Exome analysis identifies Brody myopathy in a family diagnosed with malignant hyperthermia susceptibility.

Whole exome sequencing (WES) was used to determine the primary cause of muscle disorder in a family diagnosed with a mild, undetermined myopathy and malignant hyperthermia (MH) susceptibility (MHS). WES revealed the compound heterozygous mutations, p.Ile235Asn and p.Glu982Lys, in ATP2A1, encoding the sarco(endo)plasmic reticulum Ca(2+) ATPase type 1 (SERCA1), a calcium pump, expressed in fast-twitch muscles. Recessive mutations in ATP2A1 are known to cause Brody myopathy, a rare muscle disorder characterized by exercise-induced impairment of muscle relaxation and stiffness. Analyses of affected muscles showed the absence of SERCA1, but SERCA2 upregulation in slow and fast myofibers, suggesting a compensatory mechanism that partially restores the diminished Ca(2+) transport in Brody myopathy. This compensatory adaptation to the lack of SERCA1 Ca(2+) pumping activity within the muscle explains, in part, the mild course of disease in our patient. Diagnosis of MHS in this family was secondary to a loss of SERCA1 due to disease-associated mutations. Although there are obvious differences in clinical expression and molecular mechanisms between MH and Brody myopathy, a feature common to both conditions is elevated myoplasmic Ca(2+) content. Prolonged intracellular Ca(2+) elevation is likely to have led to MHS diagnosis in vitro and postoperative MH-like symptoms in Brody patient.

Ryanodine receptor type 1 gene mutations found in the Canadian malignant hyperthermia population.

PURPOSE: Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder that is manifested on exposure of susceptible individuals to halogenated anesthetics or succinylcholine. Since MH is associated primarily with mutations in the ryanodine receptor type 1 (RYR1) gene, the purpose of this study was to determine the distribution and frequency of MH causative RyR1 mutations in the Canadian MH susceptible (MHS) population. METHODS: In this study, we screened a representative cohort of 36 unrelated Canadian MHS individuals for RYR1 mutations by sequencing complete RYR1 transcripts and selected regions of CACNA1S transcripts. We then analyzed the correlation between caffeine-halothane contracture test (CHCT) results and RYR1 genotypes within MH families. RESULTS: Eighty-six percent of patients had at least one RyR1 mutation (31 out of 36), five of which were unrelated individuals who were double-variant carriers. Fifteen of the 27 mutations identified in RYR1 were novel. Eight novel mutations, involving highly conserved amino acid residues, were predicted to be causal. Two of the mutations co-segregated with the MHS phenotype within two large independent families (a total of 79 individuals). Fourteen percent of MHS individuals (five out of 36) carried neither RYR1 nor known CACNA1S mutations. CONCLUSIONS: The distribution and frequency of MH causative RyR1 mutations in the Canadian MHS population are close to those of European MHS populations. Novel mutations described in this study will contribute to the worldwide pool of MH-associated mutations in the RYR1 gene, ultimately increasing the value of MH genetic diagnostic testing.

Ca2+ dysregulation in Ryr1(I4895T/wt) mice causes congenital myopathy with progressive formation of minicores, cores, and nemaline rods.

Ryr1(I4895T/wt) (IT/+) mice express a knockin mutation corresponding to the human I4898T EC-uncoupling mutation in the type 1 ryanodine receptor/Ca(2+) release channel (RyR1), which causes a severe form of central core disease (CCD). IT/+ mice exhibit a slowly progressive congenital myopathy, with neonatal respiratory stress, skeletal muscle weakness, impaired mobility, dorsal kyphosis, and hind limb paralysis. Lesions observed in myofibers from diseased mice undergo age-dependent transformation from minicores to cores and nemaline rods. Early ultrastructural abnormalities include sarcomeric misalignment, Z-line streaming, focal loss of cross-striations, and myofibrillar splitting and intermingling that may arise from defective myofibrillogenesis. However, manifestation of the disease phenotype is highly variable on a Sv129 genomic background. Quantitative RT-PCR shows an equimolar ratio of WT and mutant Ryr1 transcripts within IT/+ myofibers and total RyR1 protein expression levels are normal. We propose a unifying theory in which the cause of core formation lies in functional heterogeneity among RyR1 tetramers. Random combinations of normal and either leaky or EC-uncoupled RyR subunits would lead to spatial differences in Ca(2+) transients; the resulting heterogeneity of contraction among myofibrils would lead to focal, irreversible tearing and shearing, which would, over time, enlarge to form minicores, cores, and nemaline rods. The IT/+ mouse line is proposed to be a valid model of RyR1-related congenital myopathy, offering high potential for elucidation of the pathogenesis of skeletal muscle disorders arising from impaired EC coupling.

An Ryr1I4895T mutation abolishes Ca2+ release channel function and delays development in homozygous offspring of a mutant mouse line.

A heterozygous Ile4898 to Thr (I4898T) mutation in the human type 1 ryanodine receptor/Ca(2+) release channel (RyR1) leads to a severe form of central core disease. We created a mouse line in which the corresponding Ryr1(I4895T) mutation was introduced by using a "knockin" protocol. The heterozygote does not exhibit an overt disease phenotype, but homozygous (IT/IT) mice are paralyzed and die perinatally, apparently because of asphyxia. Histological analysis shows that IT/IT mice have greatly reduced and amorphous skeletal muscle. Myotubes are small, nuclei remain central, myofibrils are disarranged, and no cross striation is obvious. Many areas indicate probable degeneration, with shortened myotubes containing central stacks of pyknotic nuclei. Other manifestations of a delay in completion of late stages of embryogenesis include growth retardation and marked delay in ossification, dermatogenesis, and cardiovascular development. Electron microscopy of IT/IT muscle demonstrates appropriate targeting and positioning of RyR1 at triad junctions and a normal organization of dihydropyridine receptor (DHPR) complexes into RyR1-associated tetrads. Functional studies carried out in cultured IT/IT myotubes show that ligand-induced and DHPR-activated RyR1 Ca(2+) release is absent, although retrograde enhancement of DHPR Ca(2+) conductance is retained. IT/IT mice, in which RyR1-mediated Ca(2+) release is abolished without altering the formation of the junctional DHPR-RyR1 macromolecular complex, provide a valuable model for elucidation of the role of RyR1-mediated Ca(2+) signaling in mammalian embryogenesis.