These authors contributed equally to this project.
GNAS1 Mutation and Cbfa1 Misexpression in a Child with Severe Congenital Platelike Osteoma Cutis†
Article first published online: 1 NOV 2000
Copyright © 2000 ASBMR
Journal of Bone and Mineral Research
Volume 15, Issue 11, pages 2063–2073, November 2000
How to Cite
Yeh, G. L., Mathur, S., Wivel, A., Li, M., Gannon, F. H., Ulied, A., Audi, L., Olmsted, E. A., Kaplan, F. S. and Shore, E. M. (2000), GNAS1 Mutation and Cbfa1 Misexpression in a Child with Severe Congenital Platelike Osteoma Cutis. J Bone Miner Res, 15: 2063–2073. doi: 10.1359/jbmr.2000.15.11.2063
The Human Gene Nomenclature Committee of HUGO has recently designated RUNX2 as the approved name of Cbfa1. Other names that have been used for RUNX2 are AML3, Osf2, and PEBPαA. The nomenclature for Cbfa1/RUNX2 mRNA isoforms has not been standardized. We refer to the Cbfa1 isoforms by the first exon used for each transcript. The exon numbers are those first reported(1) and later used in additional characterization.(2) Exon 0 and exon 1 correspond to exon 2 and exon 3, respectively, of other reports.(3,4)
- Issue published online: 2 DEC 2009
- Article first published online: 1 NOV 2000
- Manuscript Accepted: 26 JUL 2000
- Manuscript Revised: 28 JUN 2000
- Manuscript Received: 10 NOV 1999
- heterotopic ossification;
- platelike osteoma cutis;
- progressive osseous heteroplasia;
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 α-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.
PLATELIKE OSTEOMA cutis (POC; Mendalian Inheritance in Man [MIM] 166350) is a disorder of heterotopic ossification of the dermis that is present at birth or appears within the first year of life and is characterized by the presence of at least one bony platelike lesion, although other areas of cutaneous ossification also may be present. POC is not associated with trauma, infection, or metabolic abnormalities. Recently, severe congenital POC affecting the scalp and face, and severe nonplatelike heterotopic ossification affecting extremities, sternal, and inguinal regions of a 7-year-old child was described.(5) In this report, we examine the molecular basis of the child's condition.
Primary cutaneous ossification with progressive involvement of subcutaneous fat and skeletal muscle is exceedingly rare. Postnatal heterotopic ossification of dermis and subcutaneous fat are common clinical features of Albright hereditary osteodystrophy (AHO). However, heterotopic ossification in AHO involves only cutaneous and subcutaneous tissues, unlike the condition of the patient described in this report, which progressed to involve deep connective tissues including skeletal muscles of the face, skull, and eyes. Progression of dermal ossification into deep connective tissue is the defining feature of progressive osseous heteroplasia (POH).(6) However, although involvement of skeletal muscle is a prominent feature of POH, it has never before been described in the face, scalp, or extraocular muscles.
The superficial clinical similarities to AHO in our patient suggested the possibility that an inactivating mutation might exist in the GNAS1 gene, which encodes the α-subunit of the stimulatory G protein of adenylyl cyclase (Gsα) and which has been identified as the underlying genetic cause of AHO.(7,8) Examination of the GNAS1 gene in our patient revealed a mutation that predicted a nonfunctional Gsα protein and a possible cause for the heterotopic ossification in this child. The identification of an inactivating mutation of GNAS1 in this patient with severe cutaneous ossification and progression into deep skeletal muscle is an unexpected finding and provides an opportunity to examine cellular events through which an inactivating mutation of GNAS1 mediates heterotopic bone formation.
Cbfa1/RUNX2 has been identified as an obligate osteogenic transcription factor.(1,9–13) In vivo forced expression of the Cbfa1 gene in nonosteoblastic cells induces the expression of osteoblast-specific genes,(4,11,12,14,15) and Cbfa1 binds cis-acting regulatory regions of genes that are regulated in osteoblast differentiation.(12,16,17) Homozygous deletion of Cbfa1 blocks osteoblastic differentiation and marrow formation, resulting in a profound defect in osteogenesis.(9,10) Mutations in Cbfa1 in humans are the cause of cleidocranial dysplasia (CCD).(1) To initiate studies to examine whether an inactivating GNAS1 mutation would influence, directly or indirectly, the expression of genes involved in osteogenesis, we investigated the expression of Cbfa1 in both lesional and nonlesional dermal fibroblasts from our POC patient and detected expression of bone-specific Cbfa1 messenger RNA (mRNA) in both cell types.
Our findings present the first example of a GNAS1 mutation in a child with severe POC and shows severe heterotopic ossification in the absence of AHO features that is caused by an inactivating GNAS1 mutation. This case also identifies downstream pathways of osteogenesis through which GNAS1 may act and establishes the GNAS1 gene as the leading candidate gene for POH.
MATERIALS AND METHODS
Unless otherwise specified, all culture media and reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD, U.S.A.). Human osteogenic sarcoma cell lines, TE 85 and U-2 OS were obtained from American Type Culture Collection (Rockville, MD, U.S.A.) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, U.S.A.) and 100 U/ml pencillin/100 μg/ml streptomycin.
Primary cell lines were established from a lesional skin biopsy of the forehead and from uninvolved skin adjacent to the surgical margin from a 7-year-old female patient with severe POC. The tissue biopsy specimens were cultured in DMEM, 10–20% FBS, and 100 U/ml penicillin per 100 μg/ml streptomycin per 1.25 μg/ml amphotericin B. Under the culture conditions used, dermal fibroblasts are expected to outgrow any osteoblast cells that might be present.(18) Control dermal fibroblasts were isolated from normal human volunteers and grown under the identical conditions. Informed consent was obtained in accordance with the standards set by the institutional review boards of the participating institutions.
A primary osteoblast cell line was established from a surgical sample of normal human trabecular bone from the distal femur of a patient with osteoarthritis, as previously described.(18) Bone fragments were washed thoroughly in phosphate-buffered saline (PBS) and penicillin G/streptomycin/amphotericin B, treated with collagenase type 3 (Worthington, Freehold, NJ, U.S.A.), and then cultured in DMEM with 10% FBS. Proliferating-stage osteoblast RNA was isolated from the cultures at 80% confluence. At confluence, the cells were induced with 5 mg/ml ascorbic acid and 1 M β-glycerol phosphate (Sigma, St. Louis, MO, U.S.A.) to induce differentiation. This treatment was repeated every 3 days until mineralization was achieved. RNA was isolated from differentiation stage cells 6–8 weeks postconfluence and from mineralized cells at 3–4 months postconfluence.
Total RNA was recovered from cells using Trizol Reagent (Life Technologies, Inc.), and poly(A)+ RNA was isolated using Micro-FastTrack reagents (Invitrogen, Carlsbad, CA, U.S.A.) according to recommended protocols. RNA was quantitated by spectrophotometry at A260/A280. The integrity of the RNA samples was verified by electrophoresis on a 1% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, and 20 mM acetic acid).
RNA samples (10 μg) were denatured at 65°C for 15 minutes and then electrophoresed through a 1% agarose gel containing 2.2 M formaldehyde in 3-(N-morpholino)propane sulfonic acid (MOPS) buffer, pH 7.0.(19) Molecular weights were estimated using RNA standards (0.28-6.58 kilobases [kb]; Promega, Madison, WI, U.S.A.). Fractionated RNA samples were transferred to Nytran Plus membranes (Schleicher and Schuell, Keene, NH, U.S.A.) and covalently UV cross-linked (Spectronics Corp., Westbury, NY, U.S.A.). Membranes were prehybridized at 42°C for 4 h in 2× Northern Pre-Hybridization Buffer and then hybridized overnight at 42°C with labeled probes (106 counts/ml) in 2× Hybridization Buffer (5Prime-3Prime, Boulder, CO, U.S.A.). Multiple tissue Northern blots (Clonetech, Palo Alto, CA, U.S.A.) were prehybridized for 90 minutes and hybridized for 1 h at 68°C in ExpressHyb solution (Clonetech). Complementary DNA probes were labeled with [α-32P]deoxycytosine triphosphate (dCTP) by random priming using Prime-It II reagents (Stragagene, La Jolla, CA, U.S.A.). Membranes were washed twice for 5 minutes at room temperature in 2× SSC (1× is 0.15 M sodium chloride; 0.15 M sodium citrate), twice at 55°C for 5–30 minutes in 2× SSC and 1% sodium dodecyl sulfate (SDS), and twice in 0.1× SSC at room temperature for 30 minutes. Blots were exposed to Phosphor Screens and analyzed on a Storm 820 PhosphorImager (Molecular Dynamics, Sunnyvale, CA, U.S.A.). Samples were normalized by probing all blots with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA (cDNA).
Northern probes for human elastin and alkaline phosphatase were constructed from polymerase chain reaction (PCR) products amplified from cDNA templates (primers were designed from published sequences: Genbank accession nos. X52896, ABO11406, and AF001443, respectively; Table 1) that were cloned in TA vector (Invitrogen). The cDNA clones for osteopontin and bone sialoprotein (received from M. Young, National Institutes of Health [NIH])(20,21) and GAPDH(22) have been described.
Reverse transcription (RT) was performed on 1 μg of total RNA using 12.5 μg/ml of oligo dT primer and 1 mg/ml RNasin (Promega), 5 U/ml SuperScript II reverse transcriptase and 1× PCR buffer (Life Technologies), 5 mM MgCl2, 10 mM dithiothreitol (DTT), and 1 mM deoxynucleoside triphosphates (dNTPs; Pharmacia, Piscataway, NJ, U.S.A.) in a final volume of 10 μl. Samples were incubated at 42°C for 1 h, followed by heat inactivation at 95°C for 10 minutes.
PCR amplification of 5 μl of each reverse-transcribed RNA sample was performed using 1× PCR buffer with 1.5 mM MgCl2 and 2.5 U of Amplitaq (Perkin Elmer, Norwalk, CT, U.S.A.) with 50 ng of each forward and reverse primer in a final volume of 20 μl. Primer pairs were designed using MacVector (Oxford Molecular, Campbell, CA, U.S.A.). Primer pairs for Cbfa1 exon 0, exon 1, and runt domain (designed from published sequences, GenBank accession nos. AF001443, AF001444, AF001445, AF001446, AF001447, AF001448, AF001449, and AF001450; Table 1) were used to amplify RT products for 40 cycles: 1 minute at 94°C, 1 minute at 64°C, and 1 minute at 74°C (PTC-200 thermal cycler; MJ Research, Watertown, MA, U.S.A.). Primer pairs (Table 1) for elastin, tissue nonspecific human alkaline phosphatase (TNSAP), osteocalcin, and osteopontin were used to amplify RT products for 34 cycles: 1 minute at 94°C, 1 minute at 60°C, and 1 minute at 72°C. Amplification of GNAS1 exon 7 used the same conditions and previously described primers.(23)
Genomic DNA analysis
Genomic DNA was recovered directly from blood or from cultured cells and purified by standard methods using proteinase K, SDS, and phenol.(19) Oligonucleotide primers that flank the exon-coding regions of the human GNAS1 gene were used to PCR-amplify genomic DNA templates using the recommended cycling conditions for each primer pair.(7,24) Each PCR reaction contained (in a final volume of 50 μl) 250 ng of genomic DNA, 0.5 μM of each primer, 0.1 mM dNTPs (Pharmacia), and 1× PCR buffer with 1.5 mM MgCl2 and 1.25 U of Taq polymerase (Life Technologies). After denaturation at 94°C for 4 minutes, the exon 7 mutation was detected in products amplified with 5′TGAGCCTGACCTTGTAGAGAGACACA and 5′GGTTATTCCAGAGGGACTGGGGTGAA for 40 cycles at 94°C for 1 minute, 67°C for 1 minute, and 72°C for 2 minutes (PTC-200 thermal cycler; MJ Research). After amplification, the PCR products were purified with Geneclean III (Bio101, La Jolla, CA, U.S.A.) and sequenced by the DNA Sequencing Core Facility of the University of Pennsylvania School of Veterinary Medicine.
Clinical evaluation of a patient with severe POC
A child was identified with severe POC of the scalp and face, a variant of POH.(5) The POC involved the forehead and preauricular and orbital regions and progressed during early childhood to involve the underlying skeletal muscle (Fig. 1). She also developed heterotopic ossification of subcutaneous tissue in her left hand, left foot, and sternal and inguinal regions bilaterally. Postnatal heterotopic ossification of dermis and subcutaneous fat often are seen in patients who have AHO. However, the child's condition progressed to involve skeletal muscle of the face, skull, and eyes, which are features not seen in AHO. No other morphological or endocrine features of AHO were present; the child's height and weight were normal, there was no brachydactyly, and the face was not round. The serum calcium, phosphorus, albumin, parathyroid hormone (PTH) level, and thyroid function tests were normal. There was no family history of ectopic ossification, short stature, obesity, brachydactyly, or hormone resistance.
Histological analysis of POC lesional tissue and dermal fibroblasts
For aesthetic reasons, the patient underwent operative excision of the forehead lesion at 7 years of age with a 5-mm margin of clinically uninvolved skin and subcutaneous tissue.(5) Specimens taken from regions of heterotopic ossification (involved skin) of the surgical excision were examined under light microscopy (Fig. 2). The epidermis appeared normal and free of heterotopic ossification or calcification. Discrete areas of mature lamellar bone were identified in the reticular dermis and underlying frontalis muscle. There were no cartilaginous cells. No increased vascularity or inflammations were seen adjacent to the heterotopic bone or elsewhere within the dermis. Gross and microscopic examination of the margins of the surgical specimen (uninvolved skin) revealed normal epidermis and dermis, without any evidence of ossification.
Primary fibroblast cell lines were established from the dermis of the involved and uninvolved skin. The fibroblasts cultured from these surgical specimens, as well as dermal fibroblasts from a normal control, were examined under light microscopy. The fibroblasts cultured from the patient's involved and uninvolved skin showed a similar uniformly fibroblastic appearance. Although similar to normal control skin fibroblasts, cells from both lesional and perilesional skin cell lines appeared to contain more cellular extensions than normal skin fibroblasts.
Mutational analysis of the GNAS1 gene
Genomic DNA from the patient was recovered from cultured lesional and uninvolved dermal fibroblasts and from peripheral blood lymphocytes. Genomic DNAs were amplified by PCR using primers to span GNAS1 exons 1–13, including splice junctions and some small introns, and the DNA sequence was determined. DNA from all tissues examined revealed heterozygosity for a 4-base pair (bp) deletion in exon 7 (Fig. 3A). This deletion causes a shift in the protein-coding reading frame and predicts a nonfunctional protein product containing 13 incorrect amino acids followed by a premature stop codon. RT-PCR analysis of RNA isolated from the patient's cultured lesional fibroblasts, using primers(23) that amplify the exon 7 region of the GNAS1 cDNA, showed that the mutated GNAS1 allele is not detected as mRNA (Fig. 3B). The same 4-bp deletion has been previously identified as an inactivating mutation of GNAS1 in some patients with AHO, and this mutation has been shown to cause haploinsufficiency of GNAS1 protein levels in heterozygotes.(23)
Neither parent of the proband had evidence of dermal ossification. Genomic DNA was recovered from peripheral blood of both parents and analysis of the GNAS1 exon 7 DNA sequence did not reveal a 4-bp deletion, suggesting that the patient's mutation was spontaneous, although germ line mosaicism in one of the parents cannot be excluded.
Cbfa1 expression in POC dermal fibroblasts
The Cbfa1 gene encodes an essential transcription factor for bone formation and osteoblast differentiation.(1,9–13) To initiate an examination of the pathways through which GNAS1 may influence the induction of osteogenesis, Cbfa1 mRNA expression was evaluated. Total RNA was isolated from dermal fibroblasts obtained from the POC involved (POC-I) and POC uninvolved (POC-U) skin and from human control osteoblast and skin fibroblast lines. RT-PCR analysis using primers that amplify the Cbfa1 runt domain (the functional DNA binding domain of the Cbfa1 protein) showed that Cbfa1 mRNA is expressed in normal osteoblasts, POC involved and uninvolved dermal fibroblasts, and in control skin (Fig. 4, lanes 3, 6, 9, and 12).
Expression of two forms of Cbfa1 transcripts with different first exons
Two forms of Cbfa1 mRNA that contain different first exons have previously been reported and suggested to have tissue-specific expression.(1,2) These Cbfa1 transcript isoforms were further characterized in order to examine their specific expression in POC dermal fibroblasts.
RT-PCR was performed on U-2 OS (human osteosarcoma) RNA using primer pairs to detect specifically the 5′ portions of two Cbfa1 mRNA isoforms (named for the first exon used by each transcript: exon 0 or exon 1). The DNA sequence of the PCR products from each reaction was determined, compared with published sequences, and found to be identical to the previously reported exon 0 and exon 1 isoforms.(2) The junction between exon 1 and exon 2 was sequenced and was the same for both the exon 0 and the exon 1 forms of Cbfa1. The splicing events predicted by the DNA sequences from the RT-PCR products are diagrammed in Fig. 5. The exon 0 isoform consists of exon 0 spliced within exon 1, excluding a 5′ segment of exon 1. The exon 1 isoform consists of the entire exon 1 spliced to exon 2. As has been noted previously,(1,2) the differences in the two Cbfa1 transcript isoforms predict that each will encode proteins that differ at their N-terminal ends; however, in vivo physiological significance of two different Cbfa1 proteins has not been determined.(4)
The expression of the two Cbfa1 mRNA isoforms in normal osteoblasts and normal skin cells was examined by RT-PCR using primer pairs that specifically detect each isoform. The Cbfa1 exon 1 isoform was detected in both osteoblasts and skin (Fig. 4, lanes 2 and 5), whereas the exon 0 isoform was detected in osteoblasts but not skin (Fig. 4, lanes 1 and 4).
Tissue-specific expression of Cbfa1 was further examined by multiple tissue Northern blot analysis. We detected no Cbfa1 mRNA in heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (data not shown), which is consistent with previous data reporting restricted tissue expression of Cbfa1.(11,12) Additionally, no expression of Cbfa1 exon 0 was detected by RT-PCR in RNAs from muscle, lung, and T lymphocytes.
These data show the expression of Cbfa1 in both bone and skin cells. However, although the exon 1 isoform was expressed in both of these cell types, the exon 0 isoform was detected specifically in cells with an osteoblastic phenotype.
Expression of a bone-specific form of Cbfa1 mRNA in POC dermal fibroblasts
To examine Cbfa1 expression in patient cells, RNAs from cultured lesional and uninvolved dermal fibroblasts were analyzed by RT-PCR for expression of the two Cbfa1 mRNA isoforms using primers specific for the Cbfa1 exon 0 and exon 1 mRNAs. The bone/skin-expressed Cbfa1 exon 1 form was detected in normal osteoblasts, POC-U and POC-I dermal fibroblasts, and control skin (Fig. 4; lanes 2, 5, 8, and 11). The bone-specific Cbfa1 exon 0 form was not detected in control skin but was expressed in normal osteoblasts, as well as in both POC-U and POC-I dermal fibroblasts (Fig. 4; lanes 1, 4, 7, and 10).
Phenotypic characterization of cultured POC skin cell lines
POC-U and POC-I skin-derived cell lines were examined for expression of mRNAs characteristic of bone or skin cells. Northern blot analysis was used to examine the expression of elastin, a marker of dermal fibroblasts,(25) and of several osteoblast transcripts: osteocalcin, osteopontin, alkaline phosphatase, and bone sialoprotein.(26) Both POC skin cell lines expressed elastin by Northern analysis (Fig. 6A). Results from RT-PCR using primers specific for elastin mRNA were consistent with these Northern analysis data and additionally confirmed the absence of elastin expression in control osteoblast cells (data not shown). Expression of osteocalcin mRNA, found in differentiated and mineralized osteoblasts,(26) could not be detected by RT-PCR in either the POC-I or POC-U cell lines (data not shown). However, both the POC-I and the POC-U cells expressed alkaline phosphatase, osteopontin, and bone sialoprotein transcripts, which are expressed by earlier proliferating stage osteoblasts.(26) These results were determined by Northern analysis (Fig. 6B) and confirmed by RT-PCR. Although these transcripts are not exclusive to bone cells,(20,21,27) the composite pattern of expression of alkaline phosphatase, osteopontin, and bone sialoprotein is characteristic of osteoblastic cells. Expression of both skin and early osteoblast markers is consistent with a hypothetical transition or multipotential state of these cells.
This study strongly suggests that severe POC is caused by an inactivating mutation in the GNAS1 gene in the patient described. The identified heterozygous 4-bp deletion within exon 7 of the GNAS1 gene encodes a protein reading frameshift leading to 13 incorrect amino acids followed by a premature stop codon. RNA analysis showed that only GNAS1 mRNA expressed from the normal allele could be detected; the mutant GNAS1 mRNA presumably is not synthesized or rapidly degraded.(28) AHO (MIM 103580) is another disorder that is caused by inactivating mutations of the GNAS1 gene and by reduced levels of Gsα activity. However, AHO, unlike POC, is characterized by short stature, obesity, round face, brachydactyly, multiple hormone resistance, and dermal ossification that does not progress to deep connective tissues.(8,29) It is of interest to note that the same 4-bp deletion identified in the described POC patient also has been found to be a mutational “hot spot” in AHO.(23,30,31) This observation suggests that AHO and POC may be part of a phenotypic spectrum of clinical disorders that are caused by inactivating GNAS1 mutations.
The GNAS1 gene encodes the α-subunit of the stimulatory G protein of adenylyl cyclase (Gsα).(32) G proteins are heterotrimeric (α-, β-, and γ-subunits) guanine nucleotide binding proteins that interact with the cytoplasmic domains of extracellular receptors to mediate many signal transduction pathways.(8,29) Although the bone-specific pathways regulated by G-protein activation remain to be elucidated, several G-protein coupled receptor agonists that influence bone formation have been identified including PTH, PTH-related protein (PTHrP), prostaglandins, and sodium fluoride.(8,33) Indian hedgehog (Ihh) stimulation of PTHrP in the growth plate of developing long bones regulates the maturation of chondrocytes and ossification.(34,35) In McCune Albright syndrome (MAS; MIM 174800), somatic activating mutations of GNAS1, which result in overstimulation of Gsα activity, lead to an arrest of osteoblast maturation and result in fibrous dysplasia of bone in the normotopic skeleton.(36) In contrast, inactivating mutations of GNAS1 in patients with AHO and in our patient with POC lead to an understimulation of Gsα-activated pathways and result in heterotopic ossification of soft connective tissues (Table 2). Considered together, these findings suggest that GNAS1 may normally function as a negative regulator of osteoblast specification.
The identification of an inactivating mutation of GNAS1 in a patient with severe cutaneous ossification and progression into deep skeletal muscle provides an opportunity to examine cellular events through which an inactivating mutation of GNAS1 mediates heterotopic bone formation. Cbfa1/RUNX2 has been identified as an obligate transcription factor for osteoblast differentiation and osteogenesis.(1,9–13) Two variants (exon 0 and exon 1 isoforms) of Cbfa1 mRNA have been described in humans.(1,2) Each of these transcripts contains different first exons and appears to involve alternate splicing, predicting the synthesis of unique protein products that differ at their N-terminal ends. Alternate splicing involving other exons within the Cbfa1 gene also has been described(2,3); however, the pattern of this downstream splicing with respect to the exon 0 and exon 1 isoforms is not well characterized.
The importance of specific Cbfa1 isoforms to cell function and/or cellular differentiation is unknown. Studies using Cbfa1−/− mice have shown effects of Cbfa1 on the expression of genes associated with the osteoblastic phenotype, including alkaline phosphatase, osteopontin,(9,10) and osteocalcin.(11) Recent studies(4) report that different Cbfa1 isoforms can stimulate osteocalcin promoter activity; however, the physiological relevance remains to be established. In mice, the Cbfa1 transcripts containing exon 0 sequences were detected in bone but not in other tissues.(11) The exon 0 isoform is important in osteoblast development and plays an essential role in osteoblast differentiation. These observations are consistent with our data showing that expression of the Cbfa1 exon 0 transcript appears to be restricted to osteoblastic cells. Expression of the Cbfa1 exon 0 isoform in the POC patient's lesional and uninvolved fibroblasts and the composite pattern of expression of alkaline phosphatase, osteopontin, and bone sialoprotein in these cells also are consistent with an osteogenic phenotype. Because skin fibroblasts also have been reported to express at least some of these markers of differentiated bone cells,(20,21,27) one possible explanation of the dermal localization of heterotopic ossification in conditions such as POC, AHO, and POH is that dermal cells may be more susceptible than other cell types to differentiation to an osteoblastic phenotype. Alternatively, mesenchymal stem cells residing in the dermis or circulating marrow stromal cells deposited in the dermis may give rise to cells of an osteoblastic phenotype when derepressed by inactivating mutations in GNAS1 as in our patient or in those who have AHO. This hypothesis is supported by recent data that showed elevation of cyclic adenosine monophosphate (cAMP; which mediates PTH/G-protein signaling) inhibits osteoblast differentiation, including increased proteolysis and decreased DNA binding of Cbfa1.(37)
In situ hybridization of mouse embryos using a probe for the Cbfa1 runt domain (which detects both isoforms) showed Cbfa1 expression in skin.(9) Our data indicate that the Cbfa1 exon 1 transcript is expressed in both bone cells and skin cells; however, the in vivo function of the exon 1 isoform remains unknown. Several studies examining the ability of the Cbfa1 isoforms to regulate promoter activity have shown that both exon 0 and exon 1 isoforms are functionally active in vitro, although the exon 1 isoform may have a lower potential to activate osteoblast-specific genes compared with the exon 0 isoform.(4,14,15) The different amino terminal ends of the proteins predicted to be translated from each isoform could be expected to affect the cellular activities of the Cbfa1 protein. Alternatively, the production of multiple isoforms of Cbfa1 could be a mechanism to regulate Cbfa1 expression at the transcriptional level.
Our data suggest that reduced levels of GNAS1 expression influence downstream events leading to an osteoblast-competent phenotype. Expression of the bone-specific Cbfa1 exon 0 isoform may be a direct effect of decreased GNAS1 expression or a consequence of osteoblastic differentiation that is influenced by GNAS1 independently of Cbfa1. Moreover, this work suggests that GNAS1 encodes a negative regulator of osteogenic lineage determination. Inactivating GNAS1 mutations, such as found in AHO and in our patient with POC, are expected to produce reduced levels of Gsα protein that may be insufficient to maintain a brake on cellular induction to osteoblast differentiation at ectopic sites. Our finding of Cbfa1 exon 0 expression in uninvolved dermal fibroblasts of our POC patient further suggests that the misexpression of the Cbfa1 exon 0 isoform may be necessary but not sufficient to form bone at an ectopic site.
POC is a disorder in which mesenchymal stem cells differentiate into bone outside the context of the normal skeleton. The question of how multipotent stem cells differentiate into multiple lineages is one that arises not just in the context of mesenchymal tissues but also in neural crest and hematopoietic tissues. Whether mesenchymal differentiation occurs on an instructive or permissive basis(38) has yet to be determined, but our data suggest that GNAS1 pathways play an important role in lineage specification in cells of mesenchymal origin.
A recent study(39) reported the presence of inactivating GNAS1 mutations and/or decreased Gsα activity in 2 patients with features of both AHO and severe POH-like progressive dermal ossification. Although it is possible that these two phenotypes in these children are caused by mutations in different genes, the presence of deep and progressive heterotopic ossification in children with variable features of AHO suggests that a common molecular mechanism may be responsible for their heterotopic ossification. Here, we report the first identification of an inactivating mutation of GNAS1 in a POC patient with severe heterotopic ossification in the absence of any AHO features.
The two major findings of this study are the discovery of an inactivating mutation of GNAS1 in a child with severe POC (a clinical variant of POH) and the discovery of the altered expression in skin cells of a bone-specific isoform of Cbfa1, an obligate osteogenic transcription factor. These findings establish molecular landmarks in the transformation of soft connective tissue into heterotopic bone and strongly suggest that GNAS1 is the leading candidate gene for other forms of primary dermal ossification such as POH, a disorder for which progression of dermal ossification to deep connective tissue is the defining feature.
The authors thank members of our laboratory, Mei-qi Xu, Leota Terry, Bob Caron, and Andrea Tiglio, for their technical contributions to this work. The authors acknowledge the support of the POH Association, the International Fibrodysplasia Ossificans Progressiva Association, the New Jersey Association of Student Councils, the Ian Cali Fund, the NIH (2-RO1-AR41916-04 and 1-RO1-AR46831-01), and the Isaac & Rose Nassau Professorship of Orthopedic Molecular Medicine.
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