The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 87, Issue 11

Scientific Articles | November 01, 2005

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; 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@ccf.org

View Disclosures and Other Information

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.

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

The Journal of Bone and Joint Surgery, Incorporated

J Bone Joint Surg Am, 2005 Nov 01;87(11):2489-2494. doi: 10.2106/JBJS.E.00160

5 Recommendations (Recommend) | 3 Comments | Saved by 3 Users Save Case

Article

Comments (0)

text A A A

Abstract

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.

Figures in this Article

McCune-Albright syndrome is a sporadic disorder that is characterized by the clinical triad of polyostotic fibrous dysplasia, café au lait skin lesions, and endocrine hyperfunction^1-5. The prominent features of McCune-Albright syndrome are due to constitutive (i.e., hormone-independent) activation of the adenylyl cyclase signaling pathway in affected tissues and unregulated synthesis of the second messenger cyclic adenosine monophosphate (cAMP). The molecular defect in McCune-Albright syndrome arises from missense mutations in exon 8 of the GNAS gene that lead to replacement of arginine 201 (Arg²⁰¹ or R201) and conversion to the gsp oncogene that encodes the stimulatory guanine nucleotide binding regulatory protein alpha (Gsa) lacking intrinsic GTPase activity^3-5. Cells containing these activating mutations are present in affected endocrine tissues, skin, and bone from patients with McCune-Albright syndrome as well as in many unaffected tissues, but they are not present in all cells of affected patients, even within affected organs. Mutation-bearing cells are distributed in a mosaic pattern, with the greatest number being present in the most abnormal areas of affected tissues, consistent with the fact that the mutation is not germline but rather is a postzygotic somatic mutation^3-5.

View Large | Download Slide(.ppt)
Add to My JBJS

Fig. 1: Illustration depicting the PNA-clamping scheme. In the scenario depicted in the upper panel, the perfectly complementary PNA will preferentially bind to the target wild-type genomic DNA, thus preventing the polymerase chain reaction primer from annealing. This effectively inhibits amplification. Conversely, in the scenario depicted in the lower panel, the PNA probe will not bind to the target DNA because of an internal mismatch in the Arg²⁰¹ codon, thus permitting the polymerase chain reaction primer to anneal. The result is amplification of the mutant GNAS amplicon.

The mosaic pattern of distribution of cells bearing the GNAS mutation and the variable number of affected cells in a tissue have hampered ready identification of the gene mutation in peripheral blood cells as mutant GNAS alleles may represent only a small proportion of the total number of GNAS alleles present in DNA. Detection of mutant GNAS genes in DNA samples was previously demonstrated to be enhanced by a protocol in which multiple rounds of nested polymerase chain reaction were performed in conjunction with restriction endonuclease treatment to digest wild-type products, thus enriching the abundance of mutant alleles for selective amplification^6,7.

More recently, Bianco et al.⁸ described a protocol involving the use of PNA-clamping^5,9-14 as a means of detecting GNAS mutations in DNA samples from fibrous dysplasia lesions (Fig. 1). Karadag et al.¹⁵ further evaluated PNA-clamping with fluorescence resonance energy transfer and noted that this technique was useful for detecting the relative percentage of cells in lesional tissue with an Arg²⁰¹ mutation. PNAs are polynucleotide mimics having a 2-aminoethylglycine backbone in lieu of the deoxyribose phosphate backbone of DNA. PNAs form PNA:DNA hybrids that are more stable than DNA:DNA hybrids are, and PNAs are more sensitive to internal base mismatches with their DNA complement. In this technique, a PNA that is perfectly complementary to the wild-type Arg²⁰¹ nucleotide sequence overlaps the binding site of the forward polymerase chain reaction primer. The PNA prevents the polymerase chain reaction primer from binding to the normal sequence and thereby blocks amplification of the wild-type allele. Because mutant GNAS alleles contain a single-base mismatch, the PNA cannot block annealing of the forward polymerase chain reaction primer to mutant GNAS alleles, thus enabling selective amplification of the mutant GNAS alleles. In the present study, we demonstrate the utility of the PNA-clamping technique for the detection of GNAS mutations in genomic DNA isolated from peripheral blood cells of subjects with McCune-Albright syndrome or fibrous dysplasia. While a negative result on analysis of peripheral blood (observed in two of the patients in the present study) does not exclude the diagnosis of McCune-Albright syndrome or fibrous dysplasia, the ability to diagnose McCune-Albright syndrome or fibrous dysplasia on the basis of blood samples could prevent many diagnostic biopsies and thereby avoid complications such as fracture, infection, or anesthetic complications.

Materials and Methods

Materials and Methods | Results | Discussion | Appendix | References

The present study was approved by the internal review board at our institution. Consent was obtained from all patients (or from their family members) prior to inclusion in the study. Peripheral blood samples were obtained from individuals with either McCune-Albright syndrome or fibrous dysplasia during the course of routine care or research evaluation (see Appendix). Whole blood was collected in tubes containing ethylenediaminetetraacetic acid (EDTA), and leukocytes were isolated with the Histopaque kit (Sigma, St. Louis, Missouri) as recommended on the package insert. DNA was then obtained by means of phenol chloroform extraction.

View Large | Download Slide(.ppt)
Add to My JBJS

Fig. 2: PNA inhibited polymerase chain reaction of the wild-type GNAS gene. Polymerase chain reaction was performed in the presence (+) or absence (-) of PNA with use of genomic DNA from subjects with McCune-Albright syndrome (lanes labeled 1 through 4) and two normal control individuals (lanes labeled C). A band of predicted size (325 base pairs) was present when polymerase chain reaction was performed in the absence of PNA; the intensity of this band was reduced by the inclusion of the PNA and was nearly absent in the DNA samples from normal control subjects. These samples were loaded on a 5% polyacrylamide gel; an inverse image is presented. The water lane represents a control with no DNA; there is no amplicon in this water lane, indicating no contamination.

A PNA probe was synthesized on a PerSeptive Biosystems Expedite instrument (PerSeptive Biosystems, Framingham, Massachusetts) by Dr. Nga Nguyen at the National Institutes of Health (Center for Biologics Evaluation and Research, Facility for Biotechnology Research, Peptide Laboratory) and was purified by means of reverse-phase high performance liquid chromatography. The sequences of the PNA (base pairs 642 to 653 for codons 199 to 202) and primers used to amplify a portion of exon 8 of GNAS were as follows:

PNA: Gly-NH₂-CGCTGCCGTGTC-HAc

Forward primer: 5'-GTTTCAGGACCTGCTTCGC-3'

Reverse primer: 5'-GCAAAGCCAAGAGCGTGAG-3'

Each polymerase chain reaction contained 200 to 500 ng of target genomic DNA, 2.5 U of AmpliTaq Gold polymerase (PerkinElmer, Boston, Massachusetts), 1 µg each of forward and reverse polymerase chain reaction primer, and 2 µg of the PNA in a 100-µl final volume. After an initial denaturation at 94°C for fifteen minutes to activate the polymerase, forty polymerase chain reaction cycles followed, consisting of a denaturation step to 94°C for thirty seconds, a PNA hybridization step at 68°C for sixty seconds, a primer annealing step at 55°C for thirty seconds, and an extension step at 72°C for sixty seconds. A final extension step was performed at 72°C for seven minutes⁸.

At the conclusion of the polymerase chain reaction, the sample was electrophoresed through a 5% polyacrylamide gel in TBE buffer, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. Bands corresponding to an expected 325-base pair (bp) product were isolated from the polyacrylamide gel and were directly sequenced by polymerase chain reaction with use of the forward primer and the Thermo Sequenase radiolabeled terminator cycle sequencing kit according to the manufacturer's instructions (Amersham Biosciences, Piscataway, New Jersey). For mixing experiments, the DNA sample from the patients with McCune-Albright syndrome contained equal amounts of wild-type and mutant GNAS alleles (i.e., 50% mutant alleles) and was mixed with increasing amounts of normal DNA to obtain the indicated percentages of mutant alleles.

View Large | Download Slide(.ppt)
Add to My JBJS

Fig. 3: Analysis of GNAS mutations in DNA isolated from peripheral blood leukocytes. Nucleotide sequence analysis of the region encompassing the arginine 201 (Arg) codon is presented for three subjects with McCune-Albright syndrome. Polymerase chain reaction performed in the absence of PNA revealed only alleles containing wild-type sequence (CGT). In contrast, when polymerase chain reaction was performed in the presence of PNA, only alleles containing GNAS mutations were amplified. Three different mutations were detected with equivalent sensitivity: CGT?CAT/arginine?histidine (arg?his), CGT?CTT/arginine?leucine (arg?leu), and CGT?TGT/arginine?cysteine (arg?cys).

Results

Materials and Methods | Results | Discussion | Appendix | References

All of the patients with McCune-Albright syndrome had café au lait skin lesions, fibrous dysplasia bone lesions, and endocrinopathy and therefore met the clinical diagnostic criteria for McCune-Albright syndrome^1-5. All of the patients with fibrous dysplasia had only fibrous dysplasia bone lesions and therefore met the clinical diagnostic criteria for fibrous dysplasia.

In the absence of PNA, a strong 325-bp polymerase chain reaction band was generated from all samples and direct sequencing of the polymerase chain reaction products demonstrated R201 mutations in only three of the thirteen patients with McCune-Albright syndrome and in none of the three patients with fibrous dysplasia. When polymerase chain reaction was performed in the presence of PNA, there was an approximately 50% to 90% reduction in the intensity of this polymerase chain reaction product (Fig. 2).

In the presence of PNA, direct sequence analysis revealed the R201 mutation in eleven of the thirteen patients with McCune-Albright syndrome (four patients with R201H, six patients with R201C, and one patient with R201L) (Fig. 3) and in all three patients with fibrous dysplasia (all with R201C). In experiments involving the use of wild-type and mutant DNA samples mixed together, we were able to determine the presence of a mutant GNAS allele in the equivalent of one cell with the mutation among 1000 to 2000 normal cells (representing a rate of detection of 0.1% to 0.05%) (Fig. 4). DNA samples from normal subjects served as a negative control and showed wild-type GNAS sequence in the absence or presence of PNA and no mutation in the presence of PNA (data not shown).

Discussion

Materials and Methods | Results | Discussion | Appendix | References

The mosaic pattern of distribution of cells bearing the GNAS mutation, and the variable number of affected cells in a tissue, has complicated the identification of mutant GNAS alleles, which may represent only a small proportion of the GNAS alleles present in DNA isolated from tissue or peripheral blood. Detection of mutant GNAS genes in DNA samples had been previously enhanced by a protocol in which multiple rounds of nested polymerase chain reaction were performed in conjunction with restriction endonuclease treatment to digest wild-type products, thus enriching the abundance of mutant alleles for selective amplification^6,7. However, this technique is not applicable for all mutation sites and has been shown to have variable sensitivity¹⁶. Bianco et al.⁸ and Karadag et al.¹⁵ previously applied the PNA-clamping technique to analyze lesional tissue from patients with McCune-Albright syndrome, which suggested to us that this approach might be useful for analyzing peripheral blood samples from patients with McCune-Albright syndrome or isolated fibrous dysplasia. While the presence of café au lait and endocrine abnormalities in association with McCune-Albright syndrome and fibrous dysplasia suggests that there might be cells in the blood with GNAS mutations, and while several investigators have shown that humoral factors are involved in hypophosphatemic rickets accompanying McCune-Albright syndrome, no one to our knowledge has been able to consistently find the mutation in peripheral blood samples, and, in general, the diagnosis is still made clinically^2,17,18. In the present study, we found that PNA-clamping provided an enhancement over standard polymerase chain reaction as well as a simple alternative to nested polymerase chain reaction schemes in the detection of GNAS mutations and that it detected GNAS mutations in genomic DNA isolated from peripheral blood leukocytes in eleven of thirteen patients with McCune-Albright syndrome and three of three patients with fibrous dysplasia. The ability to detect GNAS mutations in DNA isolated from peripheral blood of patients with McCune-Albright syndrome implies that the leukocytes contain mutant GNAS alleles and/or that cells from affected tissues are also present in the circulation.

View Large | Download Slide(.ppt)
Add to My JBJS

Fig. 4: Sensitivity of the PNA-clamping method. Nucleotide sequence analysis of the region encompassing the Arg²⁰¹ codon is presented after polymerase chain reaction of DNA containing varying amounts of genomic DNA from a subject with McCune-Albright syndrome and a normal subject. The DNA sample from the patient with McCune-Albright syndrome contained equal amounts of wild-type and mutant GNAS alleles (i.e., 50% mutant alleles) and was mixed with increasing amounts of normal DNA to obtain the indicated percentages of mutant alleles. When polymerase chain reaction was performed in the absence of PNA, sequence analysis was unable to detect the GNAS mutation unless mutant alleles constituted >2.5% of the target genomic DNA. In contrast, when polymerase chain reaction was performed in the presence of PNA, the GNAS mutation was easily detected even in DNA samples in which the mutant DNA constituted only 0.1% of the total.

Similarly, the presence of cells bearing the GNAS-activating mutation in the circulation of patients with fibrous dysplasia suggests either that the somatic mosaicism is more widespread than clinically suspected or that cells from fibrous dysplasia lesions are shed into the circulation. Additional studies will be needed to distinguish between these two alternatives, and some patients may not have detectable mutations in the blood if their involvement does not extend to the peripheral blood. In conclusion, our study adds support to the growing enthusiasm for and utility of PNA-clamping in molecular diagnostics. The ability to identify somatic mutations in cells that represent only a small percentage of the total cell mass may allow this technique to be adapted for the analysis of blood or tissues in patients with malignant disease in order to increase the sensitivity of cancer-staging and therefore improve decisions about the need for and efficacy of systemic treatment.

Appendix

Materials and Methods | Results | Discussion | Appendix | References

A table presenting clinical details of all study subjects is available with the electronic versions of this article, on our web site at (go to the article citation and click on "Supplementary Material") and on our quarterly CDROM (call our subscription department, at 781-449-9780, to order the CD-ROM).

References

Materials and Methods | Results | Discussion | Appendix | References

Albright F, Butler AM, Hampton AO, Smith P. Syndrome characterized by osteitis fibrosa disseminata, areas of pigmentation, and endocrine dysfunction, with precocious puberty in females. Report of five cases. N Engl J Med.1937;216: 727-46.216727 1937 [CrossRef]

McCune DJ. Osteitis fibrosa cystica. The case of a nine year old girl who also exhibits precocious puberty, multiple pigmentation of the skin and hyperthyroidism. Am J Dis Child.1936;52: 743-4.52743 1936

Levine MA. The McCune-Albright syndrome. The whys and wherefores of abnormal signal transduction. N Engl J Med.1991;325: 1738-40.3251738 1991 [PubMed][CrossRef]

Levine MA. Clinical implications of genetic defects in G proteins: oncogenic mutations in G alpha s as the molecular basis for the McCune-Albright syndrome. Arch Med Res.1999;30: 522-31.30522 1999 [PubMed][CrossRef]

Spiegel AM, Weinstein LS. Inherited diseases involving g proteins and g protein-coupled receptors. Annu Rev Med.2004;55: 27-39.5527 2004 [CrossRef]

Candeliere GA, Roughley PJ, Glorieux FH. 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. Bone.1997;21: 201-6.21201 1997 [PubMed][CrossRef]

Sakamoto A, Oda Y, Iwamoto Y, Tsuneyoshi M. 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. J Mol Diagn.2000;2: 67-72.267 2000 [PubMed][CrossRef]

Bianco P, Riminucci M, Majolagbe A, Kuznetsov SA, Collins MT, Mankani MH, Corsi A, Bone HG, Wientroub S, Spiegel AM, Fisher LW, Robey PG. Mutations of the GNAS1 gene, stromal cell dysfunction, and osteomalacic changes in non-McCune-Albright fibrous dysplasia of bone. J Bone Miner Res.2000;15: 120-8.15120 2000 [PubMed][CrossRef]

Behn M, Schuermann M. Sensitive detection of p53 gene mutations by a `mutant enriched' PCR-SSCP technique. Nucleic Acids Res.1998;26: 1356-8.261356 1998 [PubMed][CrossRef]

Behn M, Thiede C, Neubauer A, Pankow W, Schuermann M. Facilitated detection of oncogene mutations from exfoliated tissue material by a PNA-mediated `enriched PCR' protocol. J Pathol.2000;190: 69-75.19069 2000 [CrossRef]

Cochet O, Martin E, Fridman WH, Teillaud JL. Selective PCR amplification of functional immunoglobulin light chain from hybridoma containing the aberrant MOPC 21-derived V kappa by PNA-mediated PCR clamping. Biotechniques.1999;26: 818-20, 822.26818 1999 [PubMed]

Murdock DG, Wallace DC. PNA-mediated PCR clamping. Applications and methods. Methods Mol Biol.2002;208: 145-64.208145 2002 [PubMed]

Orum H, Nielsen PE, Egholm M, Berg RH, Buchardt O, Stanley C. Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res.1993;21: 5332-6.215332 1993 [PubMed][CrossRef]

Orum H. PCR clamping. Curr Issues Mol Biol.2000;2: 27-30.227 2000

Karadag A, Riminucci M, Bianco P, Cherman N, Kuznetsov SA, Nguyen N, Collins MT, Robey PG, Fisher LW. A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome. Nucleic Acids Res.2004;32: e63.32e63 2004 [PubMed][CrossRef]

Hannon TS, Noonan K, Steinmetz R, Eugster EA, Levine MA, Pescovitz OH. Is McCune-Albright syndrome overlooked in subjects with fibrous dysplasia of bone? J Pediatr.2003;142: 532-8.142532 2003 [PubMed][CrossRef]

Yamamoto T, Miyamoto KI, Ozono K, Taketani Y, Katai K, Miyauchi A, Shima M, Yoshikawa H, Yoh K, Takeda E, Okada S. Hypophosphatemic rickets accompanying McCune-Albright syndrome: evidence that a humoral factor causes hypophosphatemia. J Bone Miner Metab.2001;19: 287-95.19287 2001 [CrossRef]

Riminucci M, Collins MT, Fedarko NS, Cherman N, Corsi A, White KE, Waguespack S, Gupta A, Hannon T, Econs MJ, Bianco P, Gehron Robey P. FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting. J Clin Invest.2003;112: 683-92.112683 2003 [PubMed]

Topics

polymerase chain reaction ; mutation ; blood cells ; dna ; fibrous dysplasia, monostotic ; osteofibrous dysplasia

Username

Password

Access for Patients

Forgot Password?

Have a subscription to the print edition?

Not a Subscriber?

Buy this Article

Get online access for 30 days for $35

New to JBJS?

Subscribe to the online edition for 30 days for $75

Register for a FREE limited account to get full access to all CME activities, to comment on public articles, or to sign up for alerts.

Have a subscription to the print edition?

Current subscribers to The Journal of Bone & Joint Surgery in either the print or quarterly DVD formats receive free online access to JBJS.org.

Activate your online account now

Forgot your password?

Enter your username and email address. We'll send you a reminder to the email address on record.

Username:*

Email Address:*

Forgot your username or need assistance? Please contact customer service at subs@jbjs.org. If your access is provided
by your institution, please contact you librarian or administrator for username and password information. Institutional
administrators, to reset your institution's master username or password, please contact subs@jbjs.org

References

Accreditation Statement

These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.

CME Activities Associated with This Article

Submit a Comment

Please read the other comments before you post yours. Contributors must reveal any conflict of interest.
Comments are moderated and will appear on the site at the discretion of JBJS editorial staff.

* = Required Field

Comment Author(s) * (if multiple authors, separate names by comma)

Example: John Doe

Affiliation & Institution*

Comment Title*

Comment*

Cancel

Print
PDF
Email

Recipient(s) will receive an email with a link (good for 5 days) to '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'.
Your Name:*
Example: John Doe

Email Address:* CC Me:
Enter your valid email address. Example: jdoe@example.com

Recipient's Email Address:*

Separate multiple email address with semi-colons (up to 5).

Subject:* 's The Journal of Bone & Joint Surgery: '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'
Subject for your email.

Message:

(Optional, message will truncate at 1000 characters)

Processing your request... Please Wait...

Copyright © in the material you requested is held by The Journal of Bone & Joint Surgery, Inc. (unless otherwise noted). This email ability is provided as a courtesy, and by using it you agree that that you are requesting the material solely for personal, non-commercial use, and that it is subject to Journal of Bone & Joint Surgery, Inc.'s Terms of Use. The information provided in order to email this topic will not be used to send unsolicited email, nor will it be furnished to third parties. Please refer to The Journal of Bone & Joint Surgery Inc.'s Privacy Policy for further information.

Copyright © The Journal of Bone & Joint Surgery Inc. All rights reserved.
Share
Get Citation

Steven A. Lietman, Changlin Ding, Michael A. Levine; 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. The Journal of Bone & Joint Surgery. 2005 Nov;87(11):2489-2494.

Download citation file:

RIS (Zotero)

EndNote

BibTex

Medlars

ProCite

RefWorks

Reference Manager

Copyright © The Journal of Bone and Joint Surgery, Inc.
Rights & Permissions
Article Alerts

Processing your request... Please Wait.
Submit a Comment
Data Supplements