title: Assessing the pathogenicity of RYR1 variants in malignant hyperthermia.
abstract: BACKGROUND
  . Missense variants in the ryanodine receptor 1 gene ( RYR1 ) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia.
  
  
  METHODS
  . We identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein.
  We identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1.
  
  
  CONCLUSIONS
  . Segregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.
authors.affiliation: Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, UK.
authors.forename: A
authors.initials: A
authors.lastname: Merritt
authors.affiliation: Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, UK.
authors.forename: P
authors.initials: P
authors.lastname: Booms
authors.affiliation: Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, UK.
authors.forename: M-A
authors.initials: MA
authors.lastname: Shaw
authors.affiliation: Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, UK.|Malignant Hyperthermia Unit, St James's University Hospital, Leeds, UK.
authors.forename: D M
authors.initials: DM
authors.lastname: Miller
authors.affiliation: Malignant Hyperthermia Unit, St James's University Hospital, Leeds, UK.
authors.forename: C
authors.initials: C
authors.lastname: Daly
authors.affiliation: Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, UK.|Malignant Hyperthermia Unit, St James's University Hospital, Leeds, UK.
authors.forename: J G
authors.initials: JG
authors.lastname: Bilmen
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: K M
authors.initials: KM
authors.lastname: Stowell
authors.affiliation: Department of Molecular Biosciences, UC Davis, Davis, CA, USA.
authors.forename: P D
authors.initials: PD
authors.lastname: Allen
authors.affiliation: School of Biomedical Sciences, University of Leeds, Leeds, UK.
authors.forename: D S
authors.initials: DS
authors.lastname: Steele
authors.affiliation: Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, UK.|Malignant Hyperthermia Unit, St James's University Hospital, Leeds, UK.
authors.forename: P M
authors.initials: PM
authors.lastname: Hopkins
mesh_terms: D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D005190:Family; D020022:Genetic Predisposition to Disease; D014644:Genetic Variation; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D008305:Malignant Hyperthermia / Q000453:epidemiology / Q000235:genetics*; D057054:Molecular Imaging; D016296:Mutagenesis; D009154:Mutation; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism
chemical_list: C000629268:RYR1 protein, human; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium
keywords: HEK293 cells; RYR1; calcium signaling; malignant hyperthermia, diagnosis; malignant hyperthermia, genetics; malignant hyperthermia, physiopathology