Rapid diagnosis of MEN213 using unlabeled probe melting analysis and the LightCycler 480 instrument

Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant, inherited cancer syndrome. MEN2B patients have a high risk of developing medullary thyroid carcinoma, and prophylactic thyroidectomy is recommended by 6 months of age. Genetic testing can identify MEN2B patients before cancer progression. Two RET proto-oncogene mutations, in exon 15 at codon 883 (GCT>TTT) and in exon 16 at codon 918 (ATG>ACG), account for more than 98% of MEN2B cases. An assay using unlabeled probes and the LightCycler 480 instrument was developed to genotype these two common MEN2B RET mutations. Asymmetric polymerase chain reaction was used to increase ssDNA products followed by melting analysis of the unlabeled probe/ssDNA amplicon duplex. The available samples were either patient DNA of known RET genotype or artificial templates. Analysis of the codon 883 heterozygous mutation demonstrated a Delta T-m of 5.70 +/- 0.11 degrees C, while the codon 918 heterozygous mutation generated a Delta T-m of -5.72 +/- 0.11 degrees C. Samples with the targeted RET mutation genotypes were accurately detected and easily distinguishable from five other reported sequence changes using these probes. Thus, MEN2B diagnosis using unlabeled probes and the LightCycler 480 is a rapid, closed-tube method that is less time consuming and less expensive than sequencing. This assay demonstrates 100% specificity and sensitivity for the identification of RET mutations causative of MEN2B.