Mutations in the Skeletal Muscle Ryanodine Receptor (RYR1) Gene are Linked to Malignant Hyperthermia and Central-Core Disease

Human central core disease (CCD) is caused by mutations/deletions in the gene that encodes the skeletal muscle ryanodine receptor (RyR1). Previous studies have shown that CCD mutations in the NH2-terminal region of RyR1 lead to the formation of leaky SR Ca2+ release channels when expressed in myotubes derived from RyR1-knockout (dyspedic) mice, whereas a COOH-terminal mutant (I4897T) results in channels that are not leaky to Ca2+ but lack depolarization-induced Ca2+ release (termed excitation-contraction [EC] uncoupling). We show here that store depletion resulting from NH2-terminal (Y523S) and COOH-terminal (Y4795C) leaky CCD mutant release channels is eliminated after incorporation of the I4897T mutation into the channel (Y523S/I4897T and Y4795C/I4897T). In spite of normal SR Ca2+ content, myotubes expressing the double mutants lacked voltage-gated Ca2+ release and thus exhibited an EC uncoupling phenotype similar to that of I4897T-expressing myotubes. We also show that dyspedic myotubes expressing each of seven recently identified CCD mutations located in exon 102 of the RyR1 gene (G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, R4913G) behave as EC-uncoupled release channels. Interestingly, voltage-gated Ca2+ release was nearly abolished (reduced ∼90%) while caffeine-induced Ca2+ release was only marginally reduced in R4892W-expressing myotubes, indicating that this mutation preferentially disrupts voltage-sensor activation of release. These data demonstrate that CCD mutations in exon 102 disrupt release channel permeation to Ca2+ during EC coupling and that this region represents a primary molecular locus for EC uncoupling in CCD.

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Protocol for the Sequence Analysis of Ryanodine Receptor Subtype 1 Gene Transcripts from Human Leukocytes

Anesthesiology ◽

10.1097/00000542-200308000-00010 ◽

2003 ◽

Vol 99 (2) ◽

pp. 289-296 ◽

Cited By ~ 27

Author(s):

Natasha Kraev ◽

Julian C. P. Loke ◽

Alexander Kraev ◽

David H. MacLennan

Keyword(s):

Sequence Analysis ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Human Blood ◽

Messenger Rna ◽

Central Core ◽

Central Core Disease ◽

Receptor Subtype ◽

Novel Mutations ◽

Ryr1 Gene

Background The search for novel mutations in the ryanodine receptor subtype 1 (RYR1) gene causing malignant hyperthermia and central core disease is hampered by the fact that the gene contains 106 exons. Searching for novel mutations in complementary DNA (cDNA) requires an invasive muscle biopsy. Accordingly, an alternate source of RYR1 cDNA was sought for sequence analysis. Methods Leukocytes were isolated from human blood and used for extraction of RNA and reverse transcription of messenger RNA into cDNA. A detailed protocol was developed in which overlapping fragments of RYR1 cDNA were amplified by polymerase chain reaction in a series of steps and used for double-strand sequencing. Results The sequences of full-length leukocyte RYR1 cDNA obtained from four human blood samples were shown to be identical to the sequence of a human muscle RYR1 cDNA. The incidence of aberrant splicing was more pronounced in the blood-derived cDNAs, but this could be minimized by adequate sample preparation. Protocols to sequence alternatively spliced products were also developed. Several silent nucleotide polymorphisms were detected, and minor revisions were made to the RYR1 sequence. Conclusions Because there are no differences in RYR1 transcript structure between muscle and leukocytes, aside from those that may be ascribed to RNA splicing aberrations during processing, leukocytes seem to be an adequate substitute tissue for screening the RYR1 gene for previously undiscovered mutations in families with malignant hyperthermia or central core disease.

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Functional properties of ryanodine receptors carrying three amino acid substitutions identified in patients affected by multi-minicore disease and central core disease, expressed in immortalized lymphocytes

Biochemical Journal ◽

10.1042/bj20051282 ◽

2006 ◽

Vol 395 (2) ◽

pp. 259-266 ◽

Cited By ~ 43

Author(s):

Sylvie Ducreux ◽

Francesco Zorzato ◽

Ana Ferreiro ◽

Heinz Jungbluth ◽

Francesco Muntoni ◽

...

Keyword(s):

Amino Acid ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Receptor Gene ◽

Receptor Activation ◽

Central Core ◽

Central Core Disease ◽

Amino Acid Substitutions ◽

Time Data ◽

Minicore Disease

More than 80 mutations in the skeletal muscle ryanodine receptor gene have been found to be associated with autosomal dominant forms of malignant hyperthermia and central core disease, and with recessive forms of multi-minicore disease. Studies on the functional effects of pathogenic dominant mutations have shown that they mostly affect intracellular Ca2+ homoeostasis, either by rendering the channel hypersensitive to activation (malignant hyperthermia) or by altering the amount of Ca2+ released subsequent to physiological or pharmacological activation (central core disease). In the present paper, we show, for the first time, data on the functional effect of two recently identified recessive ryanodine receptor 1 amino acid substitutions, P3527S and V4849I, as well as that of R999H, another substitution that was identified in two siblings that were affected by multi-minicore disease. We studied the intracellular Ca2+ homoeostasis of EBV (Epstein–Barr virus)-transformed lymphoblastoid cells from the affected patients, their healthy relatives and control individuals. Our results show that the P3527S substitution in the homozygous state affected the amount of Ca2+ released after pharmacological activation with 4-chloro-m-cresol and caffeine, but did not affect the size of the thapsigargin-sensitive Ca2+ stores. The other substitutions had no effect on either the size of the intracellular Ca2+ stores, or on the amount of Ca2+ released after ryanodine receptor activation; however, both the P3527S and V4849I substitutions had a small but significant effect on the resting Ca2+ concentration.

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A central core disease mutation in the Ca2+-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation

AJP Cell Physiology ◽

10.1152/ajpcell.00052.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. C358-C365 ◽

Cited By ~ 3

Author(s):

Venkat R. Chirasani ◽

Le Xu ◽

Hannah G. Addis ◽

Daniel A. Pasek ◽

Nikolay V. Dokholyan ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acid ◽

Missense Mutation ◽

Ryanodine Receptor ◽

Single Channel ◽

Central Core ◽

Central Core Disease ◽

Hek293 Cells ◽

Amino Acid Residues ◽

Single Channel Recordings

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca2+-mediated activation of skeletal muscle Ca2+ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca2+ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca2+ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca2+ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca2+-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca2+-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca2+ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca2+ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca2+ of the two mutants, whereas the interaction of Ca2+ with directly interacting RyR1 amino acid residues Glu3893, Glu3967, and Thr5001 was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca2+-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.

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G.P.13.09 Identification of a point mutation in the skeletal muscle ryanodine receptor gene associated in the homozygous state to central core disease

Neuromuscular Disorders ◽

10.1016/j.nmd.2008.06.294 ◽

2008 ◽

Vol 18 (9-10) ◽

pp. 809-810

Author(s):

G. Melli ◽

L. Colleoni ◽

P. Bernasconi ◽

S. Romaggi ◽

V. Tegazzin ◽

...

Keyword(s):

Skeletal Muscle ◽

Point Mutation ◽

Ryanodine Receptor ◽

Receptor Gene ◽

Central Core ◽

Central Core Disease ◽

Homozygous State

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Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia

Journal of Neuromuscular Diseases ◽

10.3233/jnd-170210 ◽

2017 ◽

Vol 4 (2) ◽

pp. 147-158 ◽

Cited By ~ 3

Author(s):

Remai Parker ◽

Anja H. Schiemann ◽

Elaine Langton ◽

Terasa Bulger ◽

Neil Pollock ◽

...

Keyword(s):

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Functional Characterization ◽

Central Core ◽

Central Core Disease ◽

Ryanodine Receptor 1

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Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region

PLoS ONE ◽

10.1371/journal.pone.0130606 ◽

2015 ◽

Vol 10 (6) ◽

pp. e0130606 ◽

Cited By ~ 23

Author(s):

Takashi Murayama ◽

Nagomi Kurebayashi ◽

Toshiko Yamazawa ◽

Hideto Oyamada ◽

Junji Suzuki ◽

...

Keyword(s):

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Terminal Region ◽

Central Core ◽

Central Core Disease ◽

Amino Terminal ◽

Disease Mutations

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Functional Effects of Central Core Disease Mutations in the Cytoplasmic Region of the Skeletal Muscle Ryanodine Receptor

The Journal of General Physiology ◽

10.1085/jgp.118.3.277 ◽

2001 ◽

Vol 118 (3) ◽

pp. 277-290 ◽

Cited By ~ 102

Author(s):

Guillermo Avila ◽

Robert T. Dirksen

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Central Core ◽

Central Core Disease ◽

Calcium Release Channel ◽

Release Channel ◽

Voltage Gated ◽

Channel Function ◽

Store Depletion ◽

The Impact

Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca2+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH2-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca2+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca2+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 μM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca2+ release was smaller and activated at more negative potentials for myotubes expressing the NH2-terminal CCD mutations. The shift in the voltage dependence of SR Ca2+ release correlated strongly with changes in resting Ca2+, SR Ca2+ store depletion, and peak voltage–gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca2+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca2+ leak. These results demonstrate that the NH2-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca2+ leak, store depletion, and a reduction in voltage-gated Ca2+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.

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Scanning for Mutations of the Ryanodine Receptor (RYR1) Gene by Denaturing HPLC: Detection of Three Novel Malignant Hyperthermia Alleles

Clinical Chemistry ◽

10.1373/49.5.761 ◽

2003 ◽

Vol 49 (5) ◽

pp. 761-768 ◽

Cited By ~ 31

Author(s):

Angela Tammaro ◽

Adele Bracco ◽

Santolo Cozzolino ◽

Maria Esposito ◽

Antonietta Di Martino ◽

...

Keyword(s):

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Sudden Increase ◽

Gene Encoding ◽

In Vitro Contracture Test ◽

Ryr1 Gene ◽

Molecular Tests ◽

Anesthetic Agents

Abstract Background: Malignant hyperthermia (MH) is a fatal autosomal dominant pharmacogenetic disorder characterized by skeletal muscle hypertonicity that causes a sudden increase in body temperature after exposure to common anesthetic agents. The disease is genetically heterogeneous, with mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) at 19q13.1 accounting for up to 80% of the cases. To date, at least 42 RYR1 mutations have been described that cause MH and/or central core disease. Because the RYR1 gene is huge, containing 106 exons, molecular tests have focused on the regions that are more frequently mutated. Thus the causative defect has been identified in only a fraction of families as linked to chromosome 19q, whereas in others it remains undetected. Methods: We used denaturing HPLC (DHPLC) to analyze the RYR1 gene. We set up conditions to scan the 27 exons to identify both known and unknown mutations in critical regions of the protein. For each exon, we analyzed members from 52 families with positive in vitro contracture test results, but without preliminary selection by linkage analysis. Results: We identified seven different mutations in 11 MH families. Among them, three were novel MH alleles: Arg44Cys, Arg533Cys, and Val2117Leu. Conclusion: Because of its sensitivity and speed, DHPLC could be the method of choice for the detection of unknown mutations in the RYR1 gene.

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