Quantitative and Sensitive Detection of GNAS Mutations Causing McCune-Albright Syndrome with Next Generation Sequencing

Satoshi Narumi, Kumihiro Matsuo, Tomohiro Ishii, Yusuke Tanahashi, Tomonobu Hasegawa

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.

Original languageEnglish
Article numbere60525
JournalPLoS One
Volume8
Issue number3
DOIs
Publication statusPublished - 2013 Mar 25

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Polyostotic Fibrous Dysplasia
Peptide Nucleic Acids
mutation
Mutation
nucleic acids
peptides
Blood
Dilution
methodology
Mutation Rate
DNA
blood
sampling
Exons
Polymerase Chain Reaction
somatic mutation
mutants
Limit of Detection

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Quantitative and Sensitive Detection of GNAS Mutations Causing McCune-Albright Syndrome with Next Generation Sequencing. / Narumi, Satoshi; Matsuo, Kumihiro; Ishii, Tomohiro; Tanahashi, Yusuke; Hasegawa, Tomonobu.

In: PLoS One, Vol. 8, No. 3, e60525, 25.03.2013.

Research output: Contribution to journalArticle

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title = "Quantitative and Sensitive Detection of GNAS Mutations Causing McCune-Albright Syndrome with Next Generation Sequencing",
abstract = "Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1{\%} for the PNA method, 0.03{\%} for NGS and 0.01{\%} for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56{\%}, 63{\%} and 75{\%}, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.",
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