title: Identification of four novel mutations in the C-terminal membrane spanning domain of the ryanodine receptor 1: association with central core disease and alteration of calcium homeostasis.
abstract: The skeletal muscle ryanodine receptor gene (RYR1; OMIM 180901) on chromosome 19q13.1 encodes the skeletal muscle calcium release channel. To date, more than 25 missense mutations have been identified in RYR1 and are associated with central core disease (CCD; OMIM 117000) and/or the malignant hyperthermia susceptibility phenotype (MHS1; OMIM 145600). The majority of RYR1 mutations are clustered in the N-terminal hydrophilic domain of the protein. Only four mutations have been identified so far in the highly conserved C-terminal region encoding the luminal/transmembrane domain of the protein which forms the ion pore. Three of these mutations have been found to segregate with pure or mixed forms of CCD. We have screened the C-terminal domain of the RYR1 gene for mutations in 50 European patients, diagnosed clinically and/or histologically as having CCD. We have identified five missense mutations (four of them novel) in 13 index patients. The mutations cluster in exons 101 and 102 and replace amino acids which are conserved in all known vertebrate RYR genes. In order to study the functional effect of these mutations, we have immortalized B-lymphocytes from some of the patients and studied their [Ca(2+)](i) homeostasis. We show that lymphoblasts carrying the newly identified RYR1 mutations exhibit: (i) a release of calcium from intracellular stores in the absence of any pharmacological activators of RYR; (ii) significantly smaller thapsigargin-sensitive intracellular calcium stores, compared to lymphoblasts from control individuals; and (iii) a normal sensitivity of the calcium release to the RYR inhibitor dantrolene. Our data suggest the C-terminal domain of RYR1 as a hot spot for mutations leading to the CCD phenotype. If the functional alterations of mutated RYR channels observed in lymphoblastoid cells are also present in skeletal muscles this could explain the predominant symptom of CCD, i.e. chronic muscle weakness. Finally, the study of calcium homeostasis in lymphoblastoid cells naturally expressing RYR1 mutations offers a novel non-invasive approach to gain insights into the pathogenesis of MH and CCD.
authors.affiliation: Institut für Humangenetik, Biozentrum der Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Tilgen
authors.forename: F
authors.initials: F
authors.lastname: Zorzato
authors.forename: B
authors.initials: B
authors.lastname: Halliger-Keller
authors.forename: F
authors.initials: F
authors.lastname: Muntoni
authors.forename: C
authors.initials: C
authors.lastname: Sewry
authors.forename: L M
authors.initials: LM
authors.lastname: Palmucci
authors.forename: C
authors.initials: C
authors.lastname: Schneider
authors.forename: E
authors.initials: E
authors.lastname: Hauser
authors.forename: F
authors.initials: F
authors.lastname: Lehmann-Horn
authors.forename: C R
authors.initials: CR
authors.lastname: Müller
authors.forename: S
authors.initials: S
authors.lastname: Treves
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry*; D015331:Cohort Studies; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D020512:Myopathy, Central Core / Q000235:genetics* / Q000473:pathology; D016133:Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D019837:Ryanodine Receptor Calcium Release Channel; D004247:DNA; D002118:Calcium