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)
A CTIVATING MISSENSE mutations of the GNAS1 gene, encoding the alpha subunit of the stimulatory G protein, Gs, have been identified in patients with the McCuneAlbright syndrome (MAS, characterized by polyostotic fibrous dysplasia [FD], café au lait skin pigmentation, and endocrine disorders).(1-3) The reduced GTPase activity of the mutated protein leads to overstimulation of adenylyl cyclase.(4) Recent studies on the nature of FD suggest that the excess generation of cAMP resulting from activity of the mutated Gs alpha may represent a common (albeit not necessarily the only) pathogenetic mechanism for the diverse, skeletal, and nonskeletal manifestations of MAS. (5,6) Similar mutations of the GNAS1 gene also occur in non-MAS associated FD of bone. (7)(8)(9) MAS was diagnosed in an 8-year-old male presenting with precocious puberty, facial deformities, and typical café au lait spots with a "coast of Maine" profile. Extensive involvement of the cranial vault was apparent on X-ray examination, and a sample of parietal bone demonstrated changes typical of the sclerotic/pagetoid variant of FD. (10) The craniofacial lesions were apparently progressive and were treated with pamidronate which was reported to reduce bone pain as well as to normalize the levels of serum alkaline phosphatase, osteocalcin, and hydroxyproline. At the age of 13, acromegalic bone changes and growth hormone oversecretion were detected.Genomic DNA was extracted from a surgical sample of the FD parietal bone that was obtained under an institutionally approved protocol for the use of human tissue in research (National Institutes of Health Protocol #97-DK-0055). Mutation analysis was performed by sequencing the polymerase chain reaction (PCR) amplification product (exon 8) in both directions (6) and by sequencing the PCR product obtained with peptide nucleic acid (PNA) inhibition of the normal allele amplification, a novel highly sensitive method especially suited for mutation analysis of mosaic populations.(9) With both assays, a novel C → G (R201G) mutation was detected (Fig. 1).With the exception of a single case (of polyostotic FD) in which an R201S mutation was identified previously, (11) R201C and R201H have been the mutations found consistently in MAS patients (5) and in non-MAS FD of bone. (9) Thus, of the predicted missense mutations of codon 201, only R201P and R201L remain undetected to date (although R201L has been observed in an isolated, non-MAS endocrine tumor (12) ( Table 1). The diversity of the amino acids encoded by the missense mutations detected so far (basic, uncharged polar, nonpolar) demonstrates that substitution of the R201 per se, rather than the characteristics of the substituting amino acid, is the critical molecular event leading to reduced GTPase activity. It is thought that R201 is an essential component of the "timing device" that regulates GTPase. Bourne et al. (4) have reported that two of the single-point missense mutations in R201 of Gs alpha (R201C and R201H), as found in human tumors, appear to have reduced ...
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