[67] Quantitative Analysis of Activating Alpha Subunit of the G Protein Mutation by Pyrosequencing for Fibrous Dysplasia

Q Liang, MQ Wei, GH Wang, JC Fanburg-Smith, A Nelson, M Miettinen, RD Foss. Armed Forces Institute of Pathology, Washington, DC

Background: Benign fibro-osseous lesions (BFOL) frequently display overlapping histologic features. Differentiation of fibrous dysplasia (FD) from other BFOL can be difficult, even for experienced orthopedic pathologists. A somatic mutation at codon Arg²⁰¹ of the alpha subunit of the G protein has been identified in FD and is specifically absent in other BFOL. A sensitive quantitative method could potentially eliminate some of the uncertainty in the diagnosis of these BFOL. We have developed a quantitative assay using a pyrosequencing method which has a detection sensitivity of at least 5% of affected cells admixed with normal tissue. The test allows the identification of 2 most common types of mutation (Arg to His and Arg to Cys), along with the rare Arg to Leu mutation, in a single run.
Design: Twenty-five FD, 2 osteofibrous dysplasia (OD), 9 ossifying fibromas (8 gnathic and 1 metacarpal), 4 BFOL not otherwise specified, 2 desmoplastic fibroma, and 7 Paget Disease cases from our files were selected. Sequencing analysis by pyrosequencing was performed on formalin fixed, paraffin embedded tissue from these cases. Histological and immunohistochemical features (if applicable) and clinical histories were reviewed.
Results: Of the 25 FD cases, 23 (92%) were positive for alpha subunit of the G protein mutation. Nineteen of 23 positive cases have a G->A mutation (Arg->His) and 4 have a C->T mutation (Arg->Cys). Two of 4 cases of BFOL not otherwise specified cases were positive for G->A mutation. None of osteofibrous dysplasia, ossifying fibroma, desmoplastic fibroma, and Paget Disease cases were positive for this mutation.
Conclusions: Mutation analysis of the alpha subunit of the G protein by pyrosequencing has significant potential for improving discrimination between FD and other BFOL in problematic cases. Compared to other detection methods such as restriction enzymatic digestion and probe hybridization, pyrosequencing has the ability to identify several types of mutations in a single reaction. In addition, pyrosequencing method achieves greater quantification sensitivity than general sequencing. In combination with microdissection techniques, the quantification ability of pyrosequencing may enable us to correlate the mutation detection with histopathological events. We believe that pyrosequencing is a relatively easy and accurate method for activating alpha subunit of the G protein mutation detection and quantification in fibrous dysplasia.
Category: Bone & Soft Tissue

Tuesday, March 10, 2009 9:30 AM

Poster Session III # 24, Tuesday Morning