Secreted frizzled related-protein 2 (Sfrp2) deficiency decreases adult skeletal stem cell function in mice.

In a previous transcriptomic study of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells"), SFRP2 was highly over-represented in a subset of multipotent BMSCs (skeletal stem cells, SSCs), which recreate a bone/marrow organ in an in vivo ectopic bone formation assay. SFRPs modulate WNT signaling, which is essential to maintain skeletal homeostasis, but the specific role of SFRP2 in BMSCs/SSCs is unclear. Here, we evaluated Sfrp2 deficiency on BMSC/SSC function in models of skeletal organogenesis and regeneration. The skeleton of Sfrp2-deficient (KO) mice is overtly normal; but their BMSCs/SSCs exhibit reduced colony-forming efficiency, reflecting low SSC self-renewal/abundancy. Sfrp2 KO BMSCs/SSCs formed less trabecular bone than those from WT littermates in the ectopic bone formation assay. Moreover, regeneration of a cortical drilled hole defect was dramatically impaired in Sfrp2 KO mice. Sfrp2-deficient BMSCs/SSCs exhibited poor in vitro osteogenic differentiation as measured by Runx2 and Osterix expression and calcium accumulation. Interestingly, activation of the Wnt co-receptor, Lrp6, and expression of Wnt target genes, Axin2, C-myc and Cyclin D1, were reduced in Sfrp2-deficient BMSCs/SSCs. Addition of recombinant Sfrp2 restored most of these activities, suggesting that Sfrp2 acts as a Wnt agonist. We demonstrate that Sfrp2 plays a role in self-renewal of SSCs and in the recruitment and differentiation of adult SSCs during bone healing. SFRP2 is also a useful marker of BMSC/SSC multipotency, and a factor to potentially improve the quality of ex vivo expanded BMSC/SSC products.

Lineage-specific differentiation of osteogenic progenitors from pluripotent stem cells reveals the FGF1-RUNX2 association in neural crest-derived osteoprogenitors.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage-specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm-like cells, lateral plate mesoderm-like cells, and neural crest-like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest-derived OPs-a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.

Neonatal McCune-Albright Syndrome: A Unique Syndromic Profile With an Unfavorable Outcome.

Somatic gain-of-function mutations of GNAS cause a spectrum of clinical phenotypes, ranging from McCune-Albright syndrome (MAS) to isolated disease of bone, endocrine glands, and more rarely, other organs. In MAS, a syndrome classically characterized by polyostotic fibrous dysplasia (FD), café-au-lait (CAL) skin spots, and precocious puberty, the heterogenity of organ involvement, age of onset, and clinical severity of the disease are thought to reflect the variable size and the random distribution of the mutated cell clone arising from the postzygotic mutation. We report a case of neonatal MAS with hypercortisolism and cholestatic hepatobiliary dysfunction in which bone changes indirectly emanating from the disease genotype, and distinct from FD, led to a fatal outcome. Pulmonary embolism of marrow and bone fragments secondary to rib fractures was the immediate cause of death. Ribs, and all other skeletal segments, were free of changes of typical FD and fractures appeared to be the result of a mild-to-moderate degree of osteopenia. The mutated allele was abundant in the adrenal glands and liver, but not in skin, muscle, and fractured ribs, where it could only be demonstrated using a much more sensitive PNA hybridization probe-based FRET (Förster resonance energy transfer) technique. Histologically, bilateral adrenal hyperplasia and cholestatic disease matched the abundant disease genotype in the adrenals and liver. Based on this case and other sporadic reports, it appears that gain-of-function mutations of GNAS underlie a unique syndromic profile in neonates characterized by CAL skin spots, hypercortisolism, hyperthyroidism, hepatic and cardiac dysfunction, and an absence (or latency) of FD, often with a lethal outcome. Taken together, our and previous cases highlight the phenotypic severity and the diagnostic and therapeutic challenges of MAS in neonates. Furthermore, our case specifically points out how secondary bone changes, unrelated to the direct impact of the mutation, may contribute to the unfavorable outcome of very early-onset MAS.

In Vivo Formation of Stable Hyaline Cartilage by Naïve Human Bone Marrow Stromal Cells with Modified Fibrin Microbeads.

Osteoarthritic and other types of articular cartilage defects never heal on their own. Medicinal and surgical approaches are often ineffective, and the supply of autologous chondrocytes for tissue engineering is very limited. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested as an adequate cell source for cartilage reconstruction. However, the majority of studies employing BMSCs for cartilage tissue engineering have used BMSCs predifferentiated into cartilage prior to implantation. This strategy has failed to achieve formation of stable, hyaline-like cartilage, resistant to hypertrophy in vivo. We hypothesized that in vitro predifferentiation of BMSCs is not necessary when cells are combined with an adequate scaffold that supports the formation of stable cartilage in vivo. In this study, naïve (undifferentiated) human BMSCs were attached to dehydrothermally crosslinked stable fibrin microbeads (FMBs) without and with other scaffolds and implanted subcutaneously into immunocompromised mice. Optimal formation of abundant, hypertrophy-resistant, ectopic hyaline-like cartilage was achieved when BMSCs were attached to FMBs covalently coated with hyaluronic acid. The cartilage that was formed was of human origin and was stable for at least 28 weeks in vivo. Stem Cells Translational Medicine 2019;8:586-592.

Molecular profile of clonal strains of human skeletal stem/progenitor cells with different potencies.

Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are fibroblastic reticular cells, a subset of which is composed of multipotent skeletal stem cells (SSCs). SSCs/BMSCs are able to recreate a bone/marrow organ in vivo. To determine differences between clonogenic multipotent SSCs and similarly clonogenic but non-multipotent BMSCs, we established single colony-derived strains (SCDSs, initiated by individual Colony Forming Unit-Fibroblasts) and determined their differentiation capacity by vivo transplantation. In this series of human SCDSs (N=24), 20.8% formed fibrous tissue (F), 66.7% formed bone (B), and 12.5% formed a bone/marrow organ, and thus were multipotent (M). RNA isolated from 12 SCDSs just prior to transplantation was analyzed by microarray. Although highly similar, there was variability from one SCDS to another, and SCDSs did not strictly segregate into the three functional groups (F, B or M) by unsupervised hierarchical clustering. We then compared 3 F-SCDSs to 3 M-SCDSs that did segregate. Genes associated with skeletogenesis, osteoblastogeneis, hematopoiesis, and extracellular matrix were over-represented in M-SCDSs compared with F-SCDSs. These results highlight the heterogeneity of SSCs/BMSCs, even between functionally similar SCDSs, but also indicate that differences can be detected that may shed light on the character of the SSC.

Bone marrow skeletal stem/progenitor cell defects in dyskeratosis congenita and telomere biology disorders.

Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow (BM) failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the BM stromal cell population (BMSCs, also known as BM-derived mesenchymal stem cells), may contribute to the hematologic phenotype. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to BM failure in TBD.

Directed differentiation of human induced pluripotent stem cells toward bone and cartilage: in vitro versus in vivo assays.

The ability to differentiate induced pluripotent stem cells (iPSCs) into committed skeletal progenitors could allow for an unlimited autologous supply of such cells for therapeutic uses; therefore, we attempted to create novel bone-forming cells from human iPSCs using lines from two distinct tissue sources and methods of differentiation that we previously devised for osteogenic differentiation of human embryonic stem cells, and as suggested by other publications. The resulting cells were assayed using in vitro methods, and the results were compared with those obtained from in vivo transplantation assays. Our results show that true bone was formed in vivo by derivatives of several iPSC lines, but that the successful cell lines and differentiation methodologies were not predicted by the results of the in vitro assays. In addition, bone was formed equally well from iPSCs originating from skin or bone marrow stromal cells (also known as bone marrow-derived mesenchymal stem cells), suggesting that the iPSCs did not retain a "memory" of their previous life. Furthermore, one of the iPSC-derived cell lines formed verifiable cartilage in vivo, which likewise was not predicted by in vitro assays.

BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.

The bromodomain protein, BRD4, has been identified recently as a therapeutic target in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease; its loss is a prognostic signature for metastatic breast cancer. BRD4 also contributes to regulation of both cell cycle and transcription of oncogenes, HIV, and human papilloma virus (HPV). Despite its role in a broad range of biological processes, the precise molecular mechanism of BRD4 function remains unknown. We report that BRD4 is an atypical kinase that binds to the carboxyl-terminal domain (CTD) of RNA polymerase II and directly phosphorylates its serine 2 (Ser2) sites both in vitro and in vivo under conditions where other CTD kinases are inactive. Phosphorylation of the CTD Ser2 is inhibited in vivo by a BRD4 inhibitor that blocks its binding to chromatin. Our finding that BRD4 is an RNA polymerase II CTD Ser2 kinase implicates it as a regulator of eukaryotic transcription.

Denosumab treatment for fibrous dysplasia.

Fibrous dysplasia (FD) is a skeletal disease caused by somatic activating mutations of the cyclic adenosine monophosphate (cAMP)-regulating protein, α-subunit of the Gs stimulatory protein (G(s) α). These mutations lead to replacement of normal bone by proliferative osteogenic precursors, resulting in deformity, fracture, and pain. Medical treatment has been ineffective in altering the disease course. Receptor activator of NF-κB ligand (RANKL) is a cell-surface protein involved in many cellular processes, including osteoclastogenesis, and is reported to be overexpressed in FD-like bone cells. Denosumab is a humanized monoclonal antibody to RANKL approved for treatment of osteoporosis and prevention of skeletal-related events from bone metastases. We present the case of a 9-year-old boy with severe FD who was treated with denosumab for a rapidly expanding femoral lesion. Immunohistochemical staining on a pretreatment bone biopsy specimen revealed marked RANKL expression. He was started on monthly denosumab, with an initial starting dose of 1 mg/kg and planned 0.25 mg/kg dose escalations every 3 months. Over 7 months of treatment he showed marked reduction in pain, bone turnover markers (BTMs), and tumor growth rate. Denosumab did not appear to impair healing of a femoral fracture that occurred while on treatment. With initiation of treatment he developed hypophosphatemia and secondary hyperparathyroidism, necessitating supplementation with phosphorus, calcium, and calcitriol. BTMs showed rapid and sustained suppression. With discontinuation there was rapid and dramatic rebound of BTMs with cross-linked C-telopeptide (reflecting osteoclast activity) exceeding pretreatment levels, accompanied by severe hypercalcemia. In this child, denosumab lead to dramatic reduction of FD expansion and FD-related bone pain. Denosumab was associated with clinically significant disturbances of mineral metabolism both while on treatment and after discontinuation. Denosumab treatment of FD warrants further study to confirm efficacy and determine potential morbidity, as well as to determine the mechanism of RANKL in the pathogenesis of FD and related bone marrow stromal cell diseases.

Mechanism of FGF23 processing in fibrous dysplasia.

Fibroblast growth factor-23 (FGF23) is a phosphate- and vitamin D-regulating hormone derived from osteoblasts/osteocytes that circulates in both active (intact, iFGF23) and inactive (C-terminal, cFGF23) forms. O-glycosylation by O-glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and differential cleavage by furin have been shown to be involved in regulating the ratio of active to inactive FGF23. Elevated iFGF23 levels are observed in a number of hypophosphatemic disorders, such as X-linked, autosomal recessive, and autosomal dominant hypophosphatemic rickets, whereas low iFGF23 levels are found in the hyperphosphatemic disorder familial tumoral calcinosis/hyperphosphatemic hyperostosis syndrome. Fibrous dysplasia of bone (FD) is associated with increased total FGF23 levels (cFGF23 + iFGF23); however, classic hypophosphatemic rickets is uncommon. Our results suggest that it can be explained by increased FGF23 cleavage leading to an increase in inactive cFGF23 relative to active iFGF23. Given the fact that FD is caused by activating mutations in the small G-protein G(s) α that results in increased cyclic adenosine monophosphate (cAMP) levels, we postulated that there may be altered FGF23 cleavage in FD and that the mechanism may involve alterations in cAMP levels and ppGalNacT3 and furin activities. Analysis of blood specimens from patients with FD confirmed that the elevated total FGF23 levels are the result of proportionally increased cFGF23 levels, consistent with less glycosylation and enhanced cleavage by furin. Analysis of primary cell lines of normal and mutation-harboring bone marrow stromal cells (BMSCs) from patients with FD demonstrated that BMSCs harboring the causative G(s) α mutation had higher cAMP levels, lower ppGalNAcT3, and higher furin activity. These data support the model wherein glycosylation by ppGalNAcT3 inhibits FGF23 cleavage by furin and suggest that FGF23 processing is a regulated process that controls overall FGF23 activity in FD patients.

Wnt/ß-catenin signaling is differentially regulated by Gα proteins and contributes to fibrous dysplasia.

Skeletal dysplasias are common disabling disorders characterized by aberrant growth of bone and cartilage leading to abnormal skeletal structures and functions, often attributable to defects in skeletal progenitor cells. The underlying molecular and cellular mechanisms of most skeletal dysplasias remain elusive. Although the Wnt/ß-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. Here, we investigate interactions of the four major Gα protein families (Gα(s), Gα(i/o), Gα(q/11), and Gα(12/13)) with the Wnt/ß-catenin signaling pathway and identify a causative role of Wnt/ß-catenin signaling in fibrous dysplasia (FD) of bone, a disease that exhibits abnormal differentiation of skeletal progenitor cells. The activating Gα(s) mutations that cause FD potentiated Wnt/ß-catenin signaling, and removal of Gα(s) led to reduced Wnt/ß-catenin signaling and decreased bone formation. We further show that activation of Wnt/ß-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of ß-catenin levels rescued differentiation defects of FD patient-derived stromal cells. Gα proteins may act at the level of ß-catenin destruction complex assembly by binding Axin. Our results indicate that activated Gα proteins differentially regulate Wnt/ß-catenin signaling but, importantly, are not required core components of Wnt/ß-catenin signaling. Our data suggest that activated Gα proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/ß-catenin signaling strength.

A mosaic activating mutation in AKT1 associated with the Proteus syndrome.

BACKGROUND: The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS: We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots. RESULTS: Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation. CONCLUSIONS: The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).

In vivo bone formation by progeny of human embryonic stem cells.

The derivation of osteogenic cells from human embryonic stem cells (hESCs) or from induced pluripotent stem cells for bone regeneration would be a welcome alternative to the use of adult stem cells. In an attempt to promote hESC osteogenic differentiation, cells of the HSF-6 line were cultured in differentiating conditions in vitro for prolonged periods of time ranging from 7 to 14.5 weeks, followed by in vivo transplantation into immunocompromised mice in conjunction with hydroxyapatite/tricalcium phosphate ceramic powder. Twelve different medium compositions were tested, along with a number of other variables in culture parameters. In differentiating conditions, HSF-6-derived cells demonstrated an array of diverse phenotypes reminiscent of multiple tissues, but after a few passages, acquired a more uniform, fibroblast-like morphology. Eight to 16 weeks post-transplantation, a group of transplants revealed the formation of histologically proven bone of human origin, including broad areas of multiple intertwining trabeculae, which represents by far the most extensive in vivo bone formation by the hESC-derived cells described to date. Knockout-Dulbecco's modified Eagle's medium-based media with fetal bovine serum, dexamethasone, and ascorbate promoted more frequent bone formation, while media based on α-modified minimum essential medium promoted teratoma formation in 12- to 20-week-old transplants. Transcription levels of pluripotency-related (octamer binding protein 4, Nanog), osteogenesis-related (collagen type I, Runx2, alkaline phosphatase, and bone sialoprotein), and chondrogenesis-related (collagen types II and X, and aggrecan) genes were not predictive of either bone or teratoma formation. The most extensive bone was formed by the strains that, following 4 passages in monolayer conditions, were cultured for 23 to 25 extra days on the surface of hydroxyapatite/tricalcium phosphate particles, suggesting that coculturing of hESC-derived cells with osteoconductive material may increase their osteogenic potential. While none of the conditions tested in this study, and elsewhere, ensured consistent bone formation by hESC-derived cells, our results may elucidate further directions toward the construction of bone on the basis of hESCs or an individual's own induced pluripotent stem cells.

Age-dependent demise of GNAS-mutated skeletal stem cells and "normalization" of fibrous dysplasia of bone.

We studied the role of somatic mosaicism in fibrous dysplasia of bone (FD) within the context of skeletal ("mesenchymal") stem cells by assessing the frequency of mutated colony forming unit-fibroblasts (CFU-Fs) from FD lesions, and in some cases, from unaffected sites, in a series of patients. There was a tight inverse correlation between the percentage mutant CFU-F versus age, suggesting demise of mutant stem cells caused by exuberant apoptosis noted in samples from young patients. In older patients, either partially or completely normal bone/marrow histology was observed. On in vivo transplantation, FD ossicles were generated only by cell strains in which mutant CFU-Fs were identified. Strains that lacked mutant CFU-F (but were mutation positive) failed to regenerate an FD ossicle. These data indicate that GNAS mutations are only pathogenic when in clonogenic skeletal stem cells. From these data, we have evolved the novel concept of "normalization" of FD. As a lesion ages, mutant stem cells fail to self-renew, and their progeny are consumed by apoptosis, whereas residual normal stem cells survive, self-renew, and enable formation of a normal structure. This suggests that activating GNAS mutations disrupt a pathway that is required for skeletal stem cell self-renewal.

The role of type 1 and type 2 5'-deiodinase in the pathophysiology of the 3,5,3'-triiodothyronine toxicosis of McCune-Albright syndrome.

CONTEXT: McCune-Albright syndrome (MAS) is caused by mutations in GNAS (most often R201C or R201H) leading to constitutive cAMP signaling and multiple endocrine dysfunctions, including morphological and functional thyroid involvement. OBJECTIVE: The objective of the study was to characterize the clinical and molecular features of the MAS-associated thyroid disease in a large cohort of patients. DESIGN: This was a retrospective analysis. SETTING: The study was conducted at the National Institutes of Health Clinical Center. PATIENTS: The study included 100 consecutive MAS patients. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURE: Functional and morphological evaluation of the thyroid was measured. Ex vivo experiments were performed on MAS thyroid samples to study the effects of the GNAS mutations on the 5'-deiodinases. Reconstitution experiments in HEK-293 cells were performed to study the effects of GNAS mutations on the type-2 5'-deiodinase. RESULTS: Fifty-four patients had abnormal thyroid ultrasound findings. This group, compared with patients without abnormal findings, had higher T(3) to T(4) ratio, indicating an elevated 5'-deiodinase activity. Thyroid samples from MAS subjects, compared with normal tissue, showed a significant increase in both type 1 (D1) and type 2 (D2) 5'-deiodinase activity (D1 control 5.9 +/- 4.5 vs. MAS 41.7 +/- 26.8 fmol/min.mg, P < 0.001; D2 control 28.3 +/- 13.8 vs. MAS 153.1 +/- 43.7 fmol/min.mg, P < 0.001). Compared with cells transfected with the wild-type R201 allele, the basal transcriptional activity of the D2 promoter was significantly increased in both mutants (C and H) (R 10733 +/- 2855, vs. C 18548 +/- 4514, vs. H 19032 +/- 4410 RLU +/- SD, P < 0.001). CONCLUSION: Thyroid pathology is a common occurrence in MAS. Consistent with the molecular etiology of the disease, the shift in T(3) to T(4) ratio is at least in part secondary to a cAMP-mediated intrathyroidal activation of D2 and to elevated D1 activity.

GNAS transcripts in skeletal progenitors: evidence for random asymmetric allelic expression of Gs alpha.

Activating mutations of the Gsalpha gene, encoded by the guanine nucleotide-binding protein, alpha stimulating (GNAS) locus located on chromosome 20q13, underlie different clinical phenotypes characterized by skeletal lesions [fibrous dysplasia (FD) of bone], extraskeletal diseases (mainly endocrine hyperfunction and skin hyperpigmentation) and variable combinations thereof [the McCune-Albright syndrome (MAS)]. This clinical heterogeneity is commonly assumed to reflect the post-zygotic origin of the mutation. However, the pattern of imprinting of the Gsalpha gene in some human post-natal tissues suggests that parental-dependent epigenetic mechanisms may also play a role in the phenotypic effect of the mutated GNAS genotype. FD lesions are generated by mutated clonogenic osteoprogenitors that reside, along with their normal counterparts, in FD bone marrow stroma. We analyzed the allelic expression pattern of Gsalpha and other GNAS alternative transcripts in the progeny of normal and mutated clonogenic stromal cells isolated in vitro from a series of informative FD/MAS patients. We report here for the first time that the two Gsalpha alleles are unequally expressed in both normal and FD-mutated stromal clones. However, in contrast to imprinting, the ratio of Gsalpha allelic expression is randomly established in different clones from the same patient. This result suggests that a parental-independent modulation of Gsalpha expression occurs in clonogenic osteoprogenitor cells and, at the single cell level, may impact on the severity of an FD lesion. Furthermore, we show that normal and mutated clonogenic stromal cells express GNAS alternative transcripts other than the common Gsalpha, some of which may be relevant to the development of FD.

Sensitive and specific method for detecting G protein-coupled receptor mRNAs.

G protein-coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 mug of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines.

Monostotic fibrous dysplasia of the proximal femur and liposclerosing myxofibrous tumor: which one is which?

Clinical, histological, and genetic studies of two cases of isolated fibro-osseous lesions of the femur in adults show the overlap between monostotic fibrous dysplasia (MFD) of the proximal femur and the so-called liposclerosing myxofibrous tumor. The two cases highlight how the incomplete understanding of the natural history of MFD may result in diagnostic pitfalls or incorrect classification of individual lesions.

Pegvisomant for the treatment of gsp-mediated growth hormone excess in patients with McCune-Albright syndrome.

CONTEXT: GH excess affects approximately 20% of the patients with McCune-Albright syndrome (MAS). MAS is caused by sporadic, postzygotic, activating mutations in the GNAS gene, which codes for the cAMP-regulating protein, G(s)alpha (gsp oncogene). These same mutations are found in approximately one third of the sporadic cases of acromegaly. OBJECTIVE: We examined efficacy of the GH receptor antagonist, pegvisomant, in controlling gsp oncogene-mediated GH excess and skeletal disease (fibrous dysplasia of bone) associated with MAS. SETTING AND PATIENTS: Five MAS patients with GH excess were treated with 20 mg/d sc injection of pegvisomant for 12 wk in a randomized, double-blind, placebo-controlled crossover study at the National Institutes of Health. MAIN OUTCOME MEASURES: The primary measure of efficacy was normalization of IGF-I. Secondary outcome measures were reduction in serum IGF binding protein-3 (IGFBP-3), improvement of fatigue and sweating, and reduction in markers of bone metabolism and bone pain. RESULTS: Combined mean changes in serum IGF-I at 6 and 12 wk were -236.4 ng/ml (53%, P < 0.005) and -329.8 ng/ml (62%, P < 0.001), respectively. IGFBP-3 decreased by 0.8 mg/liter (24%, P < 0.01) and 2.9 mg/liter (37%, P < 0.005), respectively. There were no significant changes in signs and symptoms of acromegaly or markers of bone metabolism and bone pain, nor was there a significant change in pituitary size. Retrospective comparison of the degree of control achieved with pegvisomant vs. other medications (long-acting octreotide +/- dopamine agonist) in the same group showed that the two regimens were similarly effective. CONCLUSIONS: Pegvisomant effectively reduced IGF-I and IGFBP-3 levels in gsp-mediated GH excess but had no effect on fibrous dysplasia.

A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome.

Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gs(alpha) protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer.