title: Screening of the ryanodine 1 gene for malignant hyperthermia causative mutations by high resolution melt curve analysis.
abstract: BACKGROUND
  A 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.
  
  
  METHODS
  Genomic 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.
  
  
  RESULTS
  A 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%.
  
  
  CONCLUSIONS
  HRM curve analysis is a sensitive and cost-effective tool for the identification of nucleotide sequence variants in complex genes such as the RYR1 gene.
authors.affiliation: Department of Anaesthesiology and Intensive Care, VO Perioperativ och Intensivvård, Skåne University Hospital Lund, 205 02 Malmö, Sweden. marcus.broman@skane.se
authors.forename: Marcus
authors.initials: M
authors.lastname: Broman
authors.forename: Kai
authors.initials: K
authors.lastname: Heinecke
authors.forename: Gunilla
authors.initials: G
authors.lastname: Islander
authors.forename: Frank
authors.initials: F
authors.lastname: Schuster
authors.forename: Klaus
authors.initials: K
authors.lastname: Glahn
authors.forename: Mikael
authors.initials: M
authors.lastname: Bodelsson
authors.forename: Susan
authors.initials: S
authors.lastname: Treves
authors.forename: Clemens
authors.initials: C
authors.lastname: Müller
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D004247:DNA / Q000097:blood / Q000235:genetics; D017931:DNA Primers; D005091:Exons / Q000235:genetics; D005260:Female; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005820:Genetic Testing; D014644:Genetic Variation; D006801:Humans; D008297:Male; D008305:Malignant Hyperthermia / Q000235:genetics*; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics*; D055815:Young Adult
chemical_list: D017931:DNA Primers; D019837:Ryanodine Receptor Calcium Release Channel; D004247:DNA