A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome

Karadağ, Abdullah; Riminucci, Mara; Bianco, Paolo; Cherman, Natasha; Kuznetsov, Sergei A.; Nguyen, Nga Y.; Collins, Michael T.; Robey, Pamela Gehron; Fisher, Larry W.

doi:10.1093/nar/gnh059

Cited by 40 publications

(40 citation statements)

References 36 publications

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“…The approximate frequency of these mutations was comparable to published data. [2][3][4][5][6][7][8][9][13][14][15][16][17][18][19] Twelve of 24 fibrous dysplasia cases were further tested by a general sequencing method. Ten of 12 cases of mutation detected by pyrosequencing assay were confirmed with general sequencing.…”

Section: Resultsmentioning

confidence: 99%

“…The two most widely used methods are direct sequencing and restriction fragment length polymorphism (RFLP). [2][3][4][5][6][7][8][9][11][12][13][14] Allele-specific oligo hybridization with fluorescent probe 15 instead of radioactive probe 16,17 is also used for quantitative studies. However, this technique requires at least three pairs of labeled probes to detect only the two common mutation types.…”

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confidence: 99%

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Quantitative Analysis of Activating Alpha Subunit of the G Protein (Gsα) Mutation by Pyrosequencing in Fibrous Dysplasia and Other Bone Lesions

Liang

Wei

Hodge

et al. 2011

The Journal of Molecular Diagnostics

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Benign fibro-osseous lesions (BFOLs) frequently display overlapping histological features. The differentiation of fibrous dysplasia (FD) from other BFOLs can be difficult, even for experienced orthopedic pathologists. Accurately distinguishing FD from other BFOLs may have significant clinical and treatment implications. A somatic mutation in gene GNAS encoding the ␣ subunit of the G protein (Gs␣) involving the codon corresponding to Arg 201 has been identified in FD and is specifically absent in other BFOLs. We have developed a quantitative assay by pyrosequencing that has a detection sensitivity of 95%. The test allows the identification of the two most common types of mutation (Arg¡His and Arg¡Cys) in a single reaction, with the ability to analyze other rare mutations. Of the 24 FD cases in this series, 23 (96%) were positive for GNAS/Gs␣ mutation. Nineteen of 23 positive cases exhibited a G¡A mutation (Arg¡His), whereas four had a C¡T mutation (Arg¡Cys). One of three BFOL, not otherwise specified cases was positive for G¡A mutation. None of the osteofibrous dysplasia, ossifying fibromas, or other bone lesions were positive for this mutation. Our experience is that pyrosequencing is an easy and accurate quantification method for Gs␣ mutation detection in fibrous dysplasia. Mutation analysis of the Gs␣ by pyrosequencing has significant potential for improving discrimination between FD and other BFOLs in problematic cases. Fibrous dysplasia is a benign fibro-osseous lesion (BFOL) of the medullary cavity, which may involve one or more bones.1 Because fibrous dysplasia (FD) and other BFOLs frequently display overlapping histological features, differentiation of FD from a BFOL can be difficult, even for experienced orthopedic pathologists, particularly on small biopsy samples.2,3 A somatic mutation at codon 201 of the ␣ subunit of G protein (Gs␣), encoded by the GNAS gene, has been identified in FD (monostotic and polyostotic forms) and in multiple endocrinopathies of McCune-Albright syndrome, 4 but is specifically absent in other BFOLs, such as osteofibrous dysplasia.3,5,6 Point mutations result in replacement of Arg (CGT) at position 201 with, most commonly, His (CAT) or Cys (TGT) 4 ; and in rare cases, with Ser (AGT), 7 Leu (CTT), 8 or Gly (GGT). 9The mutated Gs␣ is constitutively activated, leading to cAMP activation and downstream physiological responses in affected tissues, including bone, skin, endocrine glands, and other tissues. 4,10,11 Because of the mosaic nature of this mutation, the distribution of mutant cells and the ratio of mutant to normal cells in a lesion or affected individual may be reflected in the severity of phenotypic expression.10,11 Interestingly, the percentage of mutant stem cells decreases as a lesion ages resulting in "normalization" of FD of bone.12 Addressing these issues require an extremely sensitive and accurate quantitative assay for Arg201 mutation detection.Many detection assays have been developed to identify the Arg201 mutations. The two most widely used methods are direct ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Quantitative Analysis of Activating Alpha Subunit of the G Protein (Gsα) Mutation by Pyrosequencing in Fibrous Dysplasia and Other Bone Lesions

Liang

Wei

Hodge

et al. 2011

The Journal of Molecular Diagnostics

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“…Stated in another way, the issue of relative frequencies of normal and mutated cells, as well as of normal and mutated progenitor cells, became relevant. More refined methods for mutation analysis (such as PNA clamping, which permits detection with an estimated sensitivity of ∼1:200 cells, as opposed to 1:4 cells for standard PCR/sequencing (36,37) ) were progressively developed. This made it possible to quantitate the mutational load in nonclonal monolayers of FD-derived stromal cells.…”

Section: Fibrous Dysplasia and Stem Cells P127mentioning

confidence: 99%

“…(42) Third, the in vivo history of the lesions and patients from which the cells are derived introduces an additional layer of experimental variability (e.g., mutational burden). (37) Creating GNAS-mutated cells can circumvent these problems. Viral vectors capable of integration into the genome of infected cells permit efficient and stable transduction of human cells ex vivo.…”

Section: Using Stem Cells For Fd Researchmentioning

confidence: 99%

Fibrous Dysplasia as a Stem Cell Disease

Riminucci

Saggio

Robey

et al. 2006

Journal of Bone and Mineral Research

109

100

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ABSTRACT:At a time when significant attention is devoted worldwide to stem cells as a potential tool for curing incurable diseases, fibrous dysplasia of bone (FD) provides a paradigm for stem cell diseases. Consideration of the time and mechanism of the causative mutations and of nature of the pluripotent cells that mutate in early embryonic development indicates that, as a disease of the entire organism, FD can be seen as a disease of pluripotent embryonic cells. As a disease of bone as an organ, in turn, FD can be seen as a disease of postnatal skeletal stem cells, which give rise to dysfunctional osteoblasts. Recognizing FD as a stem cell disease provides a novel conceptual angle and a way to generate appropriate models of the disease, which will continue to provide further insight into its natural history and pathogenesis. In addition, skeletal stem cells may represent a tool for innovative treatments. These can be conceived as directed to alter the in vivo behavior of mutated stem cells, to replace mutated cells through local transplantation, or to correct the genetic defect in the stem cells themselves. In vitro and in vivo models are currently being generated that will permit exploration of these avenues in depth.

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“…Since GNAS-activating mutations occur in a mosaic distribution, a method that can detect even a low number of mutant alleles is required. Two main methods based on a PCR have been reported: the use of a restriction enzyme with nested PCR to selectively digest the wild-type allele and a peptidic nucleic acid (PNA) primer to inhibit amplification of the wild-type allele (12)(13)(14) with or without mutant ratio determination (15).…”

Section: Introductionmentioning

confidence: 99%

Searching for somatic mutations in McCune–Albright syndrome: a comparative study of the peptidic nucleic acid versus the nested PCR method based on 148 DNA samples

et al. 2006

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Background: Activating mutations of the Gsa gene (GNAS), which encodes for the a-subunit of the stimulatory G protein, have been identified in patients with McCune-Albright syndrome (MAS). Accuracy and sensitivity in the molecular diagnosis of MAS is mandatory for optimal therapeutic strategy and adapted follow-up, especially for incomplete clinical forms of MAS. To date, the highly sensitive nested PCR method with intermediary digestion by a restriction enzyme at the mutation site is one of the most widely used techniques. This study evaluated a new diagnostic method using a peptidic nucleic acid (PNA) and compared it with the nested PCR method. Material and methods: One hundred and forty-eight DNA samples from eighty-eight patients presenting clinical symptoms compatible with MAS were included. The DNA samples were mainly obtained from peripheral blood, ovarian tissue or cyst liquid, and bone lesions. The nested PCR method required 4 days. PNA clamping required 1.5 days and utilized the higher thermal stability and specificity of PNA-DNA coupling to inhibit PCR product formation. Direct sequencing was subsequently performed in all cases. Results: The sensitivity of mutation detection was 54% (nZ80) for nested PCR and 46.6% (nZ69) for PNA (PO0.05). The 11 cases where PNA failed to detect the mutation were mainly incomplete and atypical clinical forms of MAS (nZ10/11). The cost per sample was 50 euros for PNA clamping versus 136 euros for nested PCR. Conclusion: PNA clamping is a rapid, reliable, and economical method to diagnose MAS. It should be the first-line diagnostic method, although negative results, especially for incomplete clinical forms of MAS, should be confirmed by nested PCR.

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A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome

Cited by 40 publications

References 36 publications

Quantitative Analysis of Activating Alpha Subunit of the G Protein (Gsα) Mutation by Pyrosequencing in Fibrous Dysplasia and Other Bone Lesions

Quantitative Analysis of Activating Alpha Subunit of the G Protein (Gsα) Mutation by Pyrosequencing in Fibrous Dysplasia and Other Bone Lesions

Fibrous Dysplasia as a Stem Cell Disease

Searching for somatic mutations in McCune–Albright syndrome: a comparative study of the peptidic nucleic acid versus the nested PCR method based on 148 DNA samples

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