Mutations of the GNAS1 Gene, Stromal Cell Dysfunction, and Osteomalacic Changes in Non–McCune–Albright Fibrous Dysplasia of Bone

Abstract

Activating missense mutations of the GNAS1 gene, encoding the ␣ subunit of the stimulatory G protein (Gs), have been identified in patients with the McCune-Albright syndrome (MAS; characterized by polyostotic fibrous dysplasia, café au lait skin pigmentation, and endocrine disorders). Because fibrous dysplasia (FD) of bone also commonly occurs outside of the context of typical MAS, we asked whether the same mutations could be identified routinely in non-MAS FD lesions. We analyzed a series of 8 randomly obtained, consecutive cases of non-MAS FD and identified R201 mutations in the GNAS1 gene in all of them by sequencing cDNA generated by amplification of genomic DNA using a standard primer set and by using a novel, highly sensitive method that uses a protein nucleic acid (PNA) primer to block amplification of the normal allele. Histologic findings were not distinguishable from those observed in MAS-related FD and included subtle changes in cell shape and collagen texture putatively ascribed to excess endogenous cyclic adenosine monophosphate (cAMP). Osteomalacic changes (unmineralized osteoid) were prominent in lesional FD bone. In an in vivo transplantation assay, stromal cells isolated from FD failed to recapitulate a normal ossicle; instead, they generated a miniature replica of fibrous dysplasia. These data provide evidence that occurrence of GNAS1 mutations, previously noted in individual cases of FD, is a common and perhaps constant finding in non-MAS FD. These findings support the view that FD, MAS, and nonskeletal isolated endocrine lesions associated with GNAS1 mutations represent a spectrum of phenotypic expressions (likely reflecting different patterns of somatic mosaicism) of the same basic disorder. We conclude that mechanisms underlying the development of the FD lesions, and hopefully mechanism-targeted therapeutic approaches to be developed, must also be the same in MAS and non-MAS FD. (J Bone Miner Res 2000;15:120-128)

Ossifying fibroma vs fibrous dysplasia of the jaw: molecular and immunological characterization

“…Therefore, a PNA-clamping method was employed in the present study. 15,20 Sequencing of the PCR-amplified fragments confirmed the accuracy of the PCR analysis using PNA, suggesting that the PCR analysis used in the present study may be a useful and easily available method for detecting GNAS mutations at the Arg 201 codon.…”

“…To block the amplification of the wild-type allele, a peptide nucleic acid (PNA) primer (Gly-NH2-CGCTGCCG TGTC-HAc) (FASMAC, Co Ltd, Kanagawa, Japan) was added to the first and nested-PCR reactions. 15,20 The P3 primer creates a new restriction site for EagI (CGGCCG) through the change to G in the first position of codon 200 in the normal allele; therefore, the 88-bp fragment amplified by the nested PCR is divided into 74-and 14-bp fragments upon EagI digestion. On the other hand, the allele containing a GNAS mutation at the first or second position of the Arg 201 codon is not digested by EagI.…”

“…11 Subsequently, GNAS mutations were observed in extragnathic fibrous dysplasias without McCuneAlbright syndrome, [12][13][14][15] recognizing it as a marker of fibrous dysplasia. However, the presence or absence of GNAS mutations in gnathic fibrous dysplasias has not been examined.…”

Fibrous Dysplasia as a Stem Cell Disease

“…Stated in another way, the issue of relative frequencies of normal and mutated cells, as well as of normal and mutated progenitor cells, became relevant. More refined methods for mutation analysis (such as PNA clamping, which permits detection with an estimated sensitivity of ∼1:200 cells, as opposed to 1:4 cells for standard PCR/sequencing (36,37) ) were progressively developed. This made it possible to quantitate the mutational load in nonclonal monolayers of FD-derived stromal cells.…”

GNAS mutational analysis in differentiating fibrous dysplasia and ossifying fibroma of the jaw

“…In the 13 studies that mentioned the types of the bone involved, 1,13,16,17,19,21,23,27,28,30,31,33,34 the positive rate of GNAS mutation was 84% in the extragnathic bones and 78% in the craniofacial bones.…”