title: Detection of a novel ryanodine receptor subtype 1 mutation (R328W) in a malignant hyperthermia family by sequencing of a leukocyte transcript.
abstract: BACKGROUND
  To determine whether malignant hyperthermia (MH) susceptibility in a Canadian pedigree is associated with a mutation in the ryanodine receptor subtype 1 (RYR1) gene, the complete RYR1 transcript obtained from the leukocytes of one MH-susceptible family member was sequenced, using a newly developed protocol.
  
  
  METHODS
  RNA was extracted from leukocytes and converted into complementary DNA. Overlapping fragments of RYR1 complementary DNA were amplified by the polymerase chain reaction and used for double-strand sequencing to find a single mutation likely to be causal of MH susceptibility. Inheritance of the mutation in the family was studied by restriction endonuclease analysis and/or sequencing of genomic DNA and compared to available caffeine halothane contracture test data. The mutation was introduced into rabbit RYR1 complementary DNA, the complementary DNA was expressed in human embryonic kidney line 293 cells, and Ca2+ release by the mutant Ca2+ release channel was measured following the addition of caffeine and halothane.
  
  
  RESULTS
  A novel arginine 328 to tryptophan mutation in RYR1 was detected by direct sequencing of the RYR1 transcript from leukocytes of one MH-susceptible individual. A causal role for this mutation in MH is indicated by cosegregation of the mutation with the MH-susceptible phenotype within the family and by the demonstration that the mutant channel has increased sensitivity to both caffeine and halothane.
  
  
  CONCLUSIONS
  The feasibility of using complete RYR1 transcripts from leukocytes for sequence analysis offers an efficient and noninvasive method for scanning RYR1 for novel mutations.
authors.affiliation: Malignant Hyperthermia Investigation Unit, Banting and Best Department of Medical Research, University of Toronto, Canada.
authors.forename: Julian C P
authors.initials: JC
authors.lastname: Loke
authors.forename: Natasha
authors.initials: N
authors.lastname: Kraev
authors.forename: Parveen
authors.initials: P
authors.lastname: Sharma
authors.forename: GuoGuang
authors.initials: G
authors.lastname: Du
authors.forename: Leena
authors.initials: L
authors.lastname: Patel
authors.forename: Alexander
authors.initials: A
authors.lastname: Kraev
authors.forename: David H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D018685:Anesthetics, Inhalation; D002110:Caffeine; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D006221:Halothane; D006801:Humans; D007962:Leukocytes / Q000737:chemistry*; D008305:Malignant Hyperthermia / Q000235:genetics*; D009119:Muscle Contraction / Q000187:drug effects; D018482:Muscle, Skeletal / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D010375:Pedigree; D010726:Phosphodiesterase Inhibitors; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D018685:Anesthetics, Inhalation; D018076:DNA, Complementary; D010726:Phosphodiesterase Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D012313:RNA; D006221:Halothane