The Journal of Bone and Joint Surgery (American). 2005;87:2489-2494.
doi:10.2106/JBJS.E.00160
© 2005 The Journal of Bone and Joint Surgery, Inc.
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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

Steven A. Lietman, MD1, Changlin Ding, MD1 and Michael A. Levine, MD1

1 Department of Orthopaedic Surgery/A41, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address for S.A. Lietman: lietmas{at}ccf.org

Investigation performed at the Departments of Orthopaedic Surgery, Biomedical Engineering, and Pediatrics, the Cleveland Clinic Foundation, Cleveland, Ohio, and the Ilyssa Center for Molecular and Cellular Endocrinology at the Johns Hopkins School of Medicine, Baltimore, Maryland

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from the National Institutes of Health US Public Health Service Research grants K08-AR47661 (SAL), DK34281 and DK56178 (MAL), and General Clinical Research Center Grant RR0035. Dr. Lietman is the recipient of and this work is supported by a Career Development Award from the Orthopaedic Research and Education Foundation. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

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.

Level of Evidence: Diagnostic Level I. See Instructions to Authors for a complete description of levels of evidence.


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R. D. Chapurlat
Fibrous Dysplasia of Bone and McCune-Albright Syndrome
IBMS BoneKEy, January 1, 2010; 7(1): 18 - 26.
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