Table 3 Cellular RYR1 model systems: Immortalized B-lymphocytes

Zullo A, et al. [110] 2019

R1335C, S2345R, S3098I, F4924_V4925ins

RYR1 sequence variants in myopathies: Expression and functional studies in two families

Ca²⁺ release in response to the RyR1 agonist 4-chloro-m-cresol and to thapsigargin showed that S2345R causes depletion of S/ER Ca²⁺ stores and that the compound heterozygosity for variant S3098I and the 30-nucleotide insertion increases RyR1-dependent Ca²⁺ release without affecting ER Ca²⁺ stores

Johannsen S, et al. [111] 2016

R4737W

Functional characterization of the RYR1 mutation pArg4737Trp associated with susceptibility to malignant hyperthermia

Intracellular resting calcium was slightly but significantly elevated in mutation positive cells. Calcium release following stimulation with 4-chloro-m-cresol was significantly increased in B lymphocytes carrying the R4737W mutation compared to mutation negative controls

Schiemann AH, et al. [112] 2014

R2355W, V2354M

Functional characterization of 2 known ryanodine receptor mutations causing malignant hyperthermia

We propose that R2355W is confirmed as being an MH-causative mutation and suggest that V2354M is a RYR1 mutation likely to cause MH

Lyfenko AD, et al. [66] 2007

R4214_F4216del

Two central core disease (CCD) deletions in the C-terminal region of RYR1 alter muscle excitation-contraction (EC) coupling by distinct mechanisms

ΔRQF RYR1 deletion did not significantly affect the sensitivity of lymphoblastoid cells to activation by 4-CmC. However, Ca21 release activated by a maximal concentration of 4-CmC (1 mM) was significantly reduced in ΔRQF-carrying cells

Schiemann AH, et al. 2013

R1583C, V2102L

Sequence capture and massively parallel sequencing to detect mutations associated with malignant hyperthermia

The amount of Ca²⁺ released after stimulation with 4-chloro-m-cresol from B lymphocytes of the MH-susceptible patients in the family was significantly greater compared with that of Ca²⁺ released from cells of an MH-negative family member

Attali R, et al. [113] 2013

Y3016C

Variable myopathic presentation in a single family with novel skeletal RYR1 mutation

Functional analysis on EBV immortalized cell lines showed no effect of the mutation on RyR1 pharmacological activation or the content of intracellular Ca²⁺ stores

Vukcevic M, et al. [114] 2010

R1679H, K1393R, E1058K, H382N, R2508G

Functional properties of RYR1 mutations identified in Swedish patients with malignant hyperthermia and central core disease

All B lymphoblastoid cell lines carrying RYR1 candidate mutations showed significantly increased resting cytoplasmic Ca²⁺ levels as well as a shift to lower concentrations of 4-CmC inducing calcium release compared with controls

Grievink H, et al. [115] 2010

H4833Y

Allele-specific differences in ryanodine receptor 1 mRNA expression levels may contribute to phenotypic variability in malignant hyperthermia

Allele-specific differences in RYR1 mRNA expression levels in heterozygous MHS samples, and can at least in part contribute to the observed variable penetrance and variations in MH clinical phenotypes

Zozato F, et al. [72] 2003

F4863_D4869delinsT

Clinical and functional effects of a deletion in a COOH-terminal lumenal loop of the skeletal muscle ryanodine receptor

Cells have depleted thapsigargin-sensitive intracellular Ca²⁺ stores, exhibit release of Ca²⁺ from intracellular stores in the absence of the addition of a pharmacological activator of the RYR1; and the unelicited Ca²⁺ transient from the thapsigargin-sensitive stores could be blocked by dantrolene, a specific inhibitor of the skeletal muscle RYR

Zullo A, et al. [116] 2009

R530H, R2163P, N2342S, E2371G, R2454H, C4664R

Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes

Increased acidification rate of lymphoblastoid cells in response to 4-CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N-terminal and in the central region of the protein (R530H, R2163P, N2342S, E2371G and R2454H) displayed higher activity compared with controls. Cell lines harboring RYR1(C4664R) were significantly less activated by 4-CmC

Levano S, et al. [117] 2009

D554Y, R2336H, E2404K, D2730G

Increasing the number of diagnostic mutations in malignant hyperthermia

All RYR1 mutations significantly increased resting calcium concentration and significantly affect either 4-CmC or caffeine dose-response curve to pharmacological activation Only one mutation (D2730G) showed a significant reduction in EC50 of both caffeine and 4-CmC

Anderson AA, et al. [118] 2008

H4833Y

Identification and biochemical characterization of a novel ryanodine receptor gene mutation associated with malignant hyperthermia

B lymphocytes from patients with this mutation were approximately twofold more sensitive than MH-negative cells to activation with 4-CmC. The amount of Ca²⁺ released from B lymphocytes of MH-susceptible patients was significantly greater than that released from cells of family members without this mutation

Ducreux D, et al. [119] 2006

P3527S, V4849I, R999H

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

P3527S in the homozygous state affected the amount of Ca²⁺ released after pharmacological activation with 4-CmC and caffeine but did not affect the size of the thapsigargin-sensitive Ca²⁺ stores. The other substitutions had no effect on the size of the intracellular Ca²⁺ stores, or the amount of Ca²⁺ released after ryanodine receptor activation P3527S and V4849I substitutions had a small but significant effect on the resting Ca²⁺ concentration

Tilgen N, et al. [120] 2001

R4861H, I4898T, G4899A

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

Cell showed release of Ca²⁺ from intracellular stores in the absence of any pharmacological activators of RYR, significantly smaller thapsigargin-sensitive intracellular calcium stores, compared to lymphoblasts from control individuals, and a normal sensitivity of the calcium release to the RYR inhibitor dantrolene

Girard T, et al. [121] 2001

V2168M

B-lymphocytes from malignant hyperthermia-susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators

EBV immortalized cells harboring the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-CmC and their peripheral blood leukocytes produce more interleukin-1beta after treatment with the RYR activators caffeine and 4-CmC, compared with cells from healthy controls

Hoppe K, et al. [122] 2016

R530H, C4664R, R2163P

Hypermetabolism in B–lymphocytes from malignant hyperthermia susceptible individuals

Native B–lymphocytes from MHS individuals are more sensitive to 4–CmC than those from MHN, reflecting a greater Ca²⁺ turnover. The acidification response, however, was less pronounced than in muscle cells, presumably reflecting the lower expression of RyR1 in B–lymphocytes.