title: Mutation scanning of the RET protooncogene using high-resolution melting analysis.
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-1, 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.
  
  
  CONCLUSIONS
  RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.
authors.affiliation: Advanced Technology Group, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA. rebecca.margraf@aruplab.com
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Margraf
authors.forename: Rong
authors.initials: R
authors.lastname: Mao
authors.forename: W Edward
authors.initials: WE
authors.lastname: Highsmith
authors.forename: Leonard M
authors.initials: LM
authors.lastname: Holtegaard
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
mesh_terms: D005838:Genotype; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*
chemical_list: D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human