Meiqi Xu

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

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

The fibrodysplasia ossificans progressiva R206H ACVR1 mutation activates BMP-independent chondrogenesis and zebrafish embryo ventralization

Patients with classic fibrodysplasia ossificans progressiva, a disorder characterized by extensive extraskeletal endochondral bone formation, share a recurrent mutation (R206H) within the glycine/serine-rich domain of ACVR1/ALK2, a bone morphogenetic protein type I receptor. Through a series of in vitro assays using several mammalian cell lines and chick limb bud micromass cultures, we determined that mutant R206H ACVR1 activated BMP signaling in the absence of BMP ligand and mediated BMP-independent chondrogenesis that was enhanced by BMP. We further investigated the interaction of mutant R206H ACVR1 with FKBP1A, a glycine/serine domain-binding protein that prevents leaky BMP type I receptor activation in the absence of ligand. The mutant protein exhibited reduced binding to FKBP1A in COS-7 simian kidney cell line assays, suggesting that increased BMP pathway activity in COS-7 cells with R206H ACVR1 is due, at least in part, to decreased binding of this inhibitory factor. Consistent with these findings, in vivo analyses of zebrafish embryos showed BMP-independent hyperactivation of BMP signaling in response to the R206H mutant, resulting in increased embryonic ventralization. These data support the conclusion that the mutant R206H ACVR1 receptor in FOP patients is an activating mutation that induces BMP signaling in a BMP-independent and BMP-responsive manner to promote chondrogenesis, consistent with the ectopic endochondral bone formation in these patients.

Early Diagnosis of Fibrodysplasia Ossificans Progressiva

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

Hematopoietic Stem-Cell Contribution to Ectopic Skeletogenesis

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

Diagnostic and Mutational Spectrum of Progressive Osseous Heteroplasia (POH) and Other Forms of GNAS-based Heterotopic Ossification

Progressive osseous heteroplasia (POH) is a rare, disabling disease of heterotopic ossification (HO) that progresses from skin and subcutaneous tissues into deep skeletal muscle. POH occurs in the absence of multiple developmental features of Albright hereditary osteodystrophy (AHO) or hormone resistance, clinical manifestations that are also associated with GNAS inactivation. However, occasional patients with AHO and pseudohypoparathyroidism 1a/c (PHP1a/c; AHO features plus hormone resistance) have also been described who have progressive HO. This study was undertaken to define the diagnostic and mutational spectrum of POH and progressive disorders of HO, and to distinguish them from related disorders in which HO remains confined to the skin and subcutaneous tissues. We reviewed the charts of 111 individuals who had cutaneous and subcutaneous ossification. All patients were assessed for eight characteristics: age of onset of HO, presence and location of HO, depth of HO, type of HO, progression of HO, features of AHO, PTH resistance, and GNAS mutation analysis. We found, based on clinical criteria, that POH and progressive HO syndromes are at the severe end of a phenotypic spectrum of GNAS‐inactivating conditions associated with extra‐skeletal ossification. While most individuals with superficial or progressive ossification had mutations in GNAS, there were no specific genotype‐phenotype correlations that distinguished the more progressive forms of HO (e.g., POH) from the non‐progressive forms (osteoma cutis, AHO, and PHP1a/c).

The genetics of fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare, heritable disorder with infrequent genetic transmission of the condition owing to low reproductive, fitness. The recent identification of several small, additional families with inheritance of classic features of FOP has provided the opportunity to expand positional cloning efforts to identify the mutated gene in FOP through whole-genome linkage nnalyses. Candidate gene studies using both genetic (linkage), and molecular (gene expression) approaches also are contributing to information about the genetic and cellular, causes of FOP.

Heterozygous inactivation of Gnas in adipose-derived mesenchymal progenitor cells enhances osteoblast differentiation and promotes heterotopic ossification

Human genetic disorders sharing the common feature of subcutaneous heterotopic ossification (HO) are caused by heterozygous inactivating mutations in GNAS, a gene encoding multiple transcripts including two stimulatory G proteins, the α subunit of the stimulatory G protein (Gsα) of adenylyl cyclase, and the extralong form of Gsα, XLαs. In one such disorder, progressive osseous heteroplasia (POH), bone formation initiates within subcutaneous fat before progressing to deeper tissues, suggesting that osteogenesis may involve abnormal differentiation of mesenchymal precursors that are present in adipose tissues. We determined by immunohistochemical analysis that GNAS protein expression is limited to Gsα in bone‐lining cells and to Gsα and XLαs in osteocytes. By contrast, the GNAS proteins Gsα, XLαs, and NESP55 are detected in adipocytes and in adipose stroma. Although Gnas transcripts, as assessed by quantitative RT‐PCR, show no significant changes on osteoblast differentiation of bone‐derived precursor cells, the abundance of these transcripts is enhanced by osteoblast differentiation of adipose‐derived mesenchymal progenitors. Using a mouse knockout model, we determined that heterozygous inactivation of Gnas (by disruption of the Gsα‐specific exon 1) abrogates upregulation of multiple Gnas transcripts that normally occurs with osteoblast differentiation in wild‐type adipose stromal cells. These transcriptional changes in Gnas+/− mice are accompanied by accelerated osteoblast differentiation of adipose stromal cells in vitro. In vivo, altered osteoblast differentiation in Gnas+/− mice manifests as subcutaneous HO by an intramembranous process. Taken together, these data suggest that Gnas is a key regulator of fate decisions in adipose‐derived mesenchymal progenitor cells, specifically those which are involved in bone formation.

Cellular Hypoxia Promotes Heterotopic Ossification by Amplifying BMP Signaling

Hypoxia and inflammation are implicated in the episodic induction of heterotopic endochondral ossification (HEO); however, the molecular mechanisms are unknown. HIF‐1α integrates the cellular response to both hypoxia and inflammation and is a prime candidate for regulating HEO. We investigated the role of hypoxia and HIF‐1α in fibrodysplasia ossificans progressiva (FOP), the most catastrophic form of HEO in humans. We found that HIF‐1α increases the intensity and duration of canonical bone morphogenetic protein (BMP) signaling through Rabaptin 5 (RABEP1)‐mediated retention of Activin A receptor, type I (ACVR1), a BMP receptor, in the endosomal compartment of hypoxic connective tissue progenitor cells from patients with FOP. We further show that early inflammatory FOP lesions in humans and in a mouse model are markedly hypoxic, and inhibition of HIF‐1α by genetic or pharmacologic means restores canonical BMP signaling to normoxic levels in human FOP cells and profoundly reduces HEO in a constitutively active Acvr1Q207D/+ mouse model of FOP. Thus, an inflammation and cellular oxygen‐sensing mechanism that modulates intracellular retention of a mutant BMP receptor determines, in part, its pathologic activity in FOP. Our study provides critical insight into a previously unrecognized role of HIF‐1α in the hypoxic amplification of BMP signaling and in the episodic induction of HEO in FOP and further identifies HIF‐1α as a therapeutic target for FOP and perhaps nongenetic forms of HEO.

GNAS-associated disorders of cutaneous ossification: two different clinical presentations

Progressive osseous heteroplasia (POH) is a rare genetic disorder characterized by dermal ossification during infancy and progressive ossification into deep connective tissue during childhood. POH is at the severe end of a spectrum of GNAS-associated ossification disorders that include osteoma cutis and Albright Hereditary Osteodystrophy (AHO). Here we describe two girls who have different clinical presentations that reflect the variable expression of GNAS-associated disorders of cutaneous ossification. Each girl had a novel heterozygous inactivating mutation in the GNAS gene. One girl had POH limited to the left arm with severe contractures and growth retardation resulting from progressive heterotopic ossification in the deep connective tissues. The other girl had AHO with widespread, superficial heterotopic ossification but with little functional impairment. While there is presently no treatment or prevention for GNAS-associated ossification disorders, early diagnosis is important for genetic counselling and for prevention of iatrogenic harm.

Mutational Screening of the Bone Morphogenetic Protein 4 Gene in a Family With Fibrodysplasia Ossificans Progressiva

Bone morphogenetic proteins have been proposed as candidate genes for fibrodysplasia ossificans progressiva. Bone morphogenetic protein 4 is overexpressed in cells derived from these patients. The bone morphogenetic protein 4 genes from a family showing autosomal dominant inheritance of fibrodysplasia ossificans progressiva have been screened for mutations by single strand conformation polymorphism analysis and deoxyribonucleic acid sequencing. The exon coding regions and splice junctions of the bone morphogenetic protein 4 gene have been examined for polymorphisms in all five family members. However, no mutation was discovered in these messenger ribonucleic acid and protein coding regions or in the splice junctions of affected or unaffected family members. In addition, approximately 1.5 kb of upstream flanking sequences also were examined. Neutral polymorphisms were identified in the upstream flanking region of the bone morphogenetic protein 4 gene. Although this study has not identified any mutations in the bone morphogenetic protein 4 gene that are correlated with the occurrence of fibrodysplasia ossificans progressiva, the bone morphogenetic protein 4 gene cannot yet be excluded from consideration as the genetic cause of this disorder because a mutation could be present in unexamined regulatory sequences of this gene.