Polymerase chain reaction-based technique for the selective enrichment and analysis of mosaic arg201 mutations in Gαs from patients with fibrous dysplasia of bone

Mutations in the arg201 codon of the αs G protein subunit have been associated with a variety of disorders, but analysis of such mutations has been complicated by their mosaic presentation. To overcome the problems associated with the analysis of genomic mutations that may be present in low and variable yield throughout the body, a polymerase chain reaction (PCR)-based technique has been developed that allows the selective amplification of products from the mutant allele. This technique uses site-directed mutagenesis to generate a PCR product from the normal allele that is susceptible to restriction endonuclease digestion, whereas that from the mutant allele is resistant to digestion. Consecutive and repeated cycles of amplification and digestion allow selective enrichment of the product from the mutant allele. The technique has been applied to the analysis of patients with fibrous dysplasia of bone, where the consequence of Gαs mutations may vary from monostotic to polyostotic lesions, and has been performed with DNA isolated from either bone biopsy specimens or peripheral blood leukocytes. In addition to the previously described arg → his and arg → cys substitutions, the analyses have detected a novel arg → ser substitution in one of the patients. This patient presented with a panostotic disease and may represent a unique subgroup of fibrous dysplasia.