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Technical Briefs |
1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT;2 Department of Pathology, University of Utah Medical School, Salt Lake City, UT;3 Molecular Genetics Laboratory, Mayo Clinic, Rochester, MN;
aaddress correspondence to this author at: Advanced Technology Group, ARUP, 500 Chipeta Way, Salt Lake City, UT 84108; fax 801-584-5114, e-mail rebecca.margraf{at}aruplab.com
Abstract
Background: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons.
Methods: Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen®, and the high-resolution melter, HR-1TM, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position.
Results: Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes.
Conclusion: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.
The following articles in journals at HighWire Press have cited this article:
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  M.-P. Audrezet, A. Dabricot, C. Le Marechal, and C. Ferec Validation of High-Resolution DNA Melting Analysis for Mutation Scanning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene J. Mol. Diagn., September 1, 2008; 10(5): 424 - 434. [Abstract] [Full Text] [PDF]
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R. L. Margraf, R. Mao, and C. T. Wittwer Rapid Diagnosis of MEN2B Using Unlabeled Probe Melting Analysis and the LightCycler 480 Instrument J. Mol. Diagn., March 1, 2008; 10(2): 123 - 128. [Abstract] [Full Text] [PDF]
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 A. D. Laurie, M. P. Smith, and P. M. George Detection of Factor VIII Gene Mutations by High-Resolution Melting Analysis Clin. Chem., December 1, 2007; 53(12): 2211 - 2214. [Abstract] [Full Text] [PDF]
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S. Dames, D. C. Pattison, L. K. Bromley, C. T. Wittwer, and K. V. Voelkerding Unlabeled Probes for the Detection and Typing of Herpes Simplex Virus Clin. Chem., October 1, 2007; 53(10): 1847 - 1854. [Abstract] [Full Text] [PDF]
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 C. Voegele, S.V. Tavtigian, D. de Silva, S. Cuber, A. Thomas, and F. Le Calvez-Kelm A Laboratory Information Management System (LIMS) for a high throughput genetic platform aimed at candidate gene mutation screening Bioinformatics, September 15, 2007; 23(18): 2504 - 2506. [Abstract] [Full Text] [PDF]
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S. Dames, R. L. Margraf, D. C. Pattison, C. T. Wittwer, and K. V. Voelkerding Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays J. Mol. Diagn., July 1, 2007; 9(3): 290 - 296. [Abstract] [Full Text] [PDF]
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J. G. Vandersteen, P. Bayrak-Toydemir, R. A. Palais, and C. T. Wittwer Identifying Common Genetic Variants by High-Resolution Melting Clin. Chem., July 1, 2007; 53(7): 1191 - 1198. [Abstract] [Full Text] [PDF]
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D. Fortini, A. Ciammaruconi, R. De Santis, A. Fasanella, A. Battisti, R. D'Amelio, F. Lista, A. Cassone, and A. Carattoli Optimization of High-Resolution Melting Analysis for Low-Cost and Rapid Screening of Allelic Variants of Bacillus anthracis by Multiple-Locus Variable-Number Tandem Repeat Analysis Clin. Chem., July 1, 2007; 53(7): 1377 - 1380. [Abstract] [Full Text] [PDF]
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R. L. Margraf, R. Mao, W. E. Highsmith, L. M. Holtegaard, and C. T. Wittwer RET Proto-Oncogene Genotyping Using Unlabeled Probes, the Masking Technique, and Amplicon High-Resolution Melting Analysis J. Mol. Diagn., April 1, 2007; 9(2): 184 - 196. [Abstract] [Full Text] [PDF]
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M. G. Herrmann, J. D. Durtschi, L. K. Bromley, C. T. Wittwer, and K. V. Voelkerding Instrument Comparison for Heterozygote Scanning of Single and Double Heterozygotes: A Correction and Extension of Herrmann et al., Clin Chem 2006;52:494-503 Clin. Chem., January 1, 2007; 53(1): 150 - 152. [Full Text] [PDF]
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