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. Conclusion: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

“…Although HRM curve analysis has been tested and shown to be successful in a number of research laboratories for clinical use [Chou et al, 2005;Dobrowolski et al, 2005Dobrowolski et al, , 2007aDobrowolski et al, , 2007bGrievink and Stowell, 2008;Kennerson et al, 2007;Krypuy et al, 2006;Lonie et al, 2006;Margraf et al, 2006;Poláková et al, 2008;Seipp et al, 2008;Smith et al, 2008;Willmore-Payne et al, 2006], we wanted to assess this method for detecting multiple sequence variants, as well as single and multiple variants (including both constitutional and somatic), within GC-rich fragments, as it would be applied in a diagnostic setting, with minimal expertise and time to manipulate assay design. This was achieved by screening five fragments for 13 variants in a total of 35 different combinations using the LightScanner s System and LC Green s PLUS DNA binding dye (Idaho Technology) as well as screening five GC-rich (Z65%) fragments for 12 variants in 22 combinations using the LightCycler s 480 and HRM Master dye (Roche).…”

Identifying Common Genetic Variants by High-Resolution Melting

“…The method is based on high-resolution melting (3 ) of DNA duplexes in the presence of saturating fluorescent dyes (4 ) and appears to have an accuracy equivalent or superior to other heteroduplex scanning methods (5,6 ). Applications of high-resolution melting for mutation scanning in genetics (7)(8)(9)(10), oncology (11)(12)(13)(14)(15)(16)(17), and bacterial speciation (18,19 ) have been reported.…”

Genetic mutations and expression of p53 in non-invasive breast lesions

“…High resolution melting (HRM) is a novel simple homogeneous close-tube and post-PCR method that enables genomic researchers to analyze genetic variations (SNPs, mutations and methylations) in PCR amplicons, particularly when only tiny samples are available (10)(11)(12)(13)(14)(15). In HRM-designated instruments, the decrease in fluorescence caused by the transition of dsDNA to ssDNA with an increase in temperature is carefully monitored.…”