• Marcus Broman, Kai Heinecke, Gunilla Islander, Frank Schuster, Klaus Glahn, Mikael Bodelsson, Susan Treves, and Clemens Müller.
• Department of Anaesthesiology and Intensive Care, VO Perioperativ och Intensivvård, Skåne University Hospital Lund, 205 02 Malmö, Sweden. marcus.broman@skane.se
• Anesth. Analg.. 2011 Nov 1;113(5):1120-8.

BackgroundA diagnosis of malignant hyperthermia (MH) can be determined by performing an in vitro (muscle) contracture test (IVCT) or by identifying a known MH causative mutation in the ryanodine receptor 1 gene (RYR1). Genetic diagnosis has an advantage over IVCT because it is less invasive. Direct sequencing of the very large RYR1 coding region (15.117 bases) is a laborious and expensive task. In this study, we applied the High Resolution Melting (HRM) curve analysis as a tool to screen the entire coding region of the gene.MethodsGenomic DNA was extracted from peripheral blood samples in a cohort of 16 MH-susceptible patients diagnosed by the IVCT. The total coding region of RYR1 was divided and amplified by polymerase chain reaction in 131 DNA fragments and the melting profiles were compared with those of control samples. HRM curves were evaluated by Rotor-Gene Q software and visual inspection. Fragments showing aberrant melting profiles were sequenced to identify the underlying sequence variation.ResultsA subset of 520 of 2520 DNA fragments (21%) showed significantly aberrant melting profiles. Upon sequencing, 131 known polymorphisms and 17 known or suspected mutations were found in 13 of 16 MH-susceptible patients (81%). Thus, the workload of sequencing was reduced by 79%.ConclusionHRM curve analysis is a sensitive and cost-effective tool for the identification of nucleotide sequence variants in complex genes such as the RYR1 gene.

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