A highly sensitive polymerase chain reaction method detects activating mutations of the GNAS gene in peripheral blood cells in McCune-Albright syndrome or isolated fibrous dysplasia.

BACKGROUND The somatic nature of mutations in the GNAS gene in McCune-Albright syndrome and isolated fibrous dysplasia makes their identification difficult. Conventional methods for the detection of mosaic mutations of GNAS have required polymerase chain reaction analysis of genomic DNA from affected tissues or multiple rounds of tandem polymerase chain reaction and endonuclease digestion to enrich for mutant alleles in genomic deoxyribonucleic acid (DNA) from other tissues. Peptide nucleic acid (PNA) primers specifically block synthesis from the nonmutant or wild-type allele. We therefore used PNA-clamping to detect low copy numbers of mutant GNAS alleles in DNA from peripheral blood cells from patients with McCune-Albright syndrome and fibrous dysplasia. METHODS We applied the PNA-clamping method to the analysis of genomic DNA from peripheral blood cells of thirteen patients with McCune-Albright syndrome and three patients with isolated fibrous dysplasia. Polymerase chain reaction was performed in the presence and absence of PNA, and the polymerase chain reaction products were sequenced. In the absence of PNA, a strong 325 base-pair polymerase chain reaction band was generated from all samples; in the presence of PNA, there was an approximately 50% to 90% reduction in the intensity of this polymerase chain reaction product. RESULTS In the absence of PNA, direct sequencing of the polymerase chain reaction products demonstrated R201 mutations in GNAS alleles of three of the thirteen patients with McCune-Albright syndrome and none of the three patients with fibrous dysplasia. In contrast, in the presence of PNA, R201 mutations were detected in eleven of the thirteen patients with McCune-Albright syndrome and in all three of the patients with fibrous dysplasia. In mixing experiments involving the use of wild-type and mutant DNA samples, we were able to determine the presence of a mutant GNAS allele in the equivalent of one cell in 1000 to 5000 cells. CONCLUSIONS Inclusion of a specific PNA primer in the polymerase chain reaction for GNAS exon 8 allows the selective amplification of low numbers of mutant alleles, and it permits detection of activating mutations in genomic DNA from peripheral blood cells in patients with McCune-Albright syndrome and fibrous dysplasia.

[1]  M. Collins,et al.  A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome. , 2004, Nucleic acids research.

[2]  L. Weinstein,et al.  Inherited diseases involving g proteins and g protein-coupled receptors. , 2004, Annual review of medicine.

[3]  K. White,et al.  FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting. , 2003, The Journal of clinical investigation.

[4]  M. Levine,et al.  Is McCune-Albright syndrome overlooked in subjects with fibrous dysplasia of bone? , 2003, The Journal of pediatrics.

[5]  D. Murdock,et al.  PNA-Mediated PCR Clamping , 2002 .

[6]  D. Murdock,et al.  PNA-mediated PCR clamping. Applications and methods. , 2002, Methods in molecular biology.

[7]  H. Yoshikawa,et al.  Hypophosphatemic rickets accompanying McCune–Albright syndrome: evidence that a humoral factor causes hypophosphatemia , 2001, Journal of Bone and Mineral Metabolism.

[8]  Y. Iwamoto,et al.  A comparative study of fibrous dysplasia and osteofibrous dysplasia with regard to Gsalpha mutation at the Arg201 codon: polymerase chain reaction-restriction fragment length polymorphism analysis of paraffin-embedded tissues. , 2000, The Journal of molecular diagnostics : JMD.

[9]  S. Wientroub,et al.  Mutations of the GNAS1 Gene, Stromal Cell Dysfunction, and Osteomalacic Changes in Non–McCune–Albright Fibrous Dysplasia of Bone , 2000, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[10]  A. Neubauer,et al.  Facilitated detection of oncogene mutations from exfoliated tissue material by a PNA‐mediated ‘enriched PCR’ protocol , 2000, The Journal of pathology.

[11]  M. Levine.  Clinical implications of genetic defects in G proteins: oncogenic mutations in G alpha s as the molecular basis for the McCune-Albright syndrome. , 1999, Archives of medical research.

[12]  O. Cochet,et al.  Selective PCR amplification of functional immunoglobulin light chain from hybridoma containing the aberrant MOPC 21-derived V kappa by PNA-mediated PCR clamping. , 1999, BioTechniques.

[13]  M. Schuermann,et al.  Sensitive detection of p53 gene mutations by a 'mutant enriched' PCR-SSCP technique. , 1998, Nucleic acids research.

[14]  F. Glorieux,et al.  Polymerase chain reaction-based technique for the selective enrichment and analysis of mosaic arg201 mutations in Gαs from patients with fibrous dysplasia of bone , 1997 .

[15]  F. Glorieux,et al.  Polymerase chain reaction-based technique for the selective enrichment and analysis of mosaic arg201 mutations in G alpha s from patients with fibrous dysplasia of bone. , 1997, Bone.

[16]  M. Egholm,et al.  Single base pair mutation analysis by PNA directed PCR clamping. , 1993, Nucleic acids research.

[17]  M. Levine.  The McCune-Albright syndrome. The whys and wherefores of abnormal signal transduction. , 1991, The New England journal of medicine.

[18]  L. Hovelius,et al.  Report of Five Cases , 1977 .

[19]  P. Smith,et al.  Syndrome Characterized by Osteitis Fibrosa Disseminata, Areas of Pigmentation and Endocrine Dysfunction, with Precocious Puberty in Females , 1937 .

[20]  C. Mckhann.  SOCIETY FOR PEDIATRIC RESEARCH , 1933 .

[21]  Davies Hm.  Osteitis Fibrosa Cystica. , 1915 .