Remember Me
Or use your Academic/Social account:


Or use your Academic/Social account:


You have just completed your registration at OpenAire.

Before you can login to the site, you will need to activate your account. An e-mail will be sent to you with the proper instructions.


Please note that this site is currently undergoing Beta testing.
Any new content you create is not guaranteed to be present to the final version of the site upon release.

Thank you for your patience,
OpenAire Dev Team.

Close This Message


Verify Password:
Verify E-mail:
*All Fields Are Required.
Please Verify You Are Human:
fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Merritt, A; Booms, P; Shaw, M-A; Miller, DM; Daly, C; Bilmen, JG; Stowell, KM; Allen, PD; Steele, DS; Hopkins, PM (2017)
Publisher: Oxford University Press
Languages: English
Types: Article
Background: Missense variants in the ryanodine receptor 1 gene (RYR1) are associated with malignant hyperthermia but only a minority of these have met 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 4 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. Results: 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 to 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.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 1. Hopkins PM. Malignant hyperthermia Ð pharmacology of triggering. Br J Anaesth 2011; 107:48-56
    • 2. Weiss RG, OÕConnell KM, Flucher BE, Allen PD, Grabner M, Dirksen RT. Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling. Am J Physiol Cell Physiol 2004; 287: C1094-102
    • 3. Carpenter D, Ringrose C, Leo V, et al. The role of CACNA1S in predisposition to malignant hyperthermia. BMC Med Genet 2009; 10:104
    • 4. Eltit JM, Bannister RA, Moua O, et al. Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor. Proc Natl Acad Sci U S A 2012; 109: 7923-8
    • 5. Horstick EJ, Linsley JW, Dowling JJ, et al. Stac3 is a component of the excitationcontraction coupling machinery and mutated in Native American myopathy. Nat Commun 2013;4:1952
    • 6. Schiemann AH, DŸrholt EM, Pollock N, Stowell KM. Sequence capture and massively parallel sequencing to detect mutations associated with malignant hyperthermia. Br J Anaesth 2013;110:122-7
    • 7. Kim J, Jarvik GP, Browning BL, et al. Exome sequencing reveals novel rare variants in ryanodine receptor and calcium channel genes in malignant hyperthermia families. Anesthesiology 2013;119:1054-65
    • 8. Gonsalves SG, Ng D, Johnston JJ, et al. Using exome data to identify malignant hyperthermia susceptibility mutations. Anesthesiology 2013;119:1043Ð53
    • 9. Fiszer D, Shaw M-A, Fisher NA, et al. Next generation sequencing of RYR1 and CACNA1S in malignant hyperthermia and exertional heat illness. Anesthesiology 2015: 122;1033-46
    • 10. Hopkins PM, RŸffert H, Snoeck MM, et al. The European Malignant Hyperthermia Group guidelines for the investigation of malignant hyperthermia susceptibility. Br J Anaesth 2015; 115: 531-9
    • 11. Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, MacLennan DH. Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem 1997;272:26332-9
    • 12. Sato K, Pollock N, Stowell KM. Functional studies of RYR1 mutations in the skeletal muscle ryanodine receptor using human RYR1 complementary DNA. Anesthesiology 2010;112:1350-4
    • 13. Sato K, Roesl C, Pollock N, Stowell KM. Skeletal muscle ryanodine receptor mutations associated with malignant hyperthermia showed enhanced intensity and sensitivity to triggering drugs when expressed in human embryonic kidney cells. Anesthesiology 2013;119:111-8
    • 14. Urwyler A, Deufel T, McCarthy T, West S; European Malignant Hyperthermia Group. Guidelines for molecular genetic detection of susceptibility to malignant hyperthermia. Br J Anaesth 2001;86:283-7
    • 15. M¿ller P, Clark N, M¾hle L. A SImplified method for Segregation Analysis (SISA) to determine penetrance and expression of a genetic variant in a family. Hum Mutat 2011;32:568-71
    • 16. Wallis Y, Payne S, McAnulty C, et al. Practice guidelines for the evaluation of pathogenicity and the reporting of sequence variants in clinical molecular genetics. Association for Clinical Genetic Science and the Dutch Society of Clinical Genetic Laboratory Specialists 2013: http://www.acgs.uk.com/media/774853/evaluation_and_reporting_of_sequence_varia nts_bpgs_june_2013_-_finalpdf.pdf (date last accessed 21.06.2016)
    • 17. Gillard EF, Otsu K, Fujii J, et al. A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia. Genomics 1991;11:751-5
    • 18. Robinson RL, Carpenter D, Shaw M-A, Halsall PJ, Hopkins PM. Mutations in RYR1 in malignant hyperthermia and central core disease. Hum Mutat 2006; 27: 977-89
    • 19. Hopkins PM, Halsall PJ, Ellis FR, Stewart AD. An analysis of the predictive probability of the in vitro contracture test for determining malignant hyperthermia susceptibility. Anesth Analg 1997; 84: 648-56
    • 20. Miyazaki K, Takenouchi M. Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. Biotechniques 2002;33:1033-4, 1036-8
    • 21. Deans TL, Cantor CR, Collins JJ. A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells. Cell 2007 27;130:363-72
    • 22. Duke AM, Steele DS. The presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists in skinned rat skeletal muscle fibres. Cell Calcium 2008;44:411-21
    • 23. Levano S, Vukcevic M, Singer M, Matter A, Treves S, Urwyler A, Girard T. Increasing the number of diagnostic mutations in malignant hyperthermia. Hum Mutat 2009;30:590-8
    • 24. Carpenter D, Robinson RL, Quinnell RJ, et al. Genetic variation in RYR1 and malignant hyperthermia phenotypes. Br J Anaesth 2009; 103: 538-48
    • 25. Ducreux S, Zorzato F, Ferreiro A, et al. Functional properties of ryanodine receptors carrying three amino acid substitutions identified in patients affected by multiminicore disease and central core disease, expressed in immortalized lymphocytes. Biochem J 2006;395:259-66
    • 26. Tong J, McCarthy TV, MacLennan DH. Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels. J Biol Chem 1999;274:693-702
    • 27. Lynch PJ, Tong J, Lehane M, et al. A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease. Proc Natl Acad Sci U S A 1999;96:4164-9
    • 28. Avila G, Dirksen RT. Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor. J Gen Physiol 2001;118:277-90
    • 29. Monnier N, Kozak-Ribbens G, Krivosic-Horber R, et al. Correlations between genotype and pharmacological, histological, functional, and clinical phenotypes in malignant hyperthermia susceptibility. Hum Mutat 2005;26:413-25
    • 30. Wehner M, Rueffert H, Koenig F, Olthoff D. Functional characterization of malignant hyperthermia-associated RyR1 mutations in exon 44, using the human myotube model. Neuromuscul Disord 2004;14:429-37
    • 31. Schiemann AH, Paul N, Parker R, Pollock N, Bulger TF, Stowell KM. Functional characterization of 2 known ryanodine receptor mutations causing malignant hyperthermia. Anesth Analg 2014;118:375-80
    • 32. Robinson RL, Anetseder MJ, Brancadoro V, et al. Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing? Eur J Hum Genet 2003;11:342-8
    • 33. Robinson RL, Curran JL, Ellis FR, et al. Multiple interacting gene products may influence susceptibility to malignant hyperthermia. Ann Hum Genet 2000; 64: 307-20
    • 34. Robinson R, Hopkins PM, Carsana A , et al. Several interacting genes influence the malignant hyperthermia phenotype. Hum Genet 2003; 112: 217-8
  • No related research data.
  • No similar publications.

Share - Bookmark

Cite this article