An RYR1 mutation associated with malignant hyperthermia is also associated with bleeding abnormalities.

Malignant hyperthermia is a potentially fatal hypermetabolic disorder triggered by halogenated anesthetics and the myorelaxant succinylcholine in genetically predisposed individuals. About 50% of susceptible individuals carry dominant, gain-of-function mutations in RYR1 [which encodes ryanodine receptor type 1 (RyR1)], though they have normal muscle function and no overt clinical symptoms. RyR1 is predominantly found in skeletal muscle but also at lower amounts in immune and smooth muscle cells, suggesting that RYR1 mutations may have a wider range of effects than previously suspected. Mild bleeding abnormalities have been described in patients with malignant hyperthermia carrying gain-of-function RYR1 mutations. We sought to determine the frequency and molecular basis for this symptom. We found that some patients with specific RYR1 mutations had abnormally high bleeding scores, whereas their healthy relatives did not. Knock-in mice with the malignant hyperthermia susceptibility RYR1 mutation Y522S (MHS RYR1Y522S) had longer bleeding times than their wild-type littermates. Primary vascular smooth muscle cells from RYR1Y522S knock-in mice exhibited a higher frequency of subplasmalemmal Ca(2+) sparks, leading to a more negative resting membrane potential. The bleeding defect of RYR1Y522S mice and of one patient was reversed by treatment with the RYR1 antagonist dantrolene, and Ca(2+) sparks in primary vascular smooth muscle cells from the MHS RYR1Y522S mice were blocked by ryanodine or dantrolene. Thus, RYR1 mutations may lead to prolonged bleeding by altering vascular smooth muscle cell function. The reversibility of the bleeding phenotype emphasizes the potential therapeutic value of dantrolene in the treatment of such bleeding disorders.

mTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis.

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2(+/-) and TSC2(-/-) neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.

Gain of function in the immune system caused by a ryanodine receptor 1 mutation.

Mutations in RYR1, the gene encoding ryanodine receptor 1, are linked to a variety of neuromuscular disorders including malignant hyperthermia (MH), a pharmacogenetic hypermetabolic disease caused by dysregulation of Ca(2+) in skeletal muscle. RYR1 encodes a Ca(2+) channel that is predominantly expressed in skeletal muscle sarcoplasmic reticulum, where it is involved in releasing the Ca(2+) necessary for muscle contraction. Other tissues, however, including cells of the immune system, have been shown to express ryanodine receptor 1; in dendritic cells its activation leads to increased surface expression of major histocompatibility complex II molecules and provides synergistic signals leading to cell maturation. In the present study, we investigated the impact of an MH mutation on the immune system by studying the RYR1Y522S knock-in mouse. Our results show that there are subtle but significant differences both in resting 'non-challenged' mice as well as in mice treated with antigenic stimuli, in particular the knock-in mice: (i) have dendritic cells that are more efficient at stimulating T cell proliferation, (ii) have higher levels of natural IgG1 and IgE antibodies, and (iii) are faster and more efficient at mounting a specific immune response in the early phases of immunization. We suggest that some gain-of-function MH-linked RYR1 mutations might offer selective immune advantages to their carriers. Furthermore, our results raise the intriguing possibility that pharmacological activation of RyR1 might be exploited for the development of new classes of vaccines and adjuvants.

Enhanced excitation-coupled Ca(2+) entry induces nuclear translocation of NFAT and contributes to IL-6 release from myotubes from patients with central core disease.

Prolonged depolarization of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. Skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4-dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. In this study, we directly measured excitation-coupled calcium entry by total internal reflection fluorescence microscopy in human skeletal muscle myotubes harbouring mutations in the RYR1 gene linked to malignant hyperthermia (MH) and central core disease (CCD). We found that excitation-coupled calcium entry is strongly enhanced in cells from patients with CCD compared with individuals with MH and controls. Furthermore, excitation-coupled calcium entry induces generation of reactive nitrogen species and enhances nuclear localization of NFATc1, which in turn may be responsible for the increased IL-6 released by myotubes from patients with CCD.

Fibroblast growth factor 2 and platelet-derived growth factor, but not platelet lysate, induce proliferation-dependent, functional class II major histocompatibility complex antigen in human mesenchymal stem cells.

OBJECTIVE: To document the specificity and the mechanism of induction of a novel class II major histocompatibility complex (MHC) antigen by mitogenic growth factors in human mesenchymal stem cells (MSCs) expanded in vitro for translational applications. METHODS: Expression of class II MHC molecules was measured in human MSCs and differentiated cells expanded in the presence of fibroblast growth factor 2 (FGF-2), platelet-derived growth factor BB (PDGF-BB), human platelet lysate, or interferon-γ (IFNγ). The roles of cell proliferation and growth factor-induced signaling pathways were investigated as well as the class II MHC assembly machinery and functional capacity. RESULTS: FGF-2 and, to a lesser extent, PDGF-BB induced in adult human MSCs the expression of HLA-DR (normally induced by inflammatory cytokines), which was able to stimulate CD4+ T cells via superantigen binding. In contrast to IFNγ, FGF induced HLA-DR expression only in human MSCs proliferating under its mitogenic effect and not in mouse MSCs or in differentiated human cells. Although it induced cell proliferation, human platelet lysate did not cause HLA-DR expression in human MSCs. HLA-DR expression occurred following FGF-specific binding to its receptor(s), mainly FGF receptor 1, without inducing IFNγ or tumor necrosis factor α expression. Both MAPK/ERK-1/2 and phosphatidylinositol 3-kinase/Akt controlled cell proliferation and HLA-DR expression, but only MAPK/ERK-1/2 controlled the induction of the class II MHC transcription activator protein CIITA, the major determinant of HLA-DR transcription. CONCLUSION: The induction of functional HLA-DR in proliferating progenitor MSCs is a property of human MSCs that have been expanded with mitogenic growth factors. This has potential biologic significance in the regulation and/or protection of progenitor cell subpopulations under sustained mitogenic proliferation and needs to be taken into account when expanding MSCs for use in in vivo applications.

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

BACKGROUND: A diagnosis of malignant hyperthermia susceptibility by in vitro contraction testing can often only be performed at specialized laboratories far away from where patients live. Therefore, we have designed a protocol for genetic screening of the RYR1-cDNA and for functional testing of newly identified ryanodine receptor 1 (RYR1) gene variants in B lymphocytes isolated from peripheral blood samples drawn at local primary care centers. METHODS: B lymphocytes were isolated for the extraction of RYR1-mRNA and genomic DNA and for establishment of lymphoblastoid B cell lines in 5 patients carrying yet unclassified mutations in the RYR1. The B lymphoblastoid cell lines were used to study resting cytoplasmic calcium concentration, the peak calcium transient induced by the sarco(endo)plasmic reticulum Ca-ATPase inhibitor thapsigargin, and the dose-dependent calcium release induced by the ryanodine receptor agonist 4-chloro-m-cresol. RESULTS: It was possible to extract mRNA for cDNA synthesis and to create B lymphocyte clones from all samples. All B lymphoblastoid cell lines carrying RYR1 candidate mutations showed significantly increased resting cytoplasmic calcium levels as well as a shift to lower concentrations of 4-chloro-m-cresol inducing calcium release compared with controls. CONCLUSIONS: Peripheral blood samples are stable regarding RNA and DNA extraction and establishment of lymphoblastoid B cell lines after transportation at ambient temperature over large distances by ordinary mail. Functional tests on B cells harboring the newly identified amino acid substitutions indicate that they alter intracellular Ca2+ homeostasis and are most likely causative of malignant hyperthermia.

Increasing the number of diagnostic mutations in malignant hyperthermia.

Malignant hyperthermia (MH) is an autosomal dominant disorder characterized by abnormal calcium homeostasis in skeletal muscle in response to triggering agents. Today, genetic investigations on ryanodine receptor type 1 (RYR1) gene and alpha1 subunit of the dihydropyridine receptor (DHPR) (CACNA1S) gene have improved the procedures associated with MH diagnosis. In approximately 50% of MH cases a causative RYR1 mutation was found. Molecular genetic testing based on RYR1 mutations for MH diagnosis is challenging, because the causative mutations, most of which are private, are distributed throughout the RYR1 gene. A more comprehensive genetic testing procedure is needed. Therefore, we aim to expand the genetic information related to MH and to evaluate the effect of mutations on the MH phenotype. Performing an in-depth mutation screening of the RYR1 transcript sequence in 36 unrelated MH susceptible (MHS) patients, we identified 17 novel, five rare, and eight non-disease-causing variants in 23 patients. The 13 remaining MHS patients presented no known variants, neither in RYR1 nor in the CACNA1S binding regions to RYR1. The 17 novel variants were found to affect highly conserved amino acids and were absent in 100 controls. Excellent genotype-phenotype correlations were found by investigating 21 MHS families-a total of 186 individuals. Epstein-Barr virus (EBV) lymphoblastoid cells carrying four of these novel mutations showed abnormal calcium homeostasis. The results of this study contribute to the establishment of a robust genetic testing procedure for MH diagnosis.