGFβ signaling in FOP phenotypes 58 . These findings collectively underscore the dual contributions of BMP and TGFβ pathways in the pathogenesis of this disorder, advancing our understanding of both HO and TGFβ/BMP pathway regulation. Loss-of-function mutations in BMPR2 are well-established as key contributors to pulmonary arterial hypertension (PAH) 33 , 59 . It was proposed that loss of BMPR2 functions results in enhanced TGF-β signaling cascades, leading to hyperproliferation of smooth muscle cells in the pulmonary vasculature, although the exact molecular mechanisms underlying PAH remain elusive 59 . In contrast, here we report the first gain-of-function mutation in BMPR2 , BMPR2 E376K , which we demonstrate as a causative factor for a disorder phenotypically resembling FOP. This mutation drives increased activation of BMP signaling and promotes chondro-osseous differentiation, establishing a clear link between BMPR2 gain-of-function activity and the heterotopic bone formation observed in our patient. The identification of BMPR2 E376K provides an important counterpart to the established loss-of-function mutations, offering new insights into the dual roles of BMPR2 in human disease. Together, these findings might shed light on the understanding of physiological functions BMPR2, highlighting its critical role in maintaining the balance between BMP and TGF-β signaling pathways. By studying both gain- and loss-of-function mutations, we can better understand the molecular basis of conditions like PAH and FOP, paving the way for novel therapeutic strategies to modulate BMP pathway signaling in diverse contexts. BMPR2 is a constitutively active receptor capable of phosphorylating type I receptors such as ACVR1 15 . We demonstrate that the BMPR2 E376K variant exhibits an enhanced ability to associate with ACVR1, increasing the proximity between type I and type II BMP receptors on the cell membrane of mesenchymal stem cells, even in the absence of BMP ligands. This ligand-independent interaction likely enables BMP pathway signaling through ligands such as Activin A that normally do not activate BMPR2 and ACVR1. Interestingly, when BMPR2 E376K and ACVR1 R206H were co-expressed, we observed additive effects on BMP signaling, suggesting that these mutations operate through distinct molecular mechanisms. This distinction highlights a fundamentally different mechanism by which BMPR2 E376K drives aberrant signaling. Our findings will be informative for understanding the pathophysiology of FOP that arises from mutant type I or mutant type II receptors, and for developing drugs that inhibit dysregulated BMP pathway signaling for FOP and other BMP pathway-related diseases. It has been demonstrated that Activin A, which antagonizes BMP signaling in wild-type ACVR1, paradoxically acts as an agonist in the presence of gain-of-function mutations such as ACVR1 R206H . Our findings extend this paradigm to BMPR2 E376K , demonstrating that Activin A turns on BMP signaling in cells harboring the mutant receptor. This observation underscores the intricate interplay between type I and type II BMP receptors in ligand-induced signaling and highlights a potential neofunction of those receptors in driving pathological processes. In wild-type conditions, Activin A antagonizes BMP signaling by forming non-signaling complexes with ACVR1 and its associated type II receptors, ACVR2A and ACVR2B. In contrast, it has been shown that in the presence of the ACVR1 R206H variant, Activin A facilitates receptor dimerization, leading to autoactivation of ACVR1 R206H and the BMP signaling 25 . Further studies have demonstrated that the ACVR1 R206H variant clusters with ACVR2A or ACVR2B in the presence of Activin A, enhancing self-activation of the BMP pathway 25 . Interestingly, BMPR2, a type II receptor, is dispensable for Activin A-mediated BMP signaling in the ACVR1 R206H background. Our study, however, reveals that BMPR2 E376K is also responsive to Activin A. Using embryonic stem cell models, we demonstrated that both ACVR2A and ACVR2B are somehow implicated in BMPR2 E376K -mediated BMP signaling, suggesting that these type II receptors play a role in the mutant receptor's activation. These results suggest that the mechanisms by which Activin A turn on BMP SMAD1/5/9 signaling in mutant receptor backgrounds, such as ACVR1 R206H and BMPR2 E376K , may share general principles involving specific interactions between type I and type II receptors. Further studies will be required to delineate the precise molecular mechanism underlying these interactions. Nevertheless, our findings contribute to a deeper understanding of Activin A’s functional roles in HO and its relevance in driving pathological BMP signaling. The identification of BMPR2 E376K as a neomorphic mutation opens new avenues for understanding the molecular mechanisms underlying HO. While our in vitro assays have provided compelling evidence of these effects, future validation in animal models will be crucial to confirm the causal relationship between BMPR2 mutations and heterotopic ossification. Taken together, this study highlights the pathogenic role of a novel BMPR2 mutation in heterotopic ossification through enhanced chondrogenic differentiation. The findings expand our understanding of BMP signaling dysregulation and its role in skeletal disorders. Future studies should aim to explore the molecular mechanisms in greater detail and evaluate potential therapeutic strategies to mitigate the clinical manifestations of this disorder. Materials and methods Study design The goal of this study was to identify novel causative variants associated with FOP, to validate pathogenicity of the variant and to understand molecular basis of dominant negative activities of the gain-of-function mutation. Eventually, we wanted to understand pathophysiology of the human skeletal disorder, FOP, caused by genetic alterations. In order to identify causative mutations, we conducted whole exome sequencing of the individual presented with variant FOP phenotypes. Using the dermal fibroblasts obtained from the affected individual, we determined significantly enhanced BMP signaling at the molecular and cellular levels. To validate the pathogenicity of the BMPR2 E376K variant, the mutated allele was deleted through CRISPR/Cas9 mediated silencing from the patient-derived cells, which results in abrogation of the enhanced BMP signaling. Consistently, heterotopic expression of the BMPR2 E376K induced ligand-independent SMAD1/5/9 phosphorylation and chondro-osseus differentiation. In attempt to understand the molecular basis of the dominant negative activities of the BMPR2 E376K variant, we performed immunoprecipitation assay and found that the BMPR2 E376K is associated with wildtype ACVR1, leading to activation of ligand-independent BMP signaling. We also found that combined expression of causative FOP variants ACVR1 R206H and BMPR2 E376K showed additive effects of enhanced BMP signaling, implying that ACVR1 R206H might be different molecular mechanism for activation of BMP signaling. However, similar to the case of ACVR1 R206H , treatment of Activin A is only responsive to BMPR2 E376K , but not to wildtype BMPR2, suggesting that Activin A plays an important role in dysregulation of BMP signaling and FOP phenotypes. Interestingly, we noticed that SMAD2 is phosphorylated in cells expressing BMPR2 E376K . In order to determine the responsive type I receptor for SMAD phosphorylation, we depleted an individual type I receptor in cells stably expressing BMPR2 E376K and found that TGFβR1 is responsible for SMAD2 phosphorylation in the context of BMPR2 E376K variant. We further confirmed that both BMP and TGFβ signals are important for FOP phenotypes. Dermal fibroblasts and genomic DNA samples were obtained from the individual with variant FOP phenotypes and his family members. The institutional review boards of the Seoul National University Hospital, Seoul, Republic of Korea approved the studies. Detailed experimental design, statistics, and methods are provided in the main text and Materials and methods. Subjects Genomic DNA was obtained from the proband, the sibling and his parents, and dermal fibroblast cells were derived from the proband, after obtaining written informed consent. The institutional Review Board of the Seoul National University Hospital, Seoul, South Korea, approved this study. Cell Culture, DNA construction, Mutagenesis and FOP cell line establishment Patient-derived dermal fibroblasts and BJ (wild type control) cells were grown in high-glucose and no-glutamine DMEM (GIBCO, Cat#10313) supplemented with 15% fetal bovine serum (FBS, GIBCO), Glutamax™ (GIBCO, Cat#35050-061) and non-essential amino acid (GIBCO, Cat#11140-050) and penicillin and streptomycin (GIBCO, 15140-122). Fibroblasts were incubated in 5% CO 2 and 3% O 2 at 37℃. BJ foreskin fibroblasts were obtained from ATCC. HEK293T, HeLa and U2OS cells were grown in high-glucose DMEM (GIBCO, Cat#11965) supplemented with 10% FBS (GIBCO) and 1X penicillin and streptomycin (GIBCO, Cat#15140-122) and incubated in 5% CO 2 at 37℃. C2C12 myoblasts were cultured in high-glucose, glutamine and sodium pyruvate DMEM (GIBCO, Cat#11995) supplemented with 10% FBS and 1X penicillin and streptomycin at 37℃ in 5% CO 2 humidified atmosphere 60 . Undifferentiated C2C12 cells were sparsely maintained in a polystyrene cell culture dish to prevent myogenesis induced by cell contact. C3H10T1/2 fibroblasts were cultured in high-glucose, glutamine and sodium pyruvate DMEM (GIBCO, Cat#11995) supplemented with 10% FBS and 1X penicillin and streptomycin and were incubated in 5% CO 2 at 37℃ 61 . Primary patient-derived fibroblasts and BJ cells were immortalized by expressing the catalytic subunit of human telomerase (hTERT) through lentiviral transduction and transformed by the human papilloma virus E6 and E7 protein through retroviral transduction. BMPR2 cDNA was obtained from addgene. BMPR2 cDNA was cloned to EcoRI restriction sites in pcDNA6/V5-HisABC vector using In-Fusion HD Cloning kits (Takara, Cat#638920) and pDONR223 BP vector and later pHAGE-HA-FLAG LR vector using Gateway cloning system (Thermo Fisher Scientific). C.1126G > A BMPR2 mutation was generated by QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Genomics) with the following primer: BMPR2-F: 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACTTCCTCGCTGCAGCGGC-3’, BMPR2-R: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAGACAGTTCATTCC-3’. Cell lines stably expressing BMPR2 or BMPR2 mutants were generated by lentiviral transduction as previously 62 . Antibodies Antibodies used in this study are listed below with their respective working concentration. Mouse anti-V5-Tag (Invitrogen, #R960-25, 1:1000), rabbit anti-phospho-SMAD1/5/9 (Cell Signaling Technology, #13820, 1:1000), rabbit anti–p-Smad1/5/8 [clone 41D10], CST, #9516, 1:1000), rabbit anti-SMAD1 (CST, #6944, 1:2000), rabbit anti-SMAD5 (CST, #12534, 1:2000), rabbit anti-phospho-SMAD2 (CST, #3108, 1:1000), rabbit anti-SMAD2 (CST, #5339, 1:1000), rabbit anti-SMAD4 (CST, #9515, 1:1000), rabbit anti-ID1 (SANTA CRUZ BIOTECHNOLOGY, sc-488, 1:1000), rabbit anti-ID3 (SCBT, sc-490, 1:1000), mouse anti-HA (Covance, MMS-101R, 1:1000), rabbit anti-SOX9 (CST, #82630, 1:1000), rabbit anti-BMPR2 (CST, #6979, 1:1000), rabbit anti-Osteocalcin (Merck Millipore, AB10911, 1:1000), rabbit anti-Alkaline Phosphatase (abcam, ab108337, 1:1000), rabbit anti-RUNX2 (SCBT, sc-10758, 1:1000), mouse anti–β-actin [clone 8H10D10], CST, #3700, 1:5000) and rabbit anti-GAPDH (SCBT, sc-25778, 1:1000) as a loading control. Anti-mouse secondary (Jackson ImmunoResearch, 115-035-003, 1:2500), anti-rabbit secondary (Jackson ImmunoResearch, 111-035-003, 1:2500), and anti-mouse secondary light chain specific (Jackson ImmunoResearch, 115-035-174, 1:2500), and anti-rabbit IgG, horseradish peroxidase–conjugated secondary antibody (CST, #7074, 1:5000). Osteogenic differentiation BJ and patient-derived dermal fibroblasts (8x10 4 cells/well) were seeded into 24-well cell culture plates and then cultured in DMEM (GIBCO, Cat#10313) supplemented with 15% FBS, 1% Glutamax, 1% non-essential amino acid and 1% penicillin-streptomycin at 37℃ with 5% CO 2 and 3% O 2 . To induce differentiation, growth medium was replaced into DMEM supplemented with 2% FBS after cells reached 80 ~ 90% confluence. Cells were maintained without or with recombinant human BMP2 or BMP4 (R&D SYSTEMS) and replaced with fresh medium every 2 ~ 3 days for 2 ~ 21 days. C2C12 cells were seeded into 24-well cell culture plates at a density of 4x10 4 cells/well. Cells were grown in DMEM (GIBCO, Cat#11995) supplemented with 10% FBS at 37℃ with 5% CO 2 . Cells with 80 ~ 90% confluence were replaced by osteogenic differentiation DMEM (GIBCO, Cat#11995) containing 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 µM ascorbic acid-2-phosphate (all from Sigma) supplemented with 2% FBS 63 . C2C12 were treated with BMP2, BMP4, dorsomorphin (Sigma), or SB431542 (Sigma) and maintained with replacement of fresh medium every 2 ~ 3 days for 3 ~ 21 days. Chondrogenic differentiation For chondrogenesis, C3H10T1/2 cells were cultured by micromass technique, high density dot culture. First, cells were resuspended in DMEM supplemented with 10% FBS and 1X penicillin-streptomycin at a concentration of 10 7 cells/ml and a 10 µl droplet of the cell suspension was placed in the center of a well of 12-well cell culture plates followed by incubation at 37℃ and 5% CO 2 . After 2 hours, 1 ml chondrogenic differentiation medium DMEM/F12 (GIBCO, Cat#11320) consisting of 1% FBS, 1% Insulin-Transferrin-Selenium (GIBCO), 0.1 µM dexamethasone, 0.17 mM ascorbic acid-2-phosphate, 0.35 mM proline (Sigma), and 0.15% glucose (Sigma) was added in each well and cells were maintained without or with human recombinant BMP2. The fresh medium was changed once per 2 ~ 3 days for 21 days. Whole exome sequencing and DNA analysis Written informed consent was obtained from the affected individual. The Institutional Review Board of the Seoul National University Hospital, Seoul, South Korea approved the studies. Genomic DNAs were extracted from whole blood and sequencing libraries were prepared using Twist modular library preparation kits. We used SureSelect Human All Exon V5 baits covering all exon regions (Agilent, Santa Clara, CA). Targeted sequencing was performed with 101 base pair (bp) paired-end reads on an Illumina HiSeq2500 platform (Illumina, San Diego, CA). Sequenced reads were aligned to human genome reference sequence (hg19) using Burrows-Wheeler Aligner (BWA) version 0.7.5a with the Maximum Entropy Method (MEM) algorithm. At the same time, the aligned reads were selected mapping phred quality score above 30, converted to binary alignment map (BAM) format and sorted ordering by genomic position using SAMTOOLS version 1.2. For high performance accurate variant calling, i) PCR duplicates reads were marked using MarkDuplicates of Picard tools version 1.127 ( http://broadinstitute.github.io/picard/ ). ii) Insertion and deletion (Indel) realignment were performed with known Indels from Mills and 100G gold standard using RealignerTargetCreator and IndelRealigner of Genome Analysis Tool Kit (GATK) version 3.1-1. iii) Base quality score was recalibrated using machine learning model with known single nucleotide polymorphisms (SNPs) and Indels from dbSNP138, Mills and 1000 Genome Project phase I by BaseRecalibrator and PrinReads of GATK. Manipulated BAMs were simultaneously called and genotyped of single nucleotide variants (SNVs) and Indels by GATK UnifiedGenotyper uses a Bayesian genotype likelihood model. Variants were recalibrated with reference variants such as dbSNP138, Mills Indels, HapMap and Omni using GATK VariantRecalibrator and ApplyRecalibration. Variants were annotated various information using ANNOVAR described below: i) population database such as 1000 genome phase III, ExAC and KRGDB ( http://coda.nih.go.kr/coda/KRGDB/ ), ii) disease database such as OMIM, sequencing database such as RefSeqGene, iii) in silico predictive algorithms such as FATHMM, MutationAssessor, MutationTaster, SIFT, Polyphen, GERP and Phylop for interpretation and classification of variants following ACMG guideline. Classified pathogenic or likely pathogenic variants were confirmed by Sanger sequencing. Copy number variants (CNVs) were calculated using aligned read counts in target region by in-house relative comparison method. Detected and classified pathogenic CNVs were re-confirmed by array comparative genomic hybridization (array CGH) 64 – 68 . Small interfering RNA (siRNA) siRNAs were transfected twice into cells, first by reverse transfection and 24 hours later by forward transfection using Lipofectamine RNAiMAX reagent (Invitrogen) as suggested by the manufacturer’s instructions. ACVR1 (ID#s974, s976), TGFBR1 (ID#s14071, s14073), and BMPR2 (ID#s2044, s2045, s2046) siRNAs were purchased from Thermo Fisher Scientific. Pools of two or three siRNAs were used with a final siRNA concentration of 25 nM. Luciferase reporter assay 293T cells were plated in the Falcon® 96-well white flat bottom tissue culture-treated microtest assay microplate (CORNING). In each well, 5,000 cells were plated in 100 µl 10% DMEM media. 24 hours after plating, cells were transfected with pcDNA-empty vector, pcDNA6/V5-HisA-wildtype BMPR2 or -mutant BMPR2, pGL3-BMP responsive elements-luciferase (hereafter pGL3-BRE-luc, offered from addgene plasmid #45126), and pNL1.1.TK internal control vector for the assay, using calcium phosphate transfection Kit (Invitrogen). The amounts of WT or mutant BMPR2, and pGL3-BRE-luc, and pNL1.1 from Nano-Glo® Dual-Luciferase® Reporter Assay Kit (Promega) were determined according to a protocol of calcium phosphate transfection from Clontech Laboratories; 50ng of WT BMPR2 or mutant BMPR2 and pGL3-BRE-luc and 5ng of pNL1.1.TK were used and then 2M Calcium Solution and sterile water were added in each DNA tube. The same volume of 2X HEPES-Buffered Saline (HBS) was added to Calcium-DNA mixture dropwise and incubated at room temperature. After 15 minutes, the transfection solution was carefully added to culture plate medium and maintained at 37℃ in a CO 2 incubator. The next day, the calcium phosphate-containing medium was removed from cells and replaced with fresh complete growth medium. The volume of One-Glo™ EX Luciferase assay Reagent was equally added to the culture medium volume to each well and placed on an orbital shaker at 300 rpm for 3 minutes. Luminescence was measured as integration times of 1 second by GloMax® Discover System (Promega). For measurement of NanoLuc® luciferase activity, a volume of NanoDLR™ Stop & Glo® Reagent was equally added to the original culture medium volume to each well and then luminescence was analyzed. The BRE reporter luminescence was normalized to NanoLuc® luciferase activity. Western blotting and Immunoprecipitation Cells were plated either in 60 mm or 100 mm plate with 70% confluency. The next day, plasmid DNA was transfected into HEK293T cells by Lipofectamine 2000. After 4 hours, cells were changed into serum free medium and treated with human recombinant Activin A (R&D SYSTEMS) the next day. Cells were harvested and lysed by lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, and 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Roche) and quantified by Protein Assay Dye Reagent Concentrate (Bio-Rad) and NanoDrop (Thermo Fisher Scientific). Proteins were separated by 8 ~ 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gels were blotted onto polyvinylidene difluoride (PVDF) transfer membrane with 0.45 µm pore size (Merck Millipore). Blots were blocked in 1X PBS with 0.1% Tween-20 (Sigma) containing 5% Difco™ Skim Milk (BD) for 1 hour at room temperature and incubated with the antibodies indicated in each figure at 4℃ for overnight. After the blots were washed four times in 1X PBST for 1 hour at room temperature, the membranes were incubated with the secondary antibody for 2 hours at room temperature 69 . The bands were then detected using an enhanced chemiluminescence solution (Bio-Rad) and visualized with the ChemiDoc System (Bio-Rad). The band image was analyzed with Image Lab™ Software (Version 5.2.1, Bio-Rad). For immunoprecipitation, transiently transfected HEK293T cells were lysed and sonicated in lysis buffer at 4℃. Crude lysates cleared by centrifugation at 15,000 rpm at 4℃ for 20 minutes. Supernatants were incubated with Monoclonal Anti-HA-Agarose antibody (Sigma) for 2 hours at 4℃. Immunocomplex was washed five times with lysis buffer and then SDS-PAGE and western blotting were performed 70 . Anti-mouse secondary light chain specific was incubated for 2 hours at room temperature. Real-time quantitative reverse transcription PCR Total RNA of the cells was extracted using RNeasy Mini Kit and QIAshredder (QIAGEN) and quantified using NanoDrop instrument. 1 µg of total RNA was used to cDNA synthesis using a SuperScript III First-Strand Synthesis System (Invitrogen). Gene expression was quantified by 2X qPCRBIO SyGreen Blue Mix Lo-ROX (PCRBIOSYSTEMS) performed on LightCycler® 96 (Roche) 71 . Quantification cycle (Cq) values of samples were analyzed by LightCycler® 96 Application Software (Version 1.1). Gene-specific primers are listed in Supplementary Table 1. Alizarin S staining (Mineralization assay) The mineralization was determined by staining with Alizarin Red S at 21 days after osteogenic differentiation. For preparation of solution, 2 g Alizarin Red S (Sigma) was dissolved in 100 ml distilled water and then adjusted to pH4.3 with HCl or NH 4 OH. Differentiated cells were carefully washed with PBS and fixed with 4% paraformaldehyde (Sigma). After 30 minutes carefully washed the cells with distilled water followed by prepared stain solution was enough added to the cells for 45 minutes at room temperature in the dark. The cells were washed four times with distilled water and carefully aspirated. The differentiated cells are stained darker red with calcium deposits. After photography using digital camera (Nikon), the stained cells were lysed with 10% cetypyridium chloride (sigma) dissolved in 10 mM sodium phosphate buffer (1 M NaH 2 PO 4 monobasic and 1 M Na 2 HPO 4 dibasic, pH7.0) and then quantified at 560 nm using a GloMax® Discover System. Alkaline Phosphatase (ALP) staining and activity For detection of alkaline phosphatase, cells were firstly cultured with osteogenic differentiation media for 2 or 3 days. Cells were cautiously washed with PBS and then fixed with 4% paraformaldehyde. After 1 minute, cells were rinsed with Washing Buffer (0.05% Tween 20 in PBS), subsequently treated with substrate solution which was dissolved one BCIP/NBT tablet (Sigma) in 10 ml distilled water. For staining, the cells were incubated at room temperature in the dark for 10 minutes monitoring staining progress every 2 ~ 3 minutes. Carefully aspirated the substrate solution and rinsed the cell with Washing Buffer. The higher alkaline phosphatase, the more intense the dark blue-violet. For ALP activity, cultured cells were washed with PBS and lysed with cold alkaline phosphatase reaction buffer (1 M Diethanolamine and 0.5 mM Magnesium Chloride, pH9.8, Sigma). Lysates were incubated in 0.67 M p -Nitrophenyl Phosphate (pNPP) solution (Sigma) for 30 minutes at 37℃ continuing the reaction was immediately followed by monitoring in absorbance at 405 nm. Total protein was measured by using a Micro-BCA protein assay kit (Thermo Fisher Scientific) and read at 560 nm using a GloMax® instrument. The enzymatic ALP activity was normalized to the protein content of the samples. Alcian blue staining To visualize ability of chondrogenesis, stain solution (pH1.0) was prepared with 1 g Alcian blue 8GX (Sigma) in 100 ml 0.1 M HCl. Cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature and then rinsed 3 times with PBS. Alcian blue solution was used to stain the cells at room temperature in the dark. Next day, cells were washed once with 0.1 M HCl and twice with PBS. After taking a picture, the dye was extracted by 6 M Guanidine-HCl (Sigma) for 2 hours at room temperature and then read in absorbance at 600 nm using a GloMax® instrument. Statistical analysis Statistical analyses were performed with GraphPad Prism 8 software. Data were presented as bar graphs with dot blots for mean ± standard error of measurement (SEM). For comparison between two normally distributed test groups was used the two-tailed unpaired student’s t-test. Analysis of three or more groups were performed using two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. P values were statistically considered significant in P < 0.05. The following standard symbols are used to reference P values: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Declarations Acknowledgements We are grateful to the affected individual and his family. Author contributions YK and TJC conceived the study. MJK, EJ, DK, YYK, SWC, HMR, SWJ, YY and YK designed, performed, and analyzed most of the laboratory works. CHS, FSK and TJC ascertained and recruited the proband with FOP. HRL, WYP and TJC performed whole exome sequencing and analyzed the data. FSK read and revised the manuscript. YK, MJK, EJ and TJC wrote the initial draft of the manuscript, with contributions and revision from all other authors. FUNDING This research was supported by the Genome Technology to Business Translation Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2014M3C9A2064684 to TJC); by an NRF grant funded by the Korean government (NRF-2023R1A2C3007266 and NRF-2021R1A6A1A03038890 to YK). This research was partially supported by Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (No. 2021R1A6C101A564 and RS-2024-00436674), Industrial Technology Innovation Program (RS-2024-00403190) funded by the Ministry of Trade, Industry & Energy of the Republic of Korea, and Korea Drug Development Fund (RS-2024-00463605). ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at Competing interests: The authors declare no competing interests. References Shore, E. M. & Kaplan, F. S. 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Shin, D. et al. Elucidation of molecular basis of osteolytic bone lesions in advanced multiple myeloma. Haematologica 109, 2207–2218 (2024). Additional Declarations There is no conflict of interest Supplementary Files KimetalBMPR2SupplementaryFinal.docx Supplementary Materials Cite Share Download PDF Status: Under Revision Version 1 posted Reviewer # 1 agreed at journal 13 Jan, 2026 Reviewers invited by journal 12 Jan, 2026 Submission checks completed at journal 29 Dec, 2025 Editor assigned by journal 26 Dec, 2025 First submitted to journal 26 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":1030905,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of a novel variant in a patient with FOP.\u003cstrong\u003e a\u003c/strong\u003e Radiographs of the proband at age 16 years. Arrows denote the heterotopic bone formation in the skeletal muscle. The big toes were normal in shape. \u003cstrong\u003eb\u003c/strong\u003e The pedigree chart of the \u003cem\u003eBMPR2\u003c/em\u003evariant in the family. The arrow indicates the proband. \u003cstrong\u003ec\u003c/strong\u003e Chromatogram displaying the heterozygous \u003cem\u003ede novo\u003c/em\u003ec.1126G\u0026gt;A mutation in the genomic DNA isolated from the proband and family. \u003cstrong\u003ed\u003c/strong\u003eSchematic of functional domains and the site of mutation (red asterisk) in the BMPR2. The mutation is within exon 8 in the kinase domain (KD) of BMPR2. TM, transmembrane; ECD, extracellular domain; CTD, C-terminal domain. \u003cstrong\u003ee\u003c/strong\u003eInterspecies homology of the kinase domain of BMPR2. \u003cstrong\u003ef\u003c/strong\u003e Western blots of whole cell lysates from normal (BJ fibroblast) and FOP patient-derived cells. Normal and patient cells were cultured under complete media (15% fetal bovine serum) or low serum media (2% fetal bovine serum) for inducing osteogenic differentiation. \u003cstrong\u003eg\u003c/strong\u003e Alkaline phosphatase (ALP) staining of the normal and patient-derived fibroblasts in culture for 2 days. Data are the mean ± SEM from six independent experiments (\u003cem\u003en\u003c/em\u003e = 6). \u003cstrong\u003eh\u003c/strong\u003e Alizarin red S staining indicative of calcium deposits in cell culture. Data are the mean ± SEM from five independent experiments (\u003cem\u003en\u003c/em\u003e = 5). Statistics was performed by two-tailed unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8453923/v1/056bf6b0f8dadf71ee7847d6.jpg"},{"id":100406396,"identity":"0187c43a-3089-4d1a-b0fb-cad747211373","added_by":"auto","created_at":"2026-01-16 13:01:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":770050,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional validation of pathogenicity associated with the BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant.\u003cstrong\u003e \u003c/strong\u003eTo validate the pathogenicity of BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant, patient-derived cells were edited with CRISPR-Cas9 \u003cstrong\u003ea\u003c/strong\u003e Schematic of the human \u003cem\u003eBMPR2\u003c/em\u003e gene, showing the G1126A mutation in exon 8. The G-to-A mutation leads to glutamate (blue)-to-lysine (red) change in the amino-acid sequence. In clones corrected by CRISPR-Cas9, knock-out #3 (KO #3) had a 1 base pair (bp) deletion (del, hyphen) at nucleotide 1122, with the alleles in a 1:1 ratio, resulting in a premature stop codon. Knock-out #5 (KO #5) had a 1 bp insertion of nucleotide A (insA, yellow), causing a premature stop codon. Knock-in #203 (KI #203) contained a normal nucleotide guanine at 1126 and a CTC sequence (green, a synonymous mutation) from the HDR donor template, which preserved the WT amino acid sequence. \u003cstrong\u003eb\u003c/strong\u003e Protein expression determined by Western analysis of the lysates prepared from the knock-out (#3 and #5) and knock-in (#203) clones edited by CRISPR-Cas9 in patient-derived fibroblasts. \u003cstrong\u003ec\u003c/strong\u003e Duplicate samples (#1, 2) of normal, patient-derived cells, and CRISPR-modified cells (knock-out #3, #5, and knock-in #203) were incubated in 2% low serum medium at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e and 3% O\u003csub\u003e2\u003c/sub\u003e for 5 days and were performed for ALP staining to detect alkaline phosphatase, expressed in early osteogenic differentiation. \u003cstrong\u003ed\u003c/strong\u003e Normal, patient-derived cells, and CRISPR-modified cells were maintained in 2% low serum medium without or with BMP2 or BMP4 (50 ng/ml) for 21 days and followed by Alizarin red S staining to sense calcium deposits in late osteogenic differentiation. \u003cstrong\u003ee\u003c/strong\u003e Western blot analysis to validate hyperactivated BMP signaling in HEK293T expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e mutant. HEK293T cells were transiently transfected with empty vector (EV), wild-type (WT), or BMPR2\u003csup\u003eE376K\u003c/sup\u003e. As a control, protein expression of the ACVR1\u003csup\u003eWT\u003c/sup\u003e and ACVR1\u003csup\u003eR206H\u003c/sup\u003e mutant was determined. \u003cstrong\u003ef\u003c/strong\u003e The promoter reporter assay (bottom panel) in which luciferase expression is regulated by BMP-responsive elements in HEK293T cells transfected with plasmids carrying EV, WT or BMPR2\u003csup\u003eE376K\u003c/sup\u003e. As a control, ACVR1\u003csup\u003eWT\u003c/sup\u003e or ACVR1\u003csup\u003eR260H\u003c/sup\u003e activity was measured in the same reporter assay. V5-tagged protein expression level was determined by Western blot (upper panel). Data are the mean ± SEM (left panel, \u003cem\u003en=5\u003c/em\u003e and right panel, \u003cem\u003en=4\u003c/em\u003e). Statistics was performed by ordinary one-way ANOVA with Tukey’s multiple comparisons test; ns, not significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8453923/v1/f8ed449f82e9fef58cdc9ce6.jpg"},{"id":100406762,"identity":"5ed5e887-9990-46ab-9aaa-086dda2c33b5","added_by":"auto","created_at":"2026-01-16 13:03:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":549557,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced chondrogenesis and osteogenesis of BMPR2\u003csup\u003eE376K\u003c/sup\u003e patient-derived mesenchymal stem cells. Establishment of induced mesenchymal stem cells (MSCs) from FOP patient-derived induced pluripotent stem cells (iPSCs) to assess the effect of BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant on chondrogenesis and osteogenesis. \u003cstrong\u003ea\u003c/strong\u003e Western blots of whole cell lysates from human normal MSC (Normal), FOP patient-derived MSC (Patient), and CRISPR-Cas9-edited knock-in (Patient-KI) MSC from patient-derived cells. \u003cstrong\u003eb\u003c/strong\u003e Alcian blue staining to detect chondrogenic capacity in duplicate samples (#1, 2) of normal, patient, and patient-KI MSCs for 3 weeks (left panel). Quantification of staining was determined by measuring absorption at 600 nm (right panel, \u003cem\u003en\u003c/em\u003e = 2). \u003cstrong\u003ec\u003c/strong\u003e Alizarin red S staining to detect osteogenic capacity in each of the three types of MSCs\u003csup\u003e \u003c/sup\u003efor 3 weeks (left panel). Quantification of staining was determined by measuring absorption at 560 nm (right panel, \u003cem\u003en\u003c/em\u003e = 2). \u003cstrong\u003ed\u003c/strong\u003e Comparison of the relative mRNA expression of ALP (\u003cem\u003en\u003c/em\u003e = 4) and Osteocalcin (\u003cem\u003en\u003c/em\u003e = 4) obtained from each MSC\u003csup\u003e \u003c/sup\u003eusing real-time quantitative PCR.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8453923/v1/c6afc23b2cd0b7725bc6039e.jpg"},{"id":100407029,"identity":"729c640f-5fc1-4a50-bad0-504a05553372","added_by":"auto","created_at":"2026-01-16 13:03:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":525891,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced BMP signaling driven by BMPR2\u003csup\u003eE376K\u003c/sup\u003e. \u003cstrong\u003ea\u003c/strong\u003e C3H10T1/2 cells were transfected with empty vector (EV), HA-tagged WT BMPR2 or mutant BMPR2\u003csup\u003eE376K\u003c/sup\u003e and the enhanced BMP signals were examined by Western blot analysis. The asterisk indicates a cross-reactive artifact. \u003cstrong\u003eb\u003c/strong\u003e Alcian blue staining to detect chondrogenic capacity in C3H10T1/2 cells expressing BMPR2\u003csup\u003eWT\u003c/sup\u003e or BMPR2\u003csup\u003eE376K\u003c/sup\u003e with and without BMP2 treatment for 3 weeks (left panel). Quantification of staining was determined by measuring absorption at 600 nm (right panel, \u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003ec\u003c/strong\u003e Comparison of the relative mRNA expression of Col2a1 (\u003cem\u003en\u003c/em\u003e = 4), Aggrecan (\u003cem\u003en\u003c/em\u003e = 3), and Col10a1 (\u003cem\u003en\u003c/em\u003e = 3) obtained from C3H10T1/2 cells expressing empty vector (EV), BMPR2\u003csup\u003eWT\u003c/sup\u003e, or BMPR2\u003csup\u003eE376K \u003c/sup\u003eusing real-time quantitative PCR. (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001) The mean ± SEM from independent experiments indicated. Statistics was performed by two-way ANOVA with Tukey’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8453923/v1/348d791a4f2a2204ebde10e1.jpg"},{"id":100405798,"identity":"202816f6-7ef6-4a86-9b00-1486f3d9883b","added_by":"auto","created_at":"2026-01-16 12:17:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":875931,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular basis of the dominant-negative functions of BMPR2\u003csup\u003eE376K\u003c/sup\u003e. \u003cstrong\u003ea\u003c/strong\u003e Co-immunoprecipitation of HA-tagged BMPR2\u003csup\u003eE376K\u003c/sup\u003e with wild-type (WT) V5-tagged ACVR1. \u003cstrong\u003eb\u003c/strong\u003e Addictive effect of ACVR1\u003csup\u003eR206H\u003c/sup\u003e and BMPR2\u003csup\u003eE376K\u003c/sup\u003e in enhancing BMP signaling, as assessed by Western blotting for p-SMAD1/5/9, p-SMAD2, ID1, and ID3. \u003cstrong\u003ec\u003c/strong\u003e Western blot analysis of phosphorylation of SMAD2 in HEK293T cells expressing V5-tagged WT or mutant BMPR2\u003csup\u003eE376K\u003c/sup\u003e (upper panel) or ACVR1\u003csup\u003eR206H\u003c/sup\u003e (bottom panel). \u003cstrong\u003ed\u003c/strong\u003e Expression and phosphorylation of SMAD in patient-derived fibroblasts treated with siRNA against type I receptors, ALK2 or ALK5. \u003cstrong\u003ee\u003c/strong\u003e C2C12 cells stably expressing empty vector (EV), HA-tagged BMPR2\u003csup\u003eWT\u003c/sup\u003e, or BMPR2\u003csup\u003eE376K\u003c/sup\u003e construct by lenti-viral transduction displayed protein expression for phosphorylation of SMAD1/5/9 and SMAD2 using western blotting. \u003cstrong\u003ef,\u003c/strong\u003e \u003cstrong\u003eg\u003c/strong\u003e ALP staining of C2C12 cell lines stably overexpressing HA-tagged BMPR2\u003csup\u003eWT\u003c/sup\u003e or mutant BMPR2\u003csup\u003eE376K\u003c/sup\u003e, with and without treatment with BMP4, dorsomorphin (DM), or SB431542 (SB).\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8453923/v1/3a583d57352404c0aab9d503.jpg"},{"id":100406438,"identity":"e6a22cb1-e8d6-49c7-85b2-1b38ffb97f01","added_by":"auto","created_at":"2026-01-16 13:02:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":532618,"visible":true,"origin":"","legend":"\u003cp\u003eThe ability of BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant to respond to Activin A. \u003cstrong\u003ea-b\u003c/strong\u003e Phosphorylation of SMAD1/5/9 and SMAD2 increased upon treatment with Activin A (50 ng/ml) in HEK293T cells expressing (\u003cstrong\u003ea\u003c/strong\u003e) empty vector (EV), V5-tagged ACVR1\u003csup\u003eWT\u003c/sup\u003e or mutant ACVR1\u003csup\u003eR206H\u003c/sup\u003e and (\u003cstrong\u003eb\u003c/strong\u003e) empty vector (EV), V5-tagged BMPR2\u003csup\u003eWT\u003c/sup\u003e or mutant BMPR2\u003csup\u003eE376K\u003c/sup\u003e.\u003cstrong\u003e c\u003c/strong\u003e Phosphorylation of SMAD1/5/9 and SMAD2 in response to treatment with Activin A (50 ng/ml), Follistatin (500 ng/ml), ACVR2A-Fc (1 μg/ml), or ACVR2B-Fc (1 μg/ml) in FOP patient-derived cells and CRISPR-Cas9-edited knock-in cells for 2 h.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8453923/v1/4ac3de560dd78f86c58cc53b.jpg"},{"id":100422879,"identity":"02594270-ce74-4fd8-b1c3-413b6043a9f5","added_by":"auto","created_at":"2026-01-16 14:11:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5444255,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8453923/v1/a6b4004a-904b-491c-bbfd-81d9ba7dba2d.pdf"},{"id":100406595,"identity":"a74072c8-593e-4a16-a6af-0d591c972abb","added_by":"auto","created_at":"2026-01-16 13:03:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2419690,"visible":true,"origin":"","legend":"Supplementary Materials","description":"","filename":"KimetalBMPR2SupplementaryFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8453923/v1/d96d2d24eff89cfa7bf2e827.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"A Novel Gain-of-Function Mutation in BMPR2 in a Patient with Variant Fibrodysplasia Ossificans Progressiva","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHeterotopic ossification (HO) is a pathological process characterized by the formation of bone tissue in non-skeletal regions, such as muscles, tendons, or other soft tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. While HO can occur following traumatic injuries, burns, or surgical interventions, it is also associated with rare genetic disorders\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. HO represents a significant clinical challenge, often leading to pain, restricted joint movement, and in severe cases, complete immobilization. Despite its debilitating effects, the underlying mechanisms that govern this abnormal bone formation remain incompletely understood, particularly in the context of genetic predispositions.\u003c/p\u003e \u003cp\u003eOne of the most extensively studied diseases characterized by hereditary HO is fibrodysplasia ossificans progressiva (FOP, MIM #135100). FOP is an ultra-rare and devastating genetic disorder driven by gain-of-function mutations in the activin A type I receptor (\u003cem\u003eACVR1)\u003c/em\u003e gene\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Approximately 97% of affected individuals have classic FOP defined as characteristic malformations of the great toes, progressive HO and a recurrent \u003cem\u003eACVR1\u003c/em\u003e (R206H) mutation\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The remaining\u0026thinsp;~\u0026thinsp;3% have variant FOP, which is defined by either atypical or normal great toes, progressive HO, and is driven by other gain-of-function mutations in \u003cem\u003eACVR1\u003c/em\u003e\u003csup\u003e7\u003c/sup\u003e. To date, all individuals with classic or variant FOP have gain-of-function mutations in \u003cem\u003eACVR1\u003c/em\u003e gene\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These mutations are known to dysregulate bone morphogenetic protein (BMP) signaling\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. ACVR1 functions as a BMP type I receptor, forming a heterotetrameric complex with type II receptors upon ligand binding. This complex phosphorylates receptor-regulated SMAD proteins (SMAD1/5/9), which translocate to the nucleus to regulate the expression of target genes involved in endochondral bone formation\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The R206H mutation in \u003cem\u003eACVR1\u003c/em\u003e leads to a gain-of-function alteration, resulting in activation of the receptor. Consequently, the mutant receptor exhibits increased basal activity and heightened sensitivity to BMP ligands, leading to excessive phosphorylation of SMAD1/5/8, upregulation of osteogenic target genes and aberrant BMP pathway signaling.\u003c/p\u003e \u003cp\u003eOne of the pivotal mediators of heterotopic bone formation in FOP is Activin A, a member of the TGF-β superfamily. Under normal physiological conditions, Activin A acts as an antagonist to BMP signaling through the formation of non-signaling complex (NSC) with ACVR1 receptor\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. It was proposed that Activin A dampens BMP signaling by forming a complex with ACVR1 and a type II receptor, both of which are required for initiation of BMP pathway signaling\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, in FOP patients harboring the \u003cem\u003eACVR1\u003c/em\u003e R206H mutation, Activin A paradoxically activates the mutant receptor, amplifying BMP signaling pathways\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. While the precise molecular mechanisms underlying Activin A-mediated activation of BMP signaling remain largely unresolved, significant progress has been made using cell and mouse models\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Studies have demonstrated that inhibition of Activin A with a neutralizing antibody blocks the formation of new heterotopic bone lesions, halts the growth and even reduces the size of pre-existing actively growing lesions, underscoring its essential role in driving HO\u003csup\u003e23,24\u003c/sup\u003e. Furthermore, the depletion or deletion of \u003cem\u003eACVR2A\u003c/em\u003e and \u003cem\u003eACVR2B\u003c/em\u003e markedly diminishes BMP signaling, highlighting their critical role in the neofunction of \u003cem\u003eACVR1\u003c/em\u003e\u003csup\u003e\u003cem\u003eR206H\u003c/em\u003e18,19,25\u003c/sup\u003e. Inflammation is another central driver of HO in FOP. Localized inflammatory episodes, whether triggered by trauma, infection, or spontaneous flare-ups, create a pro-inflammatory microenvironment enriched with cytokines, growth factors, and immune cells\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Notably, Activin A is an indispensable ligand for the initiation of HO in FOP, and activation of \u003cem\u003eACVR1\u003c/em\u003e\u003csup\u003e\u003cem\u003eR206H\u003c/em\u003e\u003c/sup\u003e by Activin A is required to drive the pathological ossification process\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFibro/adipogenic progenitors (FAPs) play a critical role in the pathophysiology of FOP by driving enforced differentiation into chondrocytes, a pivotal step in endochondral HO\u003csup\u003e11,28,29\u003c/sup\u003e. In FOP, mutations in the \u003cem\u003eACVR1\u003c/em\u003e gene lead to aberrant activation of BMP pathway signaling, particularly in response to Activin A, thereby skewing FAPs differentiation toward osteogenic and chondrogenic lineages. This enforced differentiation underpins the endochondral HO characteristic of FOP and validates the pathogenic role of the causative genetic alteration. Animal models, such as the \u003cem\u003eAcvr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eR206H\u003c/em\u003e\u003c/sup\u003e knock-in mouse line, have demonstrated that FAPs contribute significantly to HO through their osteochondrogenic potential, offering insights into disease mechanisms and potential therapeutic targets\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Understanding the interplay between FAPs differentiation and the pathological microenvironment in FOP is crucial for elucidating the molecular basis of HO and for developing effective treatments to mitigate disease progression\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBone morphogenetic protein receptor type 2 (BMPR2) is a critical player in the BMP signaling pathway, which regulates a range of cellular processes, including growth, differentiation, and apoptosis\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Mutations in the \u003cem\u003eBMPR2\u003c/em\u003e gene have been extensively studied in pulmonary arterial hypertension (PAH), a severe vascular disorder characterized by progressive narrowing of pulmonary arteries, ultimately leading to right heart failure. Most cases of PAH linked to BMPR2 mutations involve loss-of-function variants, which disrupt normal BMP signaling\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Consequently, endothelial cell dysfunction, excessive proliferation of smooth muscle cells, and resistance to apoptosis drive vascular remodeling central to PAH pathogenesis. However, to dates, no known human disorders are due to the gain-of-function mutation of \u003cem\u003eBMPR2\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eIn this study, we present a unique case of a patient with a phenotypic variant of FOP, characterized by progressive manifestation of systemic HO but normal great toes, who lacks mutations in the commonly implicated \u003cem\u003eACVR1\u003c/em\u003e and \u003cem\u003eGNAS\u003c/em\u003e genes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Through whole exome sequencing, we identified a novel gain-of-function mutation in the \u003cem\u003eBMPR2\u003c/em\u003e gene, which encodes a type II receptor integral to the BMP signaling pathway and functionally validated the pathogenesis of the causative mutation using multiple cell types including patient-derived dermal fibroblasts. This is the first report of a \u003cem\u003eBMPR2\u003c/em\u003e gain-of-function mutation in variant FOP, providing critical new insights into genetic diversity underlying this rare disorder. While the \u003cem\u003eACVR1\u003c/em\u003e\u003csup\u003e\u003cem\u003eR206H\u003c/em\u003e\u003c/sup\u003e mutation remains the most prevalent genetic driver of FOP, these findings highlight the need to broaden the genetic landscape of the disease, as other mutations within the BMP signaling pathway may also shape its pathophysiology. These findings expand the mechanistic understanding of FOP at the molecular level, providing new avenues for research into the interplay of Activin A, inflammation, and BMP pathway signaling in disease pathogenesis.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of a gain-of-function BMPR2 mutation in a patient presented with FOP phenotype\u003c/h2\u003e \u003cp\u003eA 16-year-old boy presented with flare-ups of the left pectoral region after treatment for dental caries. The patient was a product of a full-term pregnancy and normal vaginal delivery to a healthy Korean couple. Birth weight was 2.98 kg. No perinatal problems were encountered. Motor and cognitive development were normal. The patient recovered uneventfully from laparotomy for pyloric stenosis in childhood. Subcutaneous migrating nodules were noted over the scalp at age 2 and over the posterior neck at age 4. Flare-ups and stiffness of the neck and back developed at age 6 and progressed to the limbs. At age 19, he developed severe dizziness. Magnetic resonance imaging of the brain revealed multifocal gadolinium-enhanced tumors involving the suprasellar area, septum pellucidum, and medulla oblongata that were diagnosed as mixed germ cell tumors because the serum beta-hCG level was moderately elevated. Following radiation therapy, the tumors disappeared, and the patient has remained in complete remission for 3 years but developed diabetes insipidus. Physical examination revealed a Cumulative Analogue Joint Involvement Scale (CAJIS) score of 20/30\u003csup\u003e34\u003c/sup\u003e. The neck, back, both shoulders, elbows, hips, and the left knee were functionally ankylosed. The feet and toes appeared normal.\u003c/p\u003e \u003cp\u003eAt age 22, the patient\u0026rsquo;s height was 145 cm (z \u0026lt; -4) and weight was 57 kg. The neck, back, both shoulders, hips, and the left knee were ankylosed. Both elbows maintained only 10 to 30 degrees of flexion-extension motion, and the right knee maintained 80 degrees of motion. Radiographic examination revealed extensive HO in the back, periscapular, peripelvic regions and thighs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Ankylosis of the posterior column of the cervical vertebrae was noted. Unlike in patients with classic FOP, the individual had normal great toes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The patient had progressive HO in characteristic anatomic patterns with normal great toes and was diagnosed as having a clinical variant of FOP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe clinical manifestations were fully consistent with variant FOP based on the normal great toes\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and progressive HO in characteristic anatomic patterns\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). However, full sequencing of the coding region of \u003cem\u003eACVR1\u003c/em\u003e (MIM #102576) of the proband showed two silent sequence variations, both of which were previously reported in the normal population: c.270C\u0026thinsp;\u0026gt;\u0026thinsp;T (NCBI dbSNP rs2227861) and c.690G\u0026thinsp;\u0026gt;\u0026thinsp;A (rs1146031). The patient exhibited no clinical manifestations consistent with Progressive Osseous Heteroplasia (POH), and no mutations were identified in the \u003cem\u003eGNAS\u003c/em\u003e gene, which is recognized as the causative gene for POH\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In order to find causative mutations for the disorder, we performed whole exome sequencing on the genomic DNA from the proband and identified a heterozygous mutation in \u003cem\u003eBMPR2\u003c/em\u003e (MIM #600799), c.1126G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.E376K). Sanger sequencing and restriction enzyme digestion (data not shown) confirmed the sequence variation in the proband and absence in the genome of the parents and the sibling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). The mutation of the BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant is located in the kinase domain\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) where the sequence is highly conserved among species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The BMPR2\u003csup\u003eE376K\u003c/sup\u003e sequence variation was not found in the general populations of the 100 genome, ExAC, dbSNP, or Exome Sequencing Project databases.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHyper-activation of BMP signaling in patient-derived dermal fibroblasts\u003c/h3\u003e\n\u003cp\u003eTo gain insight into the molecular basis underlying disease phenotype, we examined the molecular consequences of the BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant by performing immunoblot analysis on lysates prepared from dermal fibroblasts obtained from the patient\u0026rsquo;s skin and a normal control (BJ cells). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef (left panel), lysates from the patient-derived cells showed robust phosphorylation of SMAD1/5/9, and resultant increased expression of ID1 and ID3\u003csup\u003e35\u0026ndash;40\u003c/sup\u003e, which were not observed in the lysates prepared from the normal control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Notably, we also observed phosphorylation of SMAD2 in the patient-derived cells, although SMAD2 phosphorylation is not typically associated with BMP pathway signaling. In addition, when cultured in differentiation media, patient-derived fibroblasts expressed bone-associated proteins\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, including osteocalcin (OCN), alkaline phosphatase (ALP), and RUNX2, even in the absence of exogenous BMP ligands (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, right panel). These results strongly suggest that BMPR2\u003csup\u003eE376K\u003c/sup\u003e induces hyper-activation of BMP pathway signaling potentially due to the gain-of-function mutation. At the cellular level, the patient-derived cells were positive for the alkaline phosphatase (ALP) staining and ALP activity\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e while normal control cells barely expressed ALP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Similarly, alizarin red S staining revealed significant calcium accumulation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e in the patient-derived cell cultures after 21 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Taken together, these findings provide compelling evidence that BMPR2\u003csup\u003eE376K\u003c/sup\u003e drives hyper-activation of BMP pathway, promoting osteogenic differentiation.\u003c/p\u003e\n\u003ch3\u003eFunctional validation of pathogenicity of the BMPR2 variant\u003c/h3\u003e\n\u003cp\u003eFunctional validation of pathogenicity of the potential causative gene mutation is critical for establishing its role in disease pathogenesis. As DNA sequence analysis showed heterozygous \u003cem\u003eBMPR2\u003c/em\u003e mutation resulting in the FOP like phenotype, we assumed that the mutation is autosomal dominant. We therefore hypothesized that deletion of the BMPR2\u003csup\u003eE376K\u003c/sup\u003e allele would normalize the increased BMP signaling. To test this, we deleted the BMPR2\u003csup\u003eE376K\u003c/sup\u003e allele using CRISPR-Cas9 in the patient-derived cells. Simultaneously, we reverted the c.1126G\u0026thinsp;\u0026gt;\u0026thinsp;A variant back to the wild-type (WT) sequence using CRISPR-Cas9 knock-in methods. Multiple single clones were isolated and the sequence of the \u003cem\u003eBMPR2\u003c/em\u003e gene in individual clones was determined by a MiSeq system (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). From the gene editing experiments, we successfully obtained allele specific knock-out (KO) and knock-in (KI) patient-derived fibroblast clones. These modified clones exhibited a loss of SMAD1/5/9 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), as well as a significant reduction in ALP staining, and calcium deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d), strongly demonstrating that BMPR2\u003csup\u003eE376K\u003c/sup\u003e is a gain-of-function mutation causing BMP pathway activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the functional dominance of BMPR2\u003csup\u003eE376K\u003c/sup\u003e, we assessed whether heterotopic expression of the mutant allele could recapitulate the molecular and cellular changes observed in patient-derived cells. To test this, HEK293T cells were transfected with an empty vector, BMPR2\u003csup\u003eWT\u003c/sup\u003e, or BMPR2\u003csup\u003eE376K\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, left panel). Only cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e led to hyperphosphorylation of SMAD1/5/9 and expression of ID1 and ID3. In addition, to test if the enhanced BMP signaling due to the expression of BMPR2\u003csup\u003eE376K\u003c/sup\u003e results in BMP responsive gene expression, we established a transcriptional reporter assay system in which luciferase expression is controlled by BMP-responsive elements\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Consistent with the gain-of-function hypothesis, cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e showed significantly elevated luciferase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, left panel). In the same experimental setting, similar results were obtained when we expressed the classic ACVR1\u003csup\u003eR206H\u003c/sup\u003e mutant in HEK293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f, right panels). Collectively, these in vitro experiments highlight the critical gain-of-function properties of the BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant and its role in hyperactivating BMP signaling.\u003c/p\u003e\n\u003ch3\u003eEnhanced chondrogenic differentiation of MSCs expressing BMPR2 variant\u003c/h3\u003e\n\u003cp\u003eHO in FOP lesions involves chondrogenic differentiation from fibroadipogenic (FAP) progenitor cells due to enhanced BMP signaling\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. As BMPR2\u003csup\u003eE376K\u003c/sup\u003e stimulates BMP pathway signaling in the absence of BMP ligands, we hypothesized that BMPR2\u003csup\u003eE376K\u003c/sup\u003e might stimulate mesenchymal cells to become chondrocytes. As a test of this concept, we first generated induced pluripotent stem cells from the normal control dermal fibroblasts (BJ cells), the patient-derived cells and the restored KI patient-derived cells (#203) (Fig. S2). Then these iPSCs were partially differentiated into MSCs (Fig. S3). Consistent with previous findings, Western blot analysis showed that only the MSC from the patient-derived cells were positive for BMP signaling, indicated by the robust phosphorylation of SMAD1/5/9 and increased expression of ID1 and ID3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). At the cellular level, only the MSC from the patient derived cells were positive for alcian blue staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and alizarin S staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), which was not the case of the MSC from normal control cells and knock-in patient-derived cells. These findings demonstrated that expression of BMPR2\u003csup\u003eE376K\u003c/sup\u003e results in enforced MSC differentiation to chondrocytes, which later undergo ossification during the endochondral ossification. We additionally confirmed that the expression of osteogenic genes, including ALP and OCN, was significantly increased compared to the normal control and knock-in patient-derived cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), indicating that BMPR2\u003csup\u003eE376K\u003c/sup\u003e expression leads to the activation of BMP signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo rule out the possibility of additional unidentified genetic alteration responsible for enhanced BMP pathway signaling in the patient-derived cells, we individually expressed an empty vector, BMPR2\u003csup\u003eWT\u003c/sup\u003e, or BMPR2\u003csup\u003eE376K\u003c/sup\u003e in the mouse MSC cell line C3H10T1/2\u003csup\u003e45\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, we found that temporal expression of BMPR2\u003csup\u003eE376K\u003c/sup\u003e led to phosphorylation of SMAD1/5/9 and expression of its downstream effectors including ID1, ID3, and SOX9. In addition, C3H10T1/2 cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e exhibited a chondrocytic phenotype, based on the Alcian blue staining\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Indeed, later in culture, expression of BMPR2\u003csup\u003eE376K\u003c/sup\u003e showed enhanced expression of COL2A1, COL10A1, and Aggrecan, all of which are highly expressed in osteoblasts\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Taken together, these findings suggest that BMPR2\u003csup\u003eE376K\u003c/sup\u003e is a gain-of-function mutation and is likely causative for the enhanced BMP signaling and HO phenotype in our patient.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMechanistic insights into the pathogenicity of the BMPR2 variant\u003c/h3\u003e\n\u003cp\u003eBMP signaling is typically activated in the presence of BMP ligands, which leads to engagement of type I and type II receptors\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Given that BMPR2\u003csup\u003eE376K\u003c/sup\u003e showed hyper-activation of BMP signaling, we hypothesized that the mutant receptor might engage with the type I receptor ACVR1\u003csup\u003eWT\u003c/sup\u003e even in the absence of BMP ligands. To test this, we transiently co-expressed V5-tagged ACVR1\u003csup\u003eWT\u003c/sup\u003e with either wild-type (WT) or mutant (E376K) HA-tagged BMPR2 in HEK293T cells. Surprisingly, we found that ACVR1\u003csup\u003eWT\u003c/sup\u003e co-immunoprecipitated with BMPR2\u003csup\u003eE376K\u003c/sup\u003e, whereas BMPR2\u003csup\u003eWT\u003c/sup\u003e did not associate with ACVR1\u003csup\u003eWT\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). These results suggest that the BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant promotes ligand-independent oligomerization with ACVR1\u003csup\u003eWT\u003c/sup\u003e, providing a molecular explanation for its ability to activate BMP signaling in a dysregulated manner. Additionally, combined expression of ACVR1\u003csup\u003eR206H\u003c/sup\u003e and BMPR2\u003csup\u003eE376K\u003c/sup\u003e showed additive effects of SMAD1/5/9 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), indicating that BMPR2\u003csup\u003eE376K\u003c/sup\u003e activates BMP signaling through a distinct molecular mechanism compared to ACVR1\u003csup\u003eR206H\u003c/sup\u003e-mediated BMP activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, we observed enhanced SMAD2 phosphorylation in the patient-derived cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Consistently, as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, HEK293T cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e, showed robust SMAD2 phosphorylation, which was not detected in cells expressing the ACVR1\u003csup\u003eR206H\u003c/sup\u003e variant or in cells expressing BMPR2\u003csup\u003eWT\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). By performing real-time quantitative PCR, we further confirmed that the expression of downstream genes\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e of SMAD2 was increased (Fig. S4), implying that SMAD2 phosphorylation is because of the presence of BMPR2\u003csup\u003eE376K\u003c/sup\u003e. To identify the type I receptor potentially interacting with BMPR2\u003csup\u003eE376K\u003c/sup\u003e and driving SMAD2 phosphorylation\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, we individually depleted seven type I receptors in patient-derived cells. Notably, depletion of TGFβR1 completely abrogated SMAD2 phosphorylation in patient-derived cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) as well as in other cells stably expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e (Fig. S5a, b). These findings implicate TGFβR1 as the key type I receptor mediating SMAD2 activation in the context of BMPR2\u003csup\u003eE376K\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the role of SMAD2 activation in cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e, we used mouse myogenic C2C12 cells\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Through lentiviral transduction, we established C2C12 cell lines which stably express empty vector, human BMPR2\u003csup\u003eWT\u003c/sup\u003e, or BMPR2\u003csup\u003eE376K\u003c/sup\u003e. As expected, SMAD1/5/9 phosphorylation and its downstream target ID\u003csup\u003e35\u003c/sup\u003e were detected only in the cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Consistently, cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e were positive for ALP staining and activity indicating osteogenic differentiation (Fig. S6a, b). Addition of BMP2 or BMP4 further increased ALP expression in both BMPR2\u003csup\u003eWT\u003c/sup\u003e and BMPR2\u003csup\u003eE376K\u003c/sup\u003e backgrounds (Fig. S6a, b). Importantly, we found that treatment with dorsomorphin, a potent inhibitor of BMP pathway signaling\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), demonstrating that the BMP signaling plays a central role in BMPR2\u003csup\u003eE376K\u003c/sup\u003e-mediated effects. Interestingly, we observed that inhibition of SMAD2-mediated TGF-β signaling using SB431542, a potent inhibitor TGF-β pathway signaling\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, also reduced the ALP staining, albeit to a lesser extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg), suggesting a possibility that BMPR2\u003csup\u003eE376K\u003c/sup\u003e-dependent SMAD2 signaling might be partially involved in the processes of accelerated chondro-osseous differentiation. Together, these results indicate a complex interplay between BMP and TGF-β signaling pathways in the development of HO driven by BMPR2\u003csup\u003eE376K\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBMPR2\u003csup\u003eE376K\u003c/sup\u003e exhibits enhanced responsiveness to Activin A\u003c/h2\u003e \u003cp\u003eActivin A has been demonstrated as a culprit ligand for the HO phenotype in the context of ACVR1\u003csup\u003eR206H\u003c/sup\u003e variant, although Activin A functions as an antagonist of signaling mediated by wildtype ACVR1\u003csup\u003e23,24\u003c/sup\u003e. It was proposed that Activin A induces clustering or dimerization of ACVR1\u003csup\u003eR206H\u003c/sup\u003e, which enhances autophosphorylation of ACVR1\u003csup\u003eR206H25\u003c/sup\u003e. On the other hand, when wild-type ACVR1 and its associated type II receptors are engaged by Activin A, it results in the formation of non-signaling complexes that sequester ACVR1 and the type II receptors, rendering them unavailable for BMP signaling\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven that the \u003cem\u003eBMPR2\u003c/em\u003e\u003csup\u003e\u003cem\u003eE376K\u003c/em\u003e\u003c/sup\u003e variant drives enhanced BMP signaling and an FOP-like phenotype, we hypothesized that Activin A might similarly turn on BMP signaling in cells expressing \u003cem\u003eBMPR2\u003c/em\u003e\u003csup\u003e\u003cem\u003eE376K\u003c/em\u003e\u003c/sup\u003e. Remarkably, similar to \u003cem\u003eACVR1\u003c/em\u003e\u003csup\u003e\u003cem\u003eR206H\u003c/em\u003e20,21\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), Activin A treatment significantly increased SMAD1/5/9 phosphorylation in cells expressing \u003cem\u003eBMPR2\u003c/em\u003e\u003csup\u003e\u003cem\u003eE376K\u003c/em\u003e\u003c/sup\u003e, but not in cells transfected with an empty vector or \u003cem\u003eBMPR2\u003c/em\u003e\u003csup\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These findings suggest that Activin A plays a pivotal role in driving the HO phenotype in our patient with the \u003cem\u003eBMPR2\u003c/em\u003e\u003csup\u003e\u003cem\u003eE376K\u003c/em\u003e\u003c/sup\u003e variant, mirroring its role in patients with the \u003cem\u003eACVR1\u003c/em\u003e\u003csup\u003e\u003cem\u003eR206H\u003c/em\u003e\u003c/sup\u003e variant. Consistently, neutralization of Activin A or BMP ligands using ACVR2A-Fc, ACVR2B-Fc, or follistatin, each of which effectively sequesters Activin A or BMPs, substantially reduced SMAD1/5/9 phosphorylation in BMPR2\u003csup\u003eE376K\u003c/sup\u003e-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). These results further support the neofunction of BMPR2\u003csup\u003eE376K\u003c/sup\u003e as a driver of enhanced responsiveness to Activin A and underscore its critical role in the pathogenesis of HO. The discovery of BMPR2\u003csup\u003eE376K\u003c/sup\u003e, and its ability to enable wildtype ACVR1 to respond to Activin A, similar to ACVR1\u003csup\u003eR206H\u003c/sup\u003e, provides an additional tool to investigate the molecular mechanisms by which these FOP-causing variants convert their complexes with Activin A and partner type II receptors into signaling complexes.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTo date, ACVR1 is the only known gene responsible for FOP\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In this study, we report that a novel gain-of-function mutation in the \u003cem\u003eBMPR2\u003c/em\u003e gene causes non-classic FOP. We showed that the BMPR2\u003csup\u003eE376K\u003c/sup\u003e is a neofunction variant much like the FOP-causing variant of ACVR1\u003csup\u003eR206H\u003c/sup\u003e and may not only be neoresponsive to Activin A in association with wildtype ACVR1 but also be implicated in SMAD2/3 phosphorylation. This discovery introduces a previously unreported mechanism of HO involving a type II receptor in the BMP signaling pathway. While FOP has historically been linked to gain-of-function mutations in the \u003cem\u003eACVR1\u003c/em\u003e gene, most notably the ACVR1\u003csup\u003eR206H\u003c/sup\u003e variant present in over 97% of cases, our findings expand the spectrum of genetic contributors to FOP-like HO phenotypes and highlight potential distinctions and overlaps in molecular mechanisms. Unlike classic FOP cases, the patient in our study presented with a variant phenotype characterized by the absence of the typical great toe malformation.\u003c/p\u003e \u003cp\u003eCellular and biochemical analysis demonstrated Activin-A responsive activation of BMP signaling in patient-derived cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e. Functional assays validated the pathogenicity of this mutation, showing its significant role in dysregulated signaling. Specifically, BMPR2\u003csup\u003eE376K\u003c/sup\u003e enhanced mesenchymal stem cell (MSC) differentiation into chondrocytes, a precursor to endochondral ossification. These findings provide a mechanistic explanation for HO observed in the patient and shed light on the broader implications of dysregulated BMP signaling. In addition to its canonical BMP pathway effects, BMPR2\u003csup\u003eE376K\u003c/sup\u003e exhibited novel properties, including the activation of SMAD2 phosphorylation and downstream gene expression. Further investigation revealed that TGFβR1 is implicated in the SMAD2 signaling. This noncanonical signaling was found to contribute to accelerated chondro-osseous differentiation, aligning with prior evidence implicating TGFβ signaling in FOP phenotypes\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. These findings collectively underscore the dual contributions of BMP and TGFβ pathways in the pathogenesis of this disorder, advancing our understanding of both HO and TGFβ/BMP pathway regulation.\u003c/p\u003e \u003cp\u003eLoss-of-function mutations in \u003cem\u003eBMPR2\u003c/em\u003e are well-established as key contributors to pulmonary arterial hypertension (PAH)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. It was proposed that loss of BMPR2 functions results in enhanced TGF-β signaling cascades, leading to hyperproliferation of smooth muscle cells in the pulmonary vasculature, although the exact molecular mechanisms underlying PAH remain elusive\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In contrast, here we report the first gain-of-function mutation in \u003cem\u003eBMPR2\u003c/em\u003e, BMPR2\u003csup\u003eE376K\u003c/sup\u003e, which we demonstrate as a causative factor for a disorder phenotypically resembling FOP. This mutation drives increased activation of BMP signaling and promotes chondro-osseous differentiation, establishing a clear link between BMPR2 gain-of-function activity and the heterotopic bone formation observed in our patient. The identification of BMPR2\u003csup\u003eE376K\u003c/sup\u003e provides an important counterpart to the established loss-of-function mutations, offering new insights into the dual roles of BMPR2 in human disease. Together, these findings might shed light on the understanding of physiological functions BMPR2, highlighting its critical role in maintaining the balance between BMP and TGF-β signaling pathways. By studying both gain- and loss-of-function mutations, we can better understand the molecular basis of conditions like PAH and FOP, paving the way for novel therapeutic strategies to modulate BMP pathway signaling in diverse contexts.\u003c/p\u003e \u003cp\u003eBMPR2 is a constitutively active receptor capable of phosphorylating type I receptors such as ACVR1\u003csup\u003e15\u003c/sup\u003e. We demonstrate that the BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant exhibits an enhanced ability to associate with ACVR1, increasing the proximity between type I and type II BMP receptors on the cell membrane of mesenchymal stem cells, even in the absence of BMP ligands. This ligand-independent interaction likely enables BMP pathway signaling through ligands such as Activin A that normally do not activate BMPR2 and ACVR1. Interestingly, when BMPR2\u003csup\u003eE376K\u003c/sup\u003e and ACVR1\u003csup\u003eR206H\u003c/sup\u003e were co-expressed, we observed additive effects on BMP signaling, suggesting that these mutations operate through distinct molecular mechanisms. This distinction highlights a fundamentally different mechanism by which BMPR2\u003csup\u003eE376K\u003c/sup\u003e drives aberrant signaling. Our findings will be informative for understanding the pathophysiology of FOP that arises from mutant type I or mutant type II receptors, and for developing drugs that inhibit dysregulated BMP pathway signaling for FOP and other BMP pathway-related diseases.\u003c/p\u003e \u003cp\u003eIt has been demonstrated that Activin A, which antagonizes BMP signaling in wild-type ACVR1, paradoxically acts as an agonist in the presence of gain-of-function mutations such as ACVR1\u003csup\u003eR206H\u003c/sup\u003e. Our findings extend this paradigm to BMPR2\u003csup\u003eE376K\u003c/sup\u003e, demonstrating that Activin A turns on BMP signaling in cells harboring the mutant receptor. This observation underscores the intricate interplay between type I and type II BMP receptors in ligand-induced signaling and highlights a potential neofunction of those receptors in driving pathological processes. In wild-type conditions, Activin A antagonizes BMP signaling by forming non-signaling complexes with ACVR1 and its associated type II receptors, ACVR2A and ACVR2B. In contrast, it has been shown that in the presence of the ACVR1\u003csup\u003eR206H\u003c/sup\u003e variant, Activin A facilitates receptor dimerization, leading to autoactivation of ACVR1\u003csup\u003eR206H\u003c/sup\u003e and the BMP signaling\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Further studies have demonstrated that the ACVR1\u003csup\u003eR206H\u003c/sup\u003e variant clusters with ACVR2A or ACVR2B in the presence of Activin A, enhancing self-activation of the BMP pathway\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Interestingly, BMPR2, a type II receptor, is dispensable for Activin A-mediated BMP signaling in the ACVR1\u003csup\u003eR206H\u003c/sup\u003e background. Our study, however, reveals that BMPR2\u003csup\u003eE376K\u003c/sup\u003e is also responsive to Activin A. Using embryonic stem cell models, we demonstrated that both ACVR2A and ACVR2B are somehow implicated in BMPR2\u003csup\u003eE376K\u003c/sup\u003e-mediated BMP signaling, suggesting that these type II receptors play a role in the mutant receptor's activation. These results suggest that the mechanisms by which Activin A turn on BMP SMAD1/5/9 signaling in mutant receptor backgrounds, such as ACVR1\u003csup\u003eR206H\u003c/sup\u003e and BMPR2\u003csup\u003eE376K\u003c/sup\u003e, may share general principles involving specific interactions between type I and type II receptors. Further studies will be required to delineate the precise molecular mechanism underlying these interactions. Nevertheless, our findings contribute to a deeper understanding of Activin A\u0026rsquo;s functional roles in HO and its relevance in driving pathological BMP signaling.\u003c/p\u003e \u003cp\u003eThe identification of BMPR2\u003csup\u003eE376K\u003c/sup\u003e as a neomorphic mutation opens new avenues for understanding the molecular mechanisms underlying HO. While our in vitro assays have provided compelling evidence of these effects, future validation in animal models will be crucial to confirm the causal relationship between \u003cem\u003eBMPR2\u003c/em\u003e mutations and heterotopic ossification. Taken together, this study highlights the pathogenic role of a novel \u003cem\u003eBMPR2\u003c/em\u003e mutation in heterotopic ossification through enhanced chondrogenic differentiation. The findings expand our understanding of BMP signaling dysregulation and its role in skeletal disorders. Future studies should aim to explore the molecular mechanisms in greater detail and evaluate potential therapeutic strategies to mitigate the clinical manifestations of this disorder.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThe goal of this study was to identify novel causative variants associated with FOP, to validate pathogenicity of the variant and to understand molecular basis of dominant negative activities of the gain-of-function mutation. Eventually, we wanted to understand pathophysiology of the human skeletal disorder, FOP, caused by genetic alterations. In order to identify causative mutations, we conducted whole exome sequencing of the individual presented with variant FOP phenotypes. Using the dermal fibroblasts obtained from the affected individual, we determined significantly enhanced BMP signaling at the molecular and cellular levels. To validate the pathogenicity of the BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant, the mutated allele was deleted through CRISPR/Cas9 mediated silencing from the patient-derived cells, which results in abrogation of the enhanced BMP signaling. Consistently, heterotopic expression of the BMPR2\u003csup\u003eE376K\u003c/sup\u003e induced ligand-independent SMAD1/5/9 phosphorylation and chondro-osseus differentiation. In attempt to understand the molecular basis of the dominant negative activities of the BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant, we performed immunoprecipitation assay and found that the BMPR2\u003csup\u003eE376K\u003c/sup\u003e is associated with wildtype ACVR1, leading to activation of ligand-independent BMP signaling. We also found that combined expression of causative FOP variants ACVR1\u003csup\u003eR206H\u003c/sup\u003e and BMPR2\u003csup\u003eE376K\u003c/sup\u003e showed additive effects of enhanced BMP signaling, implying that ACVR1\u003csup\u003eR206H\u003c/sup\u003e might be different molecular mechanism for activation of BMP signaling. However, similar to the case of ACVR1\u003csup\u003eR206H\u003c/sup\u003e, treatment of Activin A is only responsive to BMPR2\u003csup\u003eE376K\u003c/sup\u003e, but not to wildtype BMPR2, suggesting that Activin A plays an important role in dysregulation of BMP signaling and FOP phenotypes. Interestingly, we noticed that SMAD2 is phosphorylated in cells expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e. In order to determine the responsive type I receptor for SMAD phosphorylation, we depleted an individual type I receptor in cells stably expressing BMPR2\u003csup\u003eE376K\u003c/sup\u003e and found that TGFβR1 is responsible for SMAD2 phosphorylation in the context of BMPR2\u003csup\u003eE376K\u003c/sup\u003e variant. We further confirmed that both BMP and TGFβ signals are important for FOP phenotypes. Dermal fibroblasts and genomic DNA samples were obtained from the individual with variant FOP phenotypes and his family members. The institutional review boards of the Seoul National University Hospital, Seoul, Republic of Korea approved the studies. Detailed experimental design, statistics, and methods are provided in the main text and Materials and methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003eGenomic DNA was obtained from the proband, the sibling and his parents, and dermal fibroblast cells were derived from the proband, after obtaining written informed consent. The institutional Review Board of the Seoul National University Hospital, Seoul, South Korea, approved this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture, DNA construction, Mutagenesis and FOP cell line establishment\u003c/h2\u003e \u003cp\u003ePatient-derived dermal fibroblasts and BJ (wild type control) cells were grown in high-glucose and no-glutamine DMEM (GIBCO, Cat#10313) supplemented with 15% fetal bovine serum (FBS, GIBCO), Glutamax\u0026trade; (GIBCO, Cat#35050-061) and non-essential amino acid (GIBCO, Cat#11140-050) and penicillin and streptomycin (GIBCO, 15140-122). Fibroblasts were incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e and 3% O\u003csub\u003e2\u003c/sub\u003e at 37℃. BJ foreskin fibroblasts were obtained from ATCC. HEK293T, HeLa and U2OS cells were grown in high-glucose DMEM (GIBCO, Cat#11965) supplemented with 10% FBS (GIBCO) and 1X penicillin and streptomycin (GIBCO, Cat#15140-122) and incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃. C2C12 myoblasts were cultured in high-glucose, glutamine and sodium pyruvate DMEM (GIBCO, Cat#11995) supplemented with 10% FBS and 1X penicillin and streptomycin at 37℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Undifferentiated C2C12 cells were sparsely maintained in a polystyrene cell culture dish to prevent myogenesis induced by cell contact. C3H10T1/2 fibroblasts were cultured in high-glucose, glutamine and sodium pyruvate DMEM (GIBCO, Cat#11995) supplemented with 10% FBS and 1X penicillin and streptomycin and were incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃\u003csup\u003e61\u003c/sup\u003e. Primary patient-derived fibroblasts and BJ cells were immortalized by expressing the catalytic subunit of human telomerase (hTERT) through lentiviral transduction and transformed by the human papilloma virus E6 and E7 protein through retroviral transduction. BMPR2 cDNA was obtained from addgene. BMPR2 cDNA was cloned to EcoRI restriction sites in pcDNA6/V5-HisABC vector using In-Fusion HD Cloning kits (Takara, Cat#638920) and pDONR223 BP vector and later pHAGE-HA-FLAG LR vector using Gateway cloning system (Thermo Fisher Scientific). C.1126G\u0026thinsp;\u0026gt;\u0026thinsp;A BMPR2 mutation was generated by QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Genomics) with the following primer: BMPR2-F: 5\u0026rsquo;-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACTTCCTCGCTGCAGCGGC-3\u0026rsquo;, BMPR2-R: 5\u0026rsquo;-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACAGACAGTTCATTCC-3\u0026rsquo;. Cell lines stably expressing BMPR2 or BMPR2 mutants were generated by lentiviral transduction as previously\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eAntibodies used in this study are listed below with their respective working concentration. Mouse anti-V5-Tag (Invitrogen, #R960-25, 1:1000), rabbit anti-phospho-SMAD1/5/9 (Cell Signaling Technology, #13820, 1:1000), rabbit anti\u0026ndash;p-Smad1/5/8 [clone 41D10], CST, #9516, 1:1000), rabbit anti-SMAD1 (CST, #6944, 1:2000), rabbit anti-SMAD5 (CST, #12534, 1:2000), rabbit anti-phospho-SMAD2 (CST, #3108, 1:1000), rabbit anti-SMAD2 (CST, #5339, 1:1000), rabbit anti-SMAD4 (CST, #9515, 1:1000), rabbit anti-ID1 (SANTA CRUZ BIOTECHNOLOGY, sc-488, 1:1000), rabbit anti-ID3 (SCBT, sc-490, 1:1000), mouse anti-HA (Covance, MMS-101R, 1:1000), rabbit anti-SOX9 (CST, #82630, 1:1000), rabbit anti-BMPR2 (CST, #6979, 1:1000), rabbit anti-Osteocalcin (Merck Millipore, AB10911, 1:1000), rabbit anti-Alkaline Phosphatase (abcam, ab108337, 1:1000), rabbit anti-RUNX2 (SCBT, sc-10758, 1:1000), mouse anti\u0026ndash;β-actin [clone 8H10D10], CST, #3700, 1:5000) and rabbit anti-GAPDH (SCBT, sc-25778, 1:1000) as a loading control. Anti-mouse secondary (Jackson ImmunoResearch, 115-035-003, 1:2500), anti-rabbit secondary (Jackson ImmunoResearch, 111-035-003, 1:2500), and anti-mouse secondary light chain specific (Jackson ImmunoResearch, 115-035-174, 1:2500), and anti-rabbit IgG, horseradish peroxidase\u0026ndash;conjugated secondary antibody (CST, #7074, 1:5000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOsteogenic differentiation\u003c/h2\u003e \u003cp\u003eBJ and patient-derived dermal fibroblasts (8x10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded into 24-well cell culture plates and then cultured in DMEM (GIBCO, Cat#10313) supplemented with 15% FBS, 1% Glutamax, 1% non-essential amino acid and 1% penicillin-streptomycin at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e and 3% O\u003csub\u003e2\u003c/sub\u003e. To induce differentiation, growth medium was replaced into DMEM supplemented with 2% FBS after cells reached 80\u0026thinsp;~\u0026thinsp;90% confluence. Cells were maintained without or with recombinant human BMP2 or BMP4 (R\u0026amp;D SYSTEMS) and replaced with fresh medium every 2\u0026thinsp;~\u0026thinsp;3 days for 2\u0026thinsp;~\u0026thinsp;21 days. C2C12 cells were seeded into 24-well cell culture plates at a density of 4x10\u003csup\u003e4\u003c/sup\u003e cells/well. Cells were grown in DMEM (GIBCO, Cat#11995) supplemented with 10% FBS at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells with 80\u0026thinsp;~\u0026thinsp;90% confluence were replaced by osteogenic differentiation DMEM (GIBCO, Cat#11995) containing 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 \u0026micro;M ascorbic acid-2-phosphate (all from Sigma) supplemented with 2% FBS\u003csup\u003e63\u003c/sup\u003e. C2C12 were treated with BMP2, BMP4, dorsomorphin (Sigma), or SB431542 (Sigma) and maintained with replacement of fresh medium every 2\u0026thinsp;~\u0026thinsp;3 days for 3\u0026thinsp;~\u0026thinsp;21 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChondrogenic differentiation\u003c/h2\u003e \u003cp\u003eFor chondrogenesis, C3H10T1/2 cells were cultured by micromass technique, high density dot culture. First, cells were resuspended in DMEM supplemented with 10% FBS and 1X penicillin-streptomycin at a concentration of 10\u003csup\u003e7\u003c/sup\u003e cells/ml and a 10 \u0026micro;l droplet of the cell suspension was placed in the center of a well of 12-well cell culture plates followed by incubation at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. After 2 hours, 1 ml chondrogenic differentiation medium DMEM/F12 (GIBCO, Cat#11320) consisting of 1% FBS, 1% Insulin-Transferrin-Selenium (GIBCO), 0.1 \u0026micro;M dexamethasone, 0.17 mM ascorbic acid-2-phosphate, 0.35 mM proline (Sigma), and 0.15% glucose (Sigma) was added in each well and cells were maintained without or with human recombinant BMP2. The fresh medium was changed once per 2\u0026thinsp;~\u0026thinsp;3 days for 21 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWhole exome sequencing and DNA analysis\u003c/h2\u003e \u003cp\u003e Written informed consent was obtained from the affected individual. The Institutional Review Board of the Seoul National University Hospital, Seoul, South Korea approved the studies. Genomic DNAs were extracted from whole blood and sequencing libraries were prepared using Twist modular library preparation kits. We used SureSelect Human All Exon V5 baits covering all exon regions (Agilent, Santa Clara, CA). Targeted sequencing was performed with 101 base pair (bp) paired-end reads on an Illumina HiSeq2500 platform (Illumina, San Diego, CA). Sequenced reads were aligned to human genome reference sequence (hg19) using Burrows-Wheeler Aligner (BWA) version 0.7.5a with the Maximum Entropy Method (MEM) algorithm. At the same time, the aligned reads were selected mapping phred quality score above 30, converted to binary alignment map (BAM) format and sorted ordering by genomic position using SAMTOOLS version 1.2. For high performance accurate variant calling, i) PCR duplicates reads were marked using MarkDuplicates of Picard tools version 1.127 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://broadinstitute.github.io/picard/\u003c/span\u003e\u003cspan address=\"http://broadinstitute.github.io/picard/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). ii) Insertion and deletion (Indel) realignment were performed with known Indels from Mills and 100G gold standard using RealignerTargetCreator and IndelRealigner of Genome Analysis Tool Kit (GATK) version 3.1-1. iii) Base quality score was recalibrated using machine learning model with known single nucleotide polymorphisms (SNPs) and Indels from dbSNP138, Mills and 1000 Genome Project phase I by BaseRecalibrator and PrinReads of GATK. Manipulated BAMs were simultaneously called and genotyped of single nucleotide variants (SNVs) and Indels by GATK UnifiedGenotyper uses a Bayesian genotype likelihood model. Variants were recalibrated with reference variants such as dbSNP138, Mills Indels, HapMap and Omni using GATK VariantRecalibrator and ApplyRecalibration. Variants were annotated various information using ANNOVAR described below: i) population database such as 1000 genome phase III, ExAC and KRGDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://coda.nih.go.kr/coda/KRGDB/\u003c/span\u003e\u003cspan address=\"http://coda.nih.go.kr/coda/KRGDB/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ii) disease database such as OMIM, sequencing database such as RefSeqGene, iii) in silico predictive algorithms such as FATHMM, MutationAssessor, MutationTaster, SIFT, Polyphen, GERP and Phylop for interpretation and classification of variants following ACMG guideline. Classified pathogenic or likely pathogenic variants were confirmed by Sanger sequencing. Copy number variants (CNVs) were calculated using aligned read counts in target region by in-house relative comparison method. Detected and classified pathogenic CNVs were re-confirmed by array comparative genomic hybridization (array CGH)\u003csup\u003e\u003cspan additionalcitationids=\"CR65 CR66 CR67\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSmall interfering RNA (siRNA)\u003c/h2\u003e \u003cp\u003esiRNAs were transfected twice into cells, first by reverse transfection and 24 hours later by forward transfection using Lipofectamine RNAiMAX reagent (Invitrogen) as suggested by the manufacturer\u0026rsquo;s instructions. ACVR1 (ID#s974, s976), TGFBR1 (ID#s14071, s14073), and BMPR2 (ID#s2044, s2045, s2046) siRNAs were purchased from Thermo Fisher Scientific. Pools of two or three siRNAs were used with a final siRNA concentration of 25 nM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003e293T cells were plated in the Falcon\u0026reg; 96-well white flat bottom tissue culture-treated microtest assay microplate (CORNING). In each well, 5,000 cells were plated in 100 \u0026micro;l 10% DMEM media. 24 hours after plating, cells were transfected with pcDNA-empty vector, pcDNA6/V5-HisA-wildtype BMPR2 or -mutant BMPR2, pGL3-BMP responsive elements-luciferase (hereafter pGL3-BRE-luc, offered from addgene plasmid #45126), and pNL1.1.TK internal control vector for the assay, using calcium phosphate transfection Kit (Invitrogen). The amounts of WT or mutant BMPR2, and pGL3-BRE-luc, and pNL1.1 from Nano-Glo\u0026reg; Dual-Luciferase\u0026reg; Reporter Assay Kit (Promega) were determined according to a protocol of calcium phosphate transfection from Clontech Laboratories; 50ng of WT BMPR2 or mutant BMPR2 and pGL3-BRE-luc and 5ng of pNL1.1.TK were used and then 2M Calcium Solution and sterile water were added in each DNA tube. The same volume of 2X HEPES-Buffered Saline (HBS) was added to Calcium-DNA mixture dropwise and incubated at room temperature. After 15 minutes, the transfection solution was carefully added to culture plate medium and maintained at 37℃ in a CO\u003csub\u003e2\u003c/sub\u003e incubator. The next day, the calcium phosphate-containing medium was removed from cells and replaced with fresh complete growth medium. The volume of One-Glo\u0026trade; EX Luciferase assay Reagent was equally added to the culture medium volume to each well and placed on an orbital shaker at 300 rpm for 3 minutes. Luminescence was measured as integration times of 1 second by GloMax\u0026reg; Discover System (Promega). For measurement of NanoLuc\u0026reg; luciferase activity, a volume of NanoDLR\u0026trade; Stop \u0026amp; Glo\u0026reg; Reagent was equally added to the original culture medium volume to each well and then luminescence was analyzed. The BRE reporter luminescence was normalized to NanoLuc\u0026reg; luciferase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting and Immunoprecipitation\u003c/h2\u003e \u003cp\u003eCells were plated either in 60 mm or 100 mm plate with 70% confluency. The next day, plasmid DNA was transfected into HEK293T cells by Lipofectamine 2000. After 4 hours, cells were changed into serum free medium and treated with human recombinant Activin A (R\u0026amp;D SYSTEMS) the next day. Cells were harvested and lysed by lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, and 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Roche) and quantified by Protein Assay Dye Reagent Concentrate (Bio-Rad) and NanoDrop (Thermo Fisher Scientific). Proteins were separated by 8\u0026thinsp;~\u0026thinsp;15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gels were blotted onto polyvinylidene difluoride (PVDF) transfer membrane with 0.45 \u0026micro;m pore size (Merck Millipore). Blots were blocked in 1X PBS with 0.1% Tween-20 (Sigma) containing 5% Difco\u0026trade; Skim Milk (BD) for 1 hour at room temperature and incubated with the antibodies indicated in each figure at 4℃ for overnight. After the blots were washed four times in 1X PBST for 1 hour at room temperature, the membranes were incubated with the secondary antibody for 2 hours at room temperature\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. The bands were then detected using an enhanced chemiluminescence solution (Bio-Rad) and visualized with the ChemiDoc System (Bio-Rad). The band image was analyzed with Image Lab\u0026trade; Software (Version 5.2.1, Bio-Rad).\u003c/p\u003e \u003cp\u003eFor immunoprecipitation, transiently transfected HEK293T cells were lysed and sonicated in lysis buffer at 4℃. Crude lysates cleared by centrifugation at 15,000 rpm at 4℃ for 20 minutes. Supernatants were incubated with Monoclonal Anti-HA-Agarose antibody (Sigma) for 2 hours at 4℃. Immunocomplex was washed five times with lysis buffer and then SDS-PAGE and western blotting were performed\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Anti-mouse secondary light chain specific was incubated for 2 hours at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eReal-time quantitative reverse transcription PCR\u003c/h2\u003e \u003cp\u003eTotal RNA of the cells was extracted using RNeasy Mini Kit and QIAshredder (QIAGEN) and quantified using NanoDrop instrument. 1 \u0026micro;g of total RNA was used to cDNA synthesis using a SuperScript III First-Strand Synthesis System (Invitrogen). Gene expression was quantified by 2X qPCRBIO SyGreen Blue Mix Lo-ROX (PCRBIOSYSTEMS) performed on LightCycler\u0026reg; 96 (Roche)\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Quantification cycle (Cq) values of samples were analyzed by LightCycler\u0026reg; 96 Application Software (Version 1.1). Gene-specific primers are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAlizarin S staining (Mineralization assay)\u003c/h2\u003e \u003cp\u003eThe mineralization was determined by staining with Alizarin Red S at 21 days after osteogenic differentiation. For preparation of solution, 2 g Alizarin Red S (Sigma) was dissolved in 100 ml distilled water and then adjusted to pH4.3 with HCl or NH\u003csub\u003e4\u003c/sub\u003eOH. Differentiated cells were carefully washed with PBS and fixed with 4% paraformaldehyde (Sigma). After 30 minutes carefully washed the cells with distilled water followed by prepared stain solution was enough added to the cells for 45 minutes at room temperature in the dark. The cells were washed four times with distilled water and carefully aspirated. The differentiated cells are stained darker red with calcium deposits. After photography using digital camera (Nikon), the stained cells were lysed with 10% cetypyridium chloride (sigma) dissolved in 10 mM sodium phosphate buffer (1 M NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e monobasic and 1 M Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e dibasic, pH7.0) and then quantified at 560 nm using a GloMax\u0026reg; Discover System.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eAlkaline Phosphatase (ALP) staining and activity\u003c/h2\u003e \u003cp\u003eFor detection of alkaline phosphatase, cells were firstly cultured with osteogenic differentiation media for 2 or 3 days. Cells were cautiously washed with PBS and then fixed with 4% paraformaldehyde. After 1 minute, cells were rinsed with Washing Buffer (0.05% Tween 20 in PBS), subsequently treated with substrate solution which was dissolved one BCIP/NBT tablet (Sigma) in 10 ml distilled water. For staining, the cells were incubated at room temperature in the dark for 10 minutes monitoring staining progress every 2\u0026thinsp;~\u0026thinsp;3 minutes. Carefully aspirated the substrate solution and rinsed the cell with Washing Buffer. The higher alkaline phosphatase, the more intense the dark blue-violet. For ALP activity, cultured cells were washed with PBS and lysed with cold alkaline phosphatase reaction buffer (1 M Diethanolamine and 0.5 mM Magnesium Chloride, pH9.8, Sigma). Lysates were incubated in 0.67 M \u003cem\u003ep\u003c/em\u003e-Nitrophenyl Phosphate (pNPP) solution (Sigma) for 30 minutes at 37℃ continuing the reaction was immediately followed by monitoring in absorbance at 405 nm. Total protein was measured by using a Micro-BCA protein assay kit (Thermo Fisher Scientific) and read at 560 nm using a GloMax\u0026reg; instrument. The enzymatic ALP activity was normalized to the protein content of the samples.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAlcian blue staining\u003c/h2\u003e \u003cp\u003eTo visualize ability of chondrogenesis, stain solution (pH1.0) was prepared with 1 g Alcian blue 8GX (Sigma) in 100 ml 0.1 M HCl. Cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature and then rinsed 3 times with PBS. Alcian blue solution was used to stain the cells at room temperature in the dark. Next day, cells were washed once with 0.1 M HCl and twice with PBS. After taking a picture, the dye was extracted by 6 M Guanidine-HCl (Sigma) for 2 hours at room temperature and then read in absorbance at 600 nm using a GloMax\u0026reg; instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with GraphPad Prism 8 software. Data were presented as bar graphs with dot blots for mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of measurement (SEM). For comparison between two normally distributed test groups was used the two-tailed unpaired student\u0026rsquo;s t-test. Analysis of three or more groups were performed using two-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s multiple comparisons test. \u003cem\u003eP\u003c/em\u003e values were statistically considered significant in \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The following standard symbols are used to reference P values: ns, not significant; * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the affected individual and his family.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYK and TJC conceived the study. MJK, EJ, DK, YYK, SWC, HMR, SWJ, YY and YK designed, performed, and analyzed most of the laboratory works. CHS, FSK and TJC ascertained and recruited the proband with FOP. HRL, WYP and TJC performed whole exome sequencing and analyzed the data. FSK read and revised the manuscript. YK, MJK, EJ and TJC wrote the initial draft of the manuscript, with contributions and revision from all other authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Genome Technology to Business Translation Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT \u0026amp; Future Planning (NRF-2014M3C9A2064684 to TJC); by an NRF grant funded by the Korean government (NRF-2023R1A2C3007266 and NRF-2021R1A6A1A03038890 to YK). This research was partially supported by Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (No. 2021R1A6C101A564 and RS-2024-00436674), Industrial Technology Innovation Program (RS-2024-00403190) funded by the Ministry of Trade, Industry \u0026amp; Energy of the Republic of Korea, and Korea Drug Development Fund (RS-2024-00463605).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShore, E. M. \u0026amp; Kaplan, F. S. Inherited human diseases of heterotopic bone formation. \u003cem\u003eNat. Rev. 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Elucidation of molecular basis of osteolytic bone lesions in advanced multiple myeloma. \u003cem\u003eHaematologica\u003c/em\u003e 109, 2207\u0026ndash;2218 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bone-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"boneres","sideBox":"Learn more about [Bone Research](http://www.nature.com/boneres/)","snPcode":"41413","submissionUrl":"https://mts-boneres.nature.com/cgi-bin/main.plex","title":"Bone Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Gain-of-function, BMPR2, Heterotopic ossification, Fibrodysplasia Ossificans Progressiva","lastPublishedDoi":"10.21203/rs.3.rs-8453923/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8453923/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeterotopic ossification (HO), a pathological process in which bone forms in soft tissues is rare and debilitating without effective treatment. Gain-of-function mutations in \u003cem\u003eACVR1\u003c/em\u003e cause fibrodysplasia ossificans progressiva (FOP). Here we report a novel, ultrarare gain of function mutation in \u003cem\u003eBMPR2\u003c/em\u003e (c.1126G\u0026thinsp;\u0026gt;\u0026thinsp;A, p.E376K) that causes a systemic HO simulating FOP. The pathological features associated with BMPR2\u003csup\u003eE376K\u003c/sup\u003e appear reminiscent of classic FOP, yet manifest a number of distinct hallmarks, including lack of stereotypic malformation of the big toes. BMPR2\u003csup\u003eE376K\u003c/sup\u003e appears to function as a neomorph, displaying an exaggerated response to Activin A stimulation by selectively interacting with ACVR1. These findings are consistent with the central role of Activin A mediated ACVR1 signaling in FOP. Taken together, our data illustrates the complex molecular features underlying the pathophysiology of HO and highlight the importance of BMPR2 as a nexus for ACVR1 and Activin A interaction. Moreover, our findings provide a theoretical framework for developing novel therapeutic options for HO.\u003c/p\u003e","manuscriptTitle":"A Novel Gain-of-Function Mutation in BMPR2 in a Patient with Variant Fibrodysplasia Ossificans Progressiva","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 10:49:22","doi":"10.21203/rs.3.rs-8453923/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-13T09:50:11+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-13T02:59:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-30T01:29:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-26T09:10:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bone Research","date":"2025-12-26T09:10:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bone-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"boneres","sideBox":"Learn more about [Bone Research](http://www.nature.com/boneres/)","snPcode":"41413","submissionUrl":"https://mts-boneres.nature.com/cgi-bin/main.plex","title":"Bone Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c50a78c-f4a6-44f5-8bd8-b4eedf20d2b2","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":61032735,"name":"Health sciences/Pathogenesis"},{"id":61032736,"name":"Biological sciences/Physiology/Bone"}],"tags":[],"updatedAt":"2026-02-27T03:41:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 10:49:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8453923","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8453923","identity":"rs-8453923","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}