Different roles of GNAS and cAMP signaling during early and late stages of osteogenic differentiation.

Progressive osseous heteroplasia (POH) and fibrous dysplasia (FD) are genetic diseases of bone formation at opposite ends of the osteogenic spectrum: imperfect osteogenesis of the skeleton occurs in FD, while heterotopic ossification in skin, subcutaneous fat, and skeletal muscle forms in POH. POH is caused by heterozygous inactivating germline mutations in GNAS, which encodes G-protein subunits regulating the cAMP pathway, while FD is caused by GNAS somatic activating mutations. We used pluripotent mouse ES cells to examine the effects of Gnas dysregulation on osteoblast differentiation. At the earliest stages of osteogenesis, Gnas transcripts Gsα, XLαs and 1A are expressed at low levels and cAMP levels are also low. Inhibition of cAMP signaling (as in POH) by 2',5'-dideoxyadenosine enhanced osteoblast differentiation while conversely, increased cAMP signaling (as in FD), induced by forskolin, inhibited osteoblast differentiation. Notably, increased cAMP was inhibitory for osteogenesis only at early stages after osteogenic induction. Expression of osteogenic and adipogenic markers showed that increased cAMP enhanced adipogenesis and impaired osteoblast differentiation even in the presence of osteogenic factors, supporting cAMP as a critical regulator of osteoblast and adipocyte lineage commitment. Furthermore, increased cAMP signaling decreased BMP pathway signaling, indicating that G protein-cAMP pathway activation (as in FD) inhibits osteoblast differentiation, at least in part by blocking the BMP-Smad pathway, and suggesting that GNAS inactivation as occurs in POH enhances osteoblast differentiation, at least in part by stimulating BMP signaling. These data support that differences in cAMP levels during early stages of cell differentiation regulate cell fate decisions. Supporting information available online at http:/www.thieme-connect.de/ejournals/toc/hmr.

Restoration of normal BMP signaling levels and osteogenic differentiation in FOP mesenchymal progenitor cells by mutant allele-specific targeting.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of progressive heterotopic ossification for which there is presently no cure. FOP is caused by a recurrent heterozygous activating mutation (c.617G>A; R206H) of Activin receptor type IA/Activin-like kinase-2 (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor that occurs in all classically affected individuals. The FOP mutation dysregulates BMP signaling and initiates the formation of a disabling second skeleton of heterotopic bone. We generated allele-specific siRNA (ASP-RNAi) duplexes capable of specifically suppressing the expression of the mutant c.617A allele in mesenchymal progenitor cells from FOP patients and showed that this ASP-RNAi approach decreased the elevated BMP signaling that is characteristic of patient cells to levels similar to control cells and restored enhanced osteogenic differentiation to control levels. Our results provide proof-of-principle that ASP-RNAi has potential therapeutic efficacy for the treatment of FOP.

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.

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).

Mast cell involvement in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a catastrophic genetic disorder of progressive heterotopic ossification associated with dysregulated production of bone morphogenetic protein 4 (BMP4), a potent osteogenic morphogen. Postnatal heterotopic ossification in FOP is often heralded by hectic episodes of severe post-traumatic connective tissue swelling and intramuscular edema, followed by an intense and highly angiogenic fibroproliferative mass. The abrupt appearance, intense size, and rapid intrafascial spread of the edematous preosseous fibroproliferative lesions implicate a dysregulated wound response mechanism and suggest that cells and mediators involved in inflammation and tissue repair may be conscripted in the growth and progression of FOP lesions. The central and coordinate role of inflammatory mast cells and their mediators in tissue edema, wound repair, fibrogenesis, angiogenesis, and tumor invasion prompted us to investigate the potential involvement of mast cells in the pathology of FOP lesions. We show that inflammatory mast cells are present at every stage of the development of FOP lesions and are most pronounced at the highly vascular fibroproliferative stage. Mast cell density at the periphery of FOP lesional tissue is 40- to 150-fold greater than in normal control skeletal muscle or in uninvolved skeletal muscle from FOP patients and 10- to 40-fold greater than in any other inflammatory myopathy examined. These findings document mobilization and activation of inflammatory mast cells in the pathology of FOP lesions and provide a novel and previously unrecognized target for pharmacologic intervention in this extremely disabling disease.

Fibrodysplasia ossificans progressiva.

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

GNAS1 mutation and Cbfa1 misexpression in a child with severe congenital platelike osteoma cutis.

We evaluated a 7-year-old girl with severe platelike osteoma cutis (POC), a variant of progressive osseous heteroplasia (POH). The child had congenital heterotopic ossification of dermis and subcutaneous fat that progressed to involve deep skeletal muscles of the face, scalp, and eyes. Although involvement of skeletal muscle is a prominent feature of POH, heterotopic ossification has not been observed in the head, face, or extraocular muscles. The cutaneous ossification in this patient was suggestive of Albright hereditary osteodystrophy (AHO); however, none of the other characteristic features of AHO were expressed. Inactivating mutations of the GNAS1 gene, which encodes the alpha-subunit of the stimulatory G protein of adenylyl cyclase, is the cause of AHO. Mutational analysis of GNAS1 using genomic DNA of peripheral blood and of lesional and nonlesional tissue from our patient revealed a heterozygous 4-base pair (bp) deletion in exon 7, identical to mutations that have been found in some AHO patients. This 4-bp deletion in GNAS1 predicts a protein reading frameshift leading to 13 incorrect amino acids followed by a premature stop codon. To investigate pathways of osteogenesis by which GNAS1 may mediate its effects, we examined the expression of the obligate osteogenic transcription factor Cbfa1/RUNX2 in lesional and uninvolved dermal fibroblasts from our patient and discovered expression of bone-specific Cbfa1 messenger RNA (mRNA) in both cell types. These findings document severe heterotopic ossification in the absence of AHO features caused by an inactivating GNAS1 mutation and establish the GNAS1 gene as the leading candidate gene for POH.

Deficiency of the alpha-subunit of the stimulatory G protein and severe extraskeletal ossification.

Progressive osseous heteroplasia (POH) is a rare disorder characterized by dermal ossification beginning in infancy followed by increasing and extensive bone formation in deep muscle and fascia. We describe two unrelated girls with typical clinical, radiographic, and histological features of POH who also have findings of another uncommon heritable disorder, Albright hereditary osteodystrophy (AHO). One patient has mild brachydactyly but no endocrinopathy, whereas the other manifests brachydactyly, obesity, and target tissue resistance to thyrotropin and parathyroid hormone (PTH). Levels of the alpha-subunit of the G protein (Gsalpha) were reduced in erythrocyte membranes from both girls and a nonsense mutation (Q12X) in exon 1 of the GNAS1 gene was identified in genomic DNA from the mildly affected patient. Features of POH and AHO in two individuals suggest that these conditions share a similar molecular basis and pathogenesis and that isolated severe extraskeletal ossification may be another manifestation of Gsalpha deficiency.

Progressive osseous heteroplasia.

Progressive osseous heteroplasia (POH) is a recently described genetic disorder of mesenchymal differentiation characterized by dermal ossification during infancy and progressive heterotopic ossification of cutaneous, subcutaneous, and deep connective tissues during childhood. The disorder can be distinguished from fibrodysplasia ossificans progressiva (FOP) by the presence of cutaneous ossification, the absence of congenital malformations of the skeleton, the absence of inflammatory tumorlike swellings, the asymmetric mosaic distribution of lesions, the absence of predictable regional patterns of heterotopic ossification, and the predominance of intramembranous rather than endochondral ossification. POH can be distinguished from Albright hereditary osteodystrophy (AHO) by the progression of heterotopic ossification from skin and subcutaneous tissue into skeletal muscle, the presence of normal endocrine function, and the absence of a distinctive habitus associated with AHO. Although the genetic basis of POH is unknown, inactivating mutations of the GNAS1 gene are associated with AHO. The report in this issue of the JBMR of 2 patients with combined features of POH and AHO--one with classic AHO, severe POH-like features, and reduced levels of Gsalpha protein and one with mild AHO, severe POH-like features, reduced levels of Gsalpha protein, and a mutation in GNAS1--suggests that classic POH also could be caused by GNAS1 mutations. This possibility is further supported by the identification of a patient with atypical but severe platelike osteoma cutis (POC) and a mutation in GNAS1, indicating that inactivating mutations in GNAS1 may lead to severe progressive heterotopic ossification of skeletal muscle and deep connective tissue independently of AHO characteristics. These observations suggest that POH may lie at one end of a clinical spectrum of ossification disorders mediated by abnormalities in GNAS1 expression and impaired activation of adenylyl cyclase. Analysis of patients with classic POH (with no AHO features) is necessary to determine whether the molecular basis of POH is caused by inactivating mutations in the GNAS1 gene.

Linkage exclusion and mutational analysis of the noggin gene in patients with fibrodysplasia ossificans progressiva (FOP).

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder characterized by congenital malformation of the great toes and by progressive heterotopic endochondral ossification in predictable anatomical patterns. Although elevated levels of bone morphogenetic protein 4 (BMP4) occur in lymphoblastoid cells and in lesional cells of patients with FOP, mutations have not been identified in the BMP4 gene, suggesting that the mutation in FOP may reside in a BMP4-interacting factor or in another component of the BMP4 pathway. A powerful antagonist of BMP4 is the secreted polypeptide noggin. A recent case report described a heterozygous 42-bp deletion in the protein-coding region of the noggin gene in a patient with FOP. In order to determine if noggin mutations are a widespread finding in FOP, we examined 31 families with 1 or more FOP patients. Linkage analysis with an array of highly polymorphic microsatellite markers closely linked to the noggin gene was performed in four classically-affected multigenerational FOP families and excluded linkage of the noggin locus to FOP (the multipoint lod score was -2 or less throughout the entire range of markers). We sequenced the noggin gene in affected members of all four families, as well as in 18 patients with sporadic FOP, and failed to detect any mutations. Single-strand conformation polymorphism (SSCP) analysis of 4 of these patients plus an additional 9 patients also failed to reveal any mutations. Among the samples analyzed by SSCP and DNA sequencing was an independently obtained DNA sample from the identical FOP patient previously described with the 42-bp noggin deletion; no mutation was detected. Examination of the DNA sequences of 20 cloned noggin PCR products, undertaken to evaluate the possibility of a somatic mutation in the noggin gene which could be carried by a small subset of white blood cells, also failed to detect the presence of the reported 42-bp deletion. We conclude that mutations in the coding region of noggin are not associated with FOP.

Osteogenic induction in hereditary disorders of heterotopic ossification.

The formation of heterotopic bone within soft connective tissue is a common feature of at least three distinct genetic disorders of osteogenesis in humans: fibrodysplasia ossificans progressiva; progressive osseous heteroplasia; and Albright hereditary osteodystrophy. The pathobiologic characteristics of osteogenic induction, the histopathologic features of osteogenesis, the anatomic distribution of heterotopic lesions, and the developmental patterns of disease progression differ among all three conditions. The molecular and cellular basis of redirecting a mature connective tissue phenotype to form bone is a remarkable biological phenomenon with enormous implications for the control of bone regeneration, fracture healing, and disorders of osteogenesis.

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

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

Phenotypic and molecular heterogeneity in fibrodysplasia ossificans progressiva.

Fibrodysplasia (myositis) ossificans progressiva (FOP) is an extremely rare inherited disorder in which progressive ossification of major striated muscles, often following injury, is associated with abnormal skeletal patterning. Altered expression of bone morphogenetic proteins may be a contributory cause. To examine this hypothesis, we compared the patterns of expression of bone morphogenetic proteins (BMPs) mRNAs from lymphoblastoid cell lines from two small multigenerational families with autosomal dominant transmission of FOP. Although affected members of both families showed the characteristic phenotype of FOP, one family was more severely affected than the other. Expression of mRNAs for BMP-1, 2, 3, 5, and 6 mRNAs were not detected within the more severely affected family, but BMP-4 mRNA was expressed in affected but not unaffected members of this family. The results of linkage exclusion analysis using a highly polymorphic microsatellite marker near the BMP-4 gene were consistent with linkage of FOP and BMP-4 in this family. Within the less severely affected family, affected and unaffected members showed similar levels of mRNA expression of BMPs 1, 2, 4, and 5, and linkage of FOP to the BMP-4 gene was excluded. It is concluded that clinical, radiographic, and biochemical data in these two families with FOP establish clinical and molecular heterogeneity and also suggest the possibility of genetic heterogeneity.

Illustrative disorders of ectopic skeletal morphogenesis: a childhood parallax for studies in gravitational and space biology.

Heterotopic ossification is a key feature of at least three distinct genetic disorders of osteogenesis in humans: fibrodysplasia ossificans progressiva, progressive osseous heteroplasia, and Albright's hereditary osteodystrophy. All three conditions are genetic disorders of childhood, but the pathobiology of osteogenic induction, the histopathology of osteogenesis, the anatomic distribution of heterotopic lesions, and the developmental patterns of disease progression differ among the three conditions. The phenotypic distinction of these disorders is critically important in counselling patients and families as well as in advancing research to define the molecular pathophysiology of heterotopic osteogenesis in these disabling genetic disorders. Genetic disorders of tissue modeling and morphogenesis provide an important parallax to disturbances of tissue re-modeling that are of paramount importance to gravitational and space biologists as humans begin to explore and live in environments beyond the planet on which they evolved. Disorders of osteogenesis are of particular concern to space biologists due to the dramatic changes in skeletal biology in altered gravitational fields.

The human bone morphogenetic protein 4 (BMP-4) gene: molecular structure and transcriptional regulation.

Bone morphogenetic protein 4 (BMP-4) is a vital regulatory molecule that functions throughout human development in mesoderm induction, tooth development, limb formation, bone induction, and fracture repair and is overexpressed in patients who have fibrodysplasia ossificans progressiva. The human gene encoding bone morphogenetic protein 4 (BMP-4) has been isolated and its structural organization characterized. The complete DNA sequence of an 11.2 kb region has been determined. BMP-4 mRNA is transcribed from four exons, although there is evidence that alternate first exons may be used. Transcript initiation occurs at variable positions within a GA-rich region of the DNA. The promoter region is GC-rich with no obvious TATA or CAAT consensus sequences, and contains both positive and negative transcriptional regulatory elements within the 3 kb 5' flanking region of the RNA start site. Comparison of the human and murine BMP-4 genes reveals highly conserved sequences not only in the exon-coding regions but also within the introns and 5' flanking regions. BMP-4 localizes to human chromosome 14q21 by fluorescence in situ hybridization, a location more centromeric than that recently reported. These studies provide a foundation for understanding the genetic regulation of this important gene in human development.

Acute lymphocytic infiltration in an extremely early lesion of fibrodysplasia ossificans progressiva.

A 2-year-old child with fibrodysplasia ossificans progressiva underwent a muscle biopsy of a very early lesion, and had findings that showed the earliest stage ever seen in the histopathology of fibrodysplasia ossificans progressiva. This very early stage consisted of intense perivascular lymphocytic infiltration into normal appearing skeletal muscle. A nearly identical histopathologic sequence was noted in a cat with phenotypic features similar to those of fibrodysplasia ossificans progressiva in humans. These new findings represent the earliest documented changes that have ever been noted in fibrodysplasia ossificans progressiva, and provide further histopathologic support for the recent discovery that lymphocytes may play a role in the pathogenesis of heterotopic ossification in fibrodysplasia ossificans progressiva.

Characterization of bone morphogenetic protein 4 receptor in fibrodysplasia ossificans progressiva.

Bone morphogenetic protein 4, a potent osteogenic morphogen, has been implicated in fibrodysplasia ossificans progressiva because it is uniquely overexpressed in lymphoblastoid cells and preosseous fibroproliferative lesional cells of patients with fibrodysplasia ossificans progressiva. Bone morphogenetic protein 4 signals through a heteromeric complex of serine/ threonine kinase receptors (type I and type II) on the surface of responding cells. Semi-quantitative competitive reverse transcription polymerase chain reaction was used to quantitate steady state levels of messenger ribonucleic acid expression for bone morphogenetic protein 4 and the bone morphogenetic protein receptors. These data confirmed the previous finding of elevated steady state levels of bone morphogenetic protein 4 messenger ribonucleic acid in lymphoblastoid cell lines of affected individuals in a family that exhibited autosomal dominant inheritance of fibrodysplasia ossificans progressiva. There were no differences in the steady state levels of messenger ribonucleic acid for either the Type I or Type II bone morphogenetic protein 4 receptors between affected and unaffected individuals in that same family. The presence of bone morphogenetic protein 4 receptor messenger ribonucleic acid in fibrodysplasia ossificans progressiva lesional tissue and unaffected muscle tissue and demonstrates the deregulation of bone morphogenetic protein 4 messenger ribonucleic acid in fibrodysplasia ossificans progressiva. These data support the hypothesis that the molecular basis of bone morphogenetic protein 4 signaling is abnormal in fibrodysplasia ossificans progressiva.

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.

Human leukocyte antigen B27 allele is not correlated with fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva is a rare heritable disorder of connective tissue characterized by skeletal malformations and by progressive heterotopic ossification. It has been suggested that the genetic marker human leukocyte antigen B27 may be associated with fibrodysplasia ossificans progressiva, as it is with ankylosing spondylitis, another disorder with less severe hyperostosis. Genomic deoxyribonucleic acid from 23 classically affected patients with fibrodysplasia ossificans progressiva was screened for the human leukocyte antigen B27 allele by polymerase chain reaction. Only two of the 23 patients (9%) with fibrodysplasia ossificans progressiva who were examined showed the presence of the human leukocyte antigen B27 allele, an incidence that corresponds to the 8% frequency of individuals within the general population not affected with ankylosing spondylitis. These data suggest that the human leukocyte antigen B27 allele does not occur more commonly in the genotype of patients with fibrodysplasia ossificans progressiva than in the general population, and that the pathogenesis of heterotopic bone in fibrodysplasia ossificans progressiva differs from that of ankylosing spondylitis and other human leukocyte antigen B27 positive disorders.