Quantitative Analysis of Activating Alpha Subunit of the G Protein (Gsα) Mutation by Pyrosequencing in Fibrous Dysplasia and Other Bone Lesions

Abstract

Benign fibro-osseous lesions (BFOLs) frequently display overlapping histological features. The differentiation of fibrous dysplasia (FD) from other BFOLs can be difficult, even for experienced orthopedic pathologists. Accurately distinguishing FD from other BFOLs may have significant clinical and treatment implications. A somatic mutation in gene GNAS encoding the ␣ subunit of the G protein (Gs␣) involving the codon corresponding to Arg 201 has been identified in FD and is specifically absent in other BFOLs. We have developed a quantitative assay by pyrosequencing that has a detection sensitivity of 95%. The test allows the identification of the two most common types of mutation (Arg¡His and Arg¡Cys) in a single reaction, with the ability to analyze other rare mutations. Of the 24 FD cases in this series, 23 (96%) were positive for GNAS/Gs␣ mutation. Nineteen of 23 positive cases exhibited a G¡A mutation (Arg¡His), whereas four had a C¡T mutation (Arg¡Cys). One of three BFOL, not otherwise specified cases was positive for G¡A mutation. None of the osteofibrous dysplasia, ossifying fibromas, or other bone lesions were positive for this mutation. Our experience is that pyrosequencing is an easy and accurate quantification method for Gs␣ mutation detection in fibrous dysplasia. Mutation analysis of the Gs␣ by pyrosequencing has significant potential for improving discrimination between FD and other BFOLs in problematic cases. Fibrous dysplasia is a benign fibro-osseous lesion (BFOL) of the medullary cavity, which may involve one or more bones.1 Because fibrous dysplasia (FD) and other BFOLs frequently display overlapping histological features, differentiation of FD from a BFOL can be difficult, even for experienced orthopedic pathologists, particularly on small biopsy samples.2,3 A somatic mutation at codon 201 of the ␣ subunit of G protein (Gs␣), encoded by the GNAS gene, has been identified in FD (monostotic and polyostotic forms) and in multiple endocrinopathies of McCune-Albright syndrome, 4 but is specifically absent in other BFOLs, such as osteofibrous dysplasia.3,5,6 Point mutations result in replacement of Arg (CGT) at position 201 with, most commonly, His (CAT) or Cys (TGT) 4 ; and in rare cases, with Ser (AGT), 7 Leu (CTT), 8 or Gly (GGT). 9The mutated Gs␣ is constitutively activated, leading to cAMP activation and downstream physiological responses in affected tissues, including bone, skin, endocrine glands, and other tissues. 4,10,11 Because of the mosaic nature of this mutation, the distribution of mutant cells and the ratio of mutant to normal cells in a lesion or affected individual may be reflected in the severity of phenotypic expression.10,11 Interestingly, the percentage of mutant stem cells decreases as a lesion ages resulting in "normalization" of FD of bone.12 Addressing these issues require an extremely sensitive and accurate quantitative assay for Arg201 mutation detection.Many detection assays have been developed to identify the Arg201 mutations. The two most widely used methods are direct ...

Diagnostic value of investigating GNAS mutations in fibro-osseous lesions: a retrospective study of 91 cases of fibrous dysplasia and 40 other fibro-osseous lesions

“…Indeed, the sensitivity of detection of GNAS mutations varied from 23.1% (3/13) to 100% (16/16) with PCR-restriction fragment-length polymorphism, 36,37 58.3% (28/48) to 88% (8/9) with direct sequencing, 39,41 67.1% (51/76) with mutation-specific restriction enzyme digestion, 32 100% (8/8) with protein nucleic acid-mediated PCR, 44 100% (9/9) with restriction digestion analysis and direct sequencing, 38 and 96% (23/24) with pyrosequencing. 41 The most common GNAS Diagnostic value of investigating GNAS mutations F Tabareau-Delalande et al mutations by far were R201H (66.4%, 95/143) and R201C (30.8%, 44/143) on exon 8, followed by only three cases of Q227L mutation (2.1%, exon 9) and one case of R201S mutation (0.7%, exon 8). In our study, GNAS mutations were detected in 45% (23/51) cases of fibrous dysplasia (52% R201H and 48% R201C).…”

“…41 The sensitivity of these techniques is variable and GNAS mutations remain unidentified in many fibrous dysplasia lesions, regardless of the molecular method used. 28,32,39 The aim of this study was to assess the value of routine detection of GNAS mutations to differentiate fibrous dysplasia from other benign fibro-osseous lesions and low-grade osteosarcoma, by (i) evaluating the sensitivity of detection of GNAS mutation in fibrous dysplasia, (ii) confirming the specificity of this mutation in fibrous dysplasia and (iii) correlating GNAS mutation status with clinical and histopathology findings.…”

GNAS mutations are not detected in parosteal and low-grade central osteosarcomas

“…Dedifferentiation affects 16-43% of parosteal osteosarcomas with the characteristic gene amplifications being retained and can be present at the first presentation (synchronous type) or at the time of recurrence (metachronous type). [7][8][9] Bone tumors analyzed previously for GNAS alterations include fibrous dysplasia (N = 405), [10][11][12][13][14][15][16] ossifying fibroma (N = 65), 10,12,[15][16] osteofibrous dysplasia (N = 19), 10,12-13 low-grade central osteosarcoma (N = 12) 10,13-14 , and parosteal osteosarcoma (N = 10). 10 GNAS mutations have been reported in fibrous dysplasia and have not been described until recently in any other fibrous osseous lesions with the exception of a single low-grade central osteosarcoma.…”

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

“…Notably, three cases with fibrous dysplasia in this study had no detectable mutation, which indicated that the diagnosis of fibrous dysplasia could not be ruled out when no mutation could be detected, mainly because of the technical concerns regarding regular PCR and direct sequencing, which requires high quality and quantity of DNA, and also a mutant threshold of about 20% in the total population; 15 however, the somatic nature of the mutations in fibrous dysplasia may not meet this level of sensitivity in some cases, especially for the older ones, as reported by Kuznetsov et al 13 that the percentage of mutated cells within a given lesion may decrease with age. Two of the three fibrous dysplasia cases with no detectable GNAS mutations in this study were older than 40 years of age, probably caused by the decreasing percentage of the mutant cells.…”

“…13 Somatic mutations at Arg 201 and Gln 227 codon of Gsa have been identified in many fibrous dysplasia lesions, but absent in ossifying fibroma lesions, 14,15 which points to a possible role of the mutational analysis in differentiating these two conditions. Furthermore, Toyosawa's study also suggest that polymerase chain reaction (PCR) analysis with peptide nucleic acid (PNA) for GNAS mutations at the Arg 201 codon is a useful method to differentiate between fibrous dysplasia and ossifying fibroma.…”