Mutation Scanning of the RET Protooncogene Using High-Resolution Melting Analysis, Rebecca L. Margraf, 1* Rong Mao, 1,2 W. Edward Highsmith, 3 Leonard M. Holte- gaard, 3 and Carl T. Wittwer 1,2 ( 1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT; 2 Depart- ment of Pathology, University of Utah Medical School, Salt Lake City, UT; 3 Molecular Genetics Laboratory, Mayo Clinic, Rochester, MN; * address 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@aruplab.com) Background: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET proto- oncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carci- noma. The current widely used approach for RET mu- tation 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-1™, to detect heterozygous and homozygous sequence vari- ations. 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 non- pathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mu- tation-scanning assay differentiated RET sequence vari- ations from the wild-type genotype by altered deriva- tive 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 poly- morphisms 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. © 2006 American Associaton for Clinical Chemistry Mutations in the RET protooncogene (exons 10, 11, and 13–16) cause multiple endocrine neoplasia type 2 (MEN2) syndromes, autosomal dominant disorders that lead to a high lifetime risk of medullary thyroid carcinoma. Detec- tion of RET germline mutations can identify MEN2 pa- tients before disease progression, when thyroidectomy can prevent cancer development and increase survival rates. The gold standard for RET mutation detection is sequencing. Other methods, such as single-strand confor- mation polymorphism analysis, heteroduplex detection by conformation-sensitive gel electrophoresis, restriction enzyme digestion of PCR products, pyrosequencing, fluo- rescently labeled hybridization probes, and microarrays, have also been developed to detect RET mutations (1–9 ). These methods, however, can require additional post- PCR processing of the amplicon to detect mutations, may misidentify nonpathogenic polymorphisms as mutations (false positives), or may target mutation hotspots and thus miss some rare mutations (false negatives) (10 ). High- resolution melting analysis is a rapid, closed-tube muta- tion scanning assay that detects sequence variation within the PCR amplicon by use of a saturating double-stranded DNA dye, but does not require post-PCR manipulation of samples or use of expensive labeled probes (11–14 ). This technique detects mutations anywhere between the prim- ers, in contrast to more localized techniques, such as hybridization probes or restriction enzyme-based assays, that target 30 nucleotides (15 ). We obtained deidentified wild-type and RET variant genomic DNA samples from the Mayo Clinic (Rochester, MN), with Institutional Review Board approval, and amplified the samples according to the GenomiPhi TM protocol (Amersham Biosciences). Additional wild-type and RET variant cell lines were from the Coriell Institute. Sample genotypes were confirmed by sequence analysis and by comparison with the RET genomic sequence (GenBank AJ243297). All RET sequence variations tested were the result of a single nucleotide change. The RET variants that alter RET function to cause MEN2 syn- dromes are mutations, whereas variants that do not cause MEN2 syndromes are polymorphisms (2, 16 –20 ). All variant samples were heterozygous for mutations or polymorphisms unless otherwise stated. The wild-type nucleotide sequence for each of the analyzed RET codons is listed in Fig. 1, with the nucleotide change for each variant highlighted in red (bold in the text). In addition to the wild-type and RET mutant genotypes, samples with polymorphisms (exon 15, codon 904 TCCTCG; exon 11, codon 631 GACGAT; exon 14, codon 836 AGCAGT; and exon 13, codon 769 CTTCTG) were available for study (2, 16, 19). With Primer3 software (21 ), we designed primers for the 6 RET exons to create amplicons that included all known pathogenic mutations and, if possible, to exclude polymorphisms from analysis (Table 1) (16, 17, 21). For each RET exon, sample DNA (50 ng total) was amplified with the LightCycler ® FastStart DNA Master Hybridiza- tion Probe Kit (Roche Diagnostics Corp.) in a final PCR reaction volume of 10 L. The PCR reaction contained 1 FastStart master hybridization mixture, 2 mM MgCl 2 ,1 M of each primer (1.4 M of each primer for exon 16), 0.01 U/L uracil-DNA glycosylase (Roche Molecular), and 1 LCGreen ® PLUS (Idaho Technology). Thermocy- cling was performed on a LightCycler (Roche) with the following conditions: initial uracil-DNA glycosylase step (50 °C for 10 min) and polymerase activation (95 °C for 10 min), followed by 40 PCR cycles (denaturation at 95 °C for 1 s, annealing at 62 °C for 1 s, and extension at 72 °C for Technical Briefs 138 Clinical Chemistry 52, No. 1, 2006 Downloaded from https://academic.oup.com/clinchem/article/52/1/138/5626604 by guest on 06 May 2021