A 54-year-old man with a history of unimelic, post-traumatic multifocal heterotopic ossification (HO) and normal genetic analysis of ACVR1 and GNAS had variants of unknown significance (VUS) in PDLIM-7 (PDZ and LIM Domain Protein 7), the gene encoding LMP-1 (LIM Mineralization Protein-1), an intracellular protein involved in the bone morphogenetic protein (BMP) pathway signaling and ossification. In order to determine if the LMP-1 variants were plausibly responsible for the phenotype observed, a series of in vitro experiments were conducted. C2C12 cells were co-transfected with a BMP-responsive reporter as well as the LMP-1 wildtype (wt) construct or the LMP-1T161I or the LMP-1D181G constructs (herein designated as LMP-161 or LMP-181) corresponding to the coding variants detected in the patient. A significantly increased BMP-reporter activity was observed in LMP-161 or LMP-181 transfected cells compared to the wt cells. The LMP-181 variant exhibited BMP-reporter activity with a four-fold increase over the LMP-1 wt protein. Similarly, mouse pre-osteoblastic MC3T3 cells transfected with the patient\'s LMP-1 variants expressed higher levels of osteoblast markers both at mRNA and protein levels and preferentially mineralized when stimulated with recombinant BMP-2 compared to control cells. Presently, there are no pathogenic variants of LMP-1 known to induce HO in humans. Our findings suggest that the germline variants in LMP-1 detected in our patient are plausibly related to his multifocal HO (LMP1-related multifocal HO). Further observations will be required to firmly establish this gene-disease relationship.

Progressive osseous heteroplasia (POH) is a rare, debilitating disorder characterized by heterotopic ossification in the skin and muscles, resulting in contractures of the joints and progressive loss of function. While 60-70% of the POH patients have paternally inherited, inactivating pathogenic variants in GNAS, the remaining 30-40% have no known etiology. FAM111B pathogenic variants, located on chromosome 11q12.1, cause POIKTMP (hereditary fibrosing poikiloderma with tendon contractures, myopathy, and pulmonary fibrosis), a very rare, autosomal-dominant disorder with high frequency of de novo missense pathogenic variants, which affects multiple tissues and organs, causing extensive fibrosis and muscle adiposis, though the exact mechanism is unknown. To our knowledge, there are no reports of FAM111B associated with POH. We describe the first case of POH phenotype associated with a novel de novo frameshift pathogenic variant in the FAM111B and present an analysis of the protein structure and function caused by this genomic disruption.
A 15-year-old African-American male presented with generalized calcific nodules, progressive contractures, and muscle weakness leading to immobility, beginning at 6 years of age. Cutaneous examination showed generalized hard nodules varying from small to plaque-like ulcerated erupted skin lesions. Biochemical evaluation revealed 25(OH) vitamin D insufficiency (20 ng/mL), and normal levels of parathyroid hormone, FGF-23, alkaline phosphatase, calcium, and phosphorus. Skeletal survey radiographs and computed tomography (CT) of the chest, abdomen, and pelvis showed extensive soft tissue and muscle heterotopic ossifications involving shoulders, axillae, trunk, abdomen, pelvis, upper and lower extremities, in a clumped, conglomerate distribution within muscle, subcutaneous fat, and in some areas extending to the skin. There was no pulmonary fibrosis on the chest CT. The clinical and radiographic findings were most consistent with POH. A trio-clinical exome sequencing revealed a de novo heterozygous likely pathogenic variant in the FAM111B (OMIM # 615584) (c.1462delT [p.Cys488Valfs*21]). The resulted frameshift change in exon 4 replaced C-terminal region with 21 alternative amino acids. Multiple, previously reported disease-associated variants appear to localize within the trypsin-like cysteine/serine peptidase domain in which this variant occurs, supporting the functional significance of this region, though none have been previously reported to be associated with POH phenotype. Our 3D protein modeling showed obliteration of predicted protein folding and structure, and elimination of the zinc-binding domain, likely severely affecting protein function.
This is the first case of POH phenotype associated with a novel de novo pathogenic frameshift variant in FAM111B. Whether the frameshift change in FAM111B predicts POH remains unclear. Further evaluations are necessary to fully elucidate this finding and the potential role and mechanism by which the FAM111B variants contributes to POH phenotype.

UNASSIGNED: Progressive osseous heteroplasia (POH) is an ultrarare genetic disorder characterized by an inactivating mutation in the GNAS gene that causes heterotopic ossification. Inhibition of the mammalian target of the rapamycin (mTOR) signalling pathway has been proposed as a therapy for progressive bone fibrodysplasia and non-genetic forms of bone heteroplasia. Herein, we describe the impact of using Everolimus as a rescue therapy for an identical twin girl exhibiting an aggressive clinical phenotype of POH.
UNASSIGNED: Clinical evaluation of the progression of the disease during Everolimus treatment was performed periodically. Cytokine markers involved in bone metabolism and protein markers related to bone activity were analyzed to explore bone turnover activity.
UNASSIGNED: The patient received Everolimus therapy for 36 weeks. During treatment, no clinical improvement of the disease was perceived. Analysis of biochemical parameters, namely, β-CTX (r 2 = -0.576, P-value = 0.016) and PNIP (r 2 = -0.598, P-value = 0.011), indicated that bone turnover activity was significantly reduced. Additionally, bone metabolism-related biomarkers showed only a significant positive correlation with PTH levels.
UNASSIGNED: Everolimus treatment did not modify the clinical progression of the disease in an aggressive form of POH, although an impact on the protein markers studied was observed.

BACKGROUND: The GNAS gene on chromosome 20q13.3, encodes the alpha-subunit of the stimulatory G protein, which is expressed in most tissues and regulated through reciprocal genomic imprinting. Disorders of GNAS inactivation produce several different clinical phenotypes including pseudohypoparathyroidism (PHP), pseudopseudohypoparathyroidism (PPHP), progressive osseous heteroplasia (POH), and osteoma cutis (OC). The clinical and biochemical characteristics overlap of PHP subtypes and other related disorders presents challenges for differential diagnosis.
METHODS: We enrolled a total of 11 Chinese children with PHP in our study and analyzed their clinical characteristics, laboratory results, and genetic mutations.
RESULTS: Among these 11 patients, nine of them (9/11) presented with resistance to parathyroid hormone (PTH); and nine (9/11) presented with an Albright\'s hereditary osteodystrophy (AHO) phenotype. GNAS abnormalities were detected in all 11 patients, including nine cases with GNAS gene variations and two cases with GNAS methylation defects. These GNAS variations included an intronic mutation (c.212 + 3_212 + 6delAAGT), three missense mutations (c.314C > T, c.308 T > C, c.1123G > T), two deletion mutations (c.565_568delGACT*2, c.74delA), and two splicing mutations (c.721 + 1G > A, c.432 + 1G > A). Three of these mutations, namely, c.314C > T, c.1123G > T, and c.721 + 1G > A, were found to be novel. This data was then used to assign a GNAS subtype to each of these patients with six cases diagnosed as PHP1a, two cases as PHP1b, one as PPHP, and two as POH.
CONCLUSIONS: Evaluating patients with PTH resistance and AHO phenotype improved the genetic diagnosis of GNAS mutations significantly. In addition, our results suggest that when GNAS gene sequencing is negative, GNAS methylation study should be performed. Early genetic detection is required for the differential diagnosis of GNAS disorders and is critical to the clinician\'s ability to distinguish between heterotopic ossification in the POH and AHO phenotype.

[This corrects the article DOI: 10.3389/fped.2021.662669.].

Progressive osseous heteroplasia (POH; OMIM 166350) is a rare autosomal-dominant genetic disorder in which extra-skeletal bone forms within skin and muscle tissue. POH is one of the clinical manifestations of an inactivating mutation in the GNAS gene. GNAS gene alterations are difficult matter to address, as GNAS alleles show genetic imprinting and produce several transcript products, and the same mutation may lead to strikingly different phenotypes. Also, most of the publications concerning POH patients are either clinical depictions of a case (or a case series), descriptions of their genetic background, or a tentative correlation of both clinical and molecular findings. Treatment for POH is rarely addressed, and POH still lacks therapeutic options. We describe a unique case of POH in two monochorionic twins, who presented an almost asymptomatic vs. the severe clinical course, despite sharing the same mutation and genetic background. We also report the results of the therapeutic interventions currently available for heterotopic ossification in the patient with the severe course. This article not only critically supports the assumption that the POH course is strongly influenced by factors beyond genetic background but also remarks the lack of options for patients suffering an orphan disease, even after testing drugs with promising in vitro results.

Heterotopic ossification (HO), the formation of bone outside of the skeleton, occurs in response to severe trauma and in rare genetic diseases such as progressive osseous heteroplasia (POH). In POH, which is caused by inactivation of GNAS, a gene that encodes the alpha stimulatory subunit of G proteins (Gsα), HO typically initiates within subcutaneous soft tissues before progressing to deeper connective tissues. To mimic POH, we used conditional Gnas-null mice which form HO in subcutaneous tissues upon Gnas inactivation. In response to Gnas inactivation, we determined that prior to detection of heterotopic bone, dermal adipose tissue changed dramatically, with progressively decreased adipose tissue volume and increased density of extracellular matrix over time. Upon depletion of the adipose tissue, heterotopic bone progressively formed in those locations. To investigate the potential relevance of the tissue microenvironment for HO formation, we implanted Gnas-null or control mesenchymal progenitor cells into Gnas-null or control host subcutaneous tissues. We found that mutant cells in a Gnas-null tissue environment induced a robust HO response while little/no HO was detected in control hosts. Additionally, a Gnas-null tissue environment appeared to support the recruitment of control cells to heterotopic bone, although control cell implants were associated with less HO formation compared to mutant cells. Our data support that Gnas inactivation alters the tissue microenvironment to influence mutant and wild-type progenitor cells to contribute to HO formation.

GNAS is one of the most complex gene loci in the human genome and encodes multiple gene products including Gsα, XLαs, NESP55, A/B, and AS transcripts. XLαs, the extra-large G protein ɑ-subunit, is paternally expressed. XLɑs and Gsɑ share the common 2-13 exons with different promoters and first exons. Therefore, XLɑs contains most of the functional domains of Gsα including receptor and effector binding sites. In vitro studies suggest a \"Gsɑ\"-like function of XLɑs regarding the stimulation of cAMP generation in response to receptor activation with different cellular actions. However, it is unclear whether XLαs has an important physiological function in humans. Pseudopseudohypoparathyroidism (PPHP) and progressive osseous heteroplasia (POH) are caused by paternally inherited mutations of GNAS. Maternal uniparental disomy of chromosome 20 [UPD(20)mat] lacks paternal chromosome 20. Therefore, the phenotypes of these diseases may be secondary to the abnormal functions of XLɑs, at least partly. From the phenotypes of human diseases like PPHP, POH, and UPD(20)mat, as well as some animal models with deficient XLɑs functions, it could be seen that XLɑs is involved in the growth and development of the mammalian fetus, plays a different role in glucose, lipid, and energy metabolism when compared with Gsɑ, and could prevent heterotopic ossification in humans and mice. More in vivo and in vitro studies, especially the development of conditional XLɑs knockout mice, are needed to clarify the physiopathologic roles and related signal pathways of XLɑs.

Skeletal development is exquisitely controlled both spatially and temporally by cell signaling networks. Gαs is the stimulatory α-subunit in a heterotrimeric G protein complex transducing the signaling of G-protein-coupled receptors (GPCRs), responsible for controlling both skeletal development and homeostasis. Gαs, encoded by the GNAS gene in humans, plays critical roles in skeletal development and homeostasis by regulating commitment, differentiation and maturation of skeletal cells. Gαs-mediated signaling interacts with the Wnt and Hedgehog signaling pathways, both crucial regulators of skeletal development, remodeling and injury repair. Genetic mutations that disrupt Gαs functions cause human disorders with severe skeletal defects, such as fibrous dysplasia of bone and heterotopic bone formation. This chapter focuses on the crucial roles of Gαs signaling during skeletal development and homeostasis, and the pathological mechanisms underlying skeletal diseases caused by GNAS mutations.

Heterotopic ossification (HO) occurs when soft tissues are inappropriately converted to bony tissue. Several human diseases result in HO with few reliable treatment options. Animal models that naturally produce dermal ectopic bone (i.e., osteoderms), such as crocodilians, have never been utilized as models for studying these disorders in humans. Here, a histological evaluation and staging criteria for osteoderm development is described for the first time in the American alligator (Alligator mississipiensis). Differential staining and immunohistochemistry of alligator scales depict a progressive change during development, where woven bone forms from the differentiated dermis. Bone formation proceeds via intramembranous ossification, which is initiated in part by endothelial cell precursors that undergo endothelial-to-mesenchymal transition and eventually acquire an osteoblast phenotype. As such, the development of osteoderms in the American alligator bears morphological and mechanistic similarities to HO in humans, presenting a potential model for future study of soft tissue mineralization pathologies and providing insight into the morphological and molecular development of osteoderms in other vertebrate lineages. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:56-76, 2018. © 2017 Wiley Periodicals, Inc.