title: Establishing high resolution melting analysis: method validation and evaluation for c-RET proto-oncogene mutation screening.
abstract: BACKGROUND
  Reliable and effective primary screening of mutation carriers is the key condition for common diagnostic use. The objective of this study is to validate the method high resolution melting (HRM) analysis for routine primary mutation screening and accomplish its optimization, evaluation and validation. Due to their heterozygous nature, germline point mutations of c-RET proto-oncogene, associated to multiple endocrine neoplasia type 2 (MEN2), are suitable for HRM analysis. Early identification of mutation carriers has a major impact on patients' survival due to early onset of medullary thyroid carcinoma (MTC) and resistance to conventional therapy.
  
  
  METHODS
  The authors performed a series of validation assays according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines for validation of analytical procedures, along with appropriate design and optimization experiments. After validated evaluation of HRM, the method was utilized for primary screening of 28 pathogenic c-RET mutations distributed among nine exons of c-RET gene.
  
  
  RESULTS
  Validation experiments confirm the repeatability, robustness, accuracy and reproducibility of HRM. All c-RET gene pathogenic variants were detected with no occurrence of false-positive/false-negative results.
  
  
  CONCLUSIONS
  The data provide basic information about design, establishment and validation of HRM for primary screening of genetic variants in order to distinguish heterozygous point mutation carriers among the wild-type sequence carriers. HRM analysis is a powerful and reliable tool for rapid and cost-effective primary screening, e.g., of c-RET gene germline and/or sporadic mutations and can be used as a first line potential diagnostic tool.
authors.affiliation: Faculty of Natural Sciences, Commenius University, Mlynsk á dolina, Bratislava, Slovakia.
authors.forename: Martin
authors.initials: M
authors.lastname: Benej
authors.forename: Bela
authors.initials: B
authors.lastname: Bendlova
authors.forename: Eliska
authors.initials: E
authors.lastname: Vaclavikova
authors.forename: Martina
authors.initials: M
authors.lastname: Poturnajova
mesh_terms: D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret