Rapid screening for mutations associated with malignant hyperthermia using high-resolution melting curve analysis.

OBJECTIVES: The diagnosis of malignant hyperthermia (MH) is based on clinical signs or laboratory testing. The gold standard laboratory test is the in vitro contracture test, although it is invasive, expensive, and only performed at specialized centers. Genetic diagnosis is another option, although direct mutation screening is a laborious task. Therefore, we evaluated whether high-resolution melting (HRM) curve analysis could be used as a rapid screening tool to target MH-associated mutations.

MATERIALS AND METHODS: The feasibility of HRM analysis was evaluated using plasmids that were constructed by cloning wild-type or mutated versions of the ryanodine receptor 1 ( RYR1 ) gene into the pCR2.1 plasmid. We obtained engineered plasmids and patient DNA extracted from blood samples with known wild-type or mutated sequences that are associated with MH. Amplicon lengths were kept relatively short (<250 bp) to improve discrimination between the engineered and patient plasmids. Real-time polymerase chain reaction (PCR) cycling and HRM analysis of the engineered plasmids and patient DNA were performed using the LightCycler 480 System (Roche).

RESULTS: The HRM results were clearly different from those obtained using real-time PCR. Furthermore, the HRM analysis provided sufficient resolution to identify two single-nucleotide variants in the tested RYR1 exons.

CONCLUSION: We conclude that HRM analysis can provide high resolution for identifying single-nucleotide variants in RYR1 , which might be useful for predicting the risk of MH in the preanesthesia setting.