An autosomal dominant congenital myopathy with cores and rods is associated with a neomutation in the RYR1 gene encoding the skeletal muscle ryanodine receptor

Central core disease (CCD) and nemaline myopathy (NM) are congenital myopathies for which differential diagnosis is often based on the presence either of cores or rods. Missense mutations in the skeletal muscle ryanodine receptor gene (RYR1) have been identified in some families with CCD. Mutations in the alpha-tropomyosin and alpha-actin genes have been associated with most dominant forms of NM. Analysis of the RYR1 cDNA in a French family identified a novel Y4796C mutation that lies in the C-terminal channel-forming domain of the RyR1 protein. This mutation was linked not only to a severe and penetrant form of CCD, but also to the presence of rods in the muscle fibres and to the malignant hyperthermia susceptibility (MHS) phenotype. The Y4796C mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. Expression of the mutant RYR1 cDNA produced channels with increased caffeine sensitivity and a significantly reduced maximal level of Ca(2+) release. Single-cell Ca(2+) analysis showed that the resting cytoplasmic level was increased by 60% in cells expressing the mutant channel. These data support the view that the rate of Ca(2+) leakage is increased in the mutant channel. The resulting chronic elevation in myoplasmic concentration is likely to be responsible for the severe expression of the disease. Haplotyping analysis indicated that the mutation arose as a neomutation in the proband. This first report of a neomutation in the RYR1 gene has strong implications for genetic linkage studies of MHS or CCD, two diseases characterized by a genetic heterogeneity.     

Recessive RYR1 mutations in a patient with severe congenital nemaline myopathy with ophthalomoplegia identified through massively parallel sequencing

Nemaline myopathy (NM) is a group of congenital myopathies, characterized by the presence of distinct rod-like inclusions "nemaline bodies" in the sarcoplasm of skeletal muscle fibers. To date, ACTA1, NEB, TPM3, TPM2, TNNT1, and CFL2 have been found to cause NM. We have identified recessive RYR1 mutations in a patient with severe congenital NM, through high-throughput screening of congenital myopathy/muscular dystrophy-related genes using massively parallel sequencing with target gene capture. The patient manifested fetal akinesia, neonatal severe hypotonia with muscle weakness, respiratory insufficiency, swallowing disturbance, and ophthalomoplegia. Skeletal muscle histology demonstrated nemaline bodies and small type 1 fibers, but without central cores or minicores. Congenital myopathies, a molecularly, histopathologically, and clinically heterogeneous group of disorders are considered to be a good candidate for massively parallel sequencing.     

Mutations in RYR1 in malignant hyperthermia and central core disease

The RYR1 gene encodes the skeletal muscle isoform ryanodine receptor and is fundamental to the process of excitation-contraction coupling and skeletal muscle calcium homeostasis. Mapping to chromosome 19q13.2, the gene comprises 106 exons and encodes a protein of 5,038 amino acids. Mutations in the gene have been found in association with several diseases: the pharmacogenetic disorder, malignant hyperthermia (MH); and three congenital myopathies, including central core disease (CCD), multiminicore disease (MmD), and in an isolated case of a congenital myopathy characterized on histology by cores and rods. The majority of gene mutations reported are missense changes identified in cases of MH and CCD. In vitro analysis has confirmed that alteration of normal calcium homeostasis is a functional consequence of some of these changes. Genotype-phenotype correlation studies performed using data from MH and CCD patients have also suggested that mutations may be associated with a range of disease severity phenotypes. This review aims to summarize the current understanding of RYR1 mutations reported in association with MH and CCD and the present viewpoint on the use of mutation data to aid clinical diagnosis of these conditions.     

Familial and sporadic forms of central core disease are associated with mutations in the C-terminal domain of the skeletal muscle ryanodine receptor.

Central core disease (CCD) is an autosomal dominant congenital myopathy. Diagnosis is based on the presence of cores in skeletal muscles. CCD has been linked to the gene encoding the ryanodine receptor (RYR1) and is considered to be an allelic disease of malignant hyperthermia susceptibility. However, the report of a recessive form of transmission together with a variable clinical presentation has raised the question of the genetic heterogeneity of the disease. Analyzing a panel of 34 families exclusively recruited on the basis of both clinically and morphologically expressed CCD, 12 different mutations of the C-terminal domain of RYR1 have been identified in 16 unrelated families. Morphological analysis of the patients' muscles showed different aspects of cores, all of them associated with mutations in the C-terminal region of RYR1. Furthermore, we characterized the presence of neomutations in the RyR1 gene in four families. This indicates that neomutations into the RyR1 gene are not a rare event and must be taken into account for genetic studies of families that present with congenital myopathies type 'central core disease'. Three mutations led to the deletion in frame of amino acids. This is the first report of amino acid deletions in RYR1 associated with CCD. According to a four-transmembrane domain model, the mutations concentrated mostly in the myoplasmic and luminal loops linking, respectively, transmembrane domains T1 and T2 or T3 and T4 of RYR1.     

Mutations of tropomyosin 3 (TPM3) are common and associated with type 1 myofiber hypotrophy in congenital fiber type disproportion

Congenital fiber type disproportion (CFTD) is a rare congenital myopathy characterized by hypotonia and generalized muscle weakness. Pathologic diagnosis of CFTD is based on the presence of type 1 fiber hypotrophy of at least 12% in the absence of other notable pathological findings. Mutations of the ACTA1 and SEPN1 genes have been identified in a small percentage of CFTD cases. The muscle tropomyosin 3 gene, TPM3, is mutated in rare cases of nemaline myopathy that typically exhibit type 1 fiber hypotrophy with nemaline rods, and recently mutations in the TPM3 gene were also found to cause CFTD. We screened the TPM3 gene in patients with a clinical diagnosis of CFTD, nemaline myopathy, and with undefined congenital myopathies. Mutations in TPM3 were identified in 6 out of 13 patients with CFTD, as well as in one case of nemaline myopathy. Review of muscle biopsies from patients with diagnoses of CFTD revealed that patients with a TPM3 mutation all displayed marked disproportion of fiber size, without type 1 fiber predominance. Several mutation‐negative cases exhibited other abnormalities, such as central nuclei and central cores. These results support the utility of the CFTD diagnosis in directing the course of genetic testing. Hum Mutat 30:1–8, 2009. © 2009 Wiley‐Liss, Inc.     

Nebulin (NEB) mutations in a childhood onset distal myopathy with rods and cores uncovered by next generation sequencing

Recessive nebulin (NEB) mutations are a common cause of nemaline myopathy (NM), typically characterized by generalized weakness of early-onset and nemaline rods on muscle biopsy. Exceptional adult cases with additional cores and an isolated distal weakness have been reported. The large NEB gene with 183 exons has been an obstacle for the genetic work-up. Here we report a childhood-onset case with distal weakness and a core-rod myopathy, associated with recessive NEB mutations identified by next generation sequencing (NGS). This 6-year-old boy presented with a history of gross-motor difficulties following a normal early development. He had distal leg weakness with bilateral foot drop, as well as axial muscle weakness, scoliosis and spinal rigidity; additionally he required nocturnal respiratory support. Muscle magnetic resonance (MR) imaging showed distal involvement in the medial and anterior compartment of the lower leg. A muscle biopsy featured both rods and cores. Initial targeted testing identified a heterozygous Nebulin exon 55 deletion. Further analysis using NGS revealed a frameshifting 4 bp duplication, c.24372_24375dup (P.Val8126fs), on the opposite allele. This case illustrates that NEB mutations can cause childhood onset distal NM, with additional cores on muscle biopsy and proves the diagnostic utility of NGS for myopathies, particularly when large genes are implicated.     

A new mutation in the skeletal ryanodine receptor gene (RYR1) is potentially causative of malignant hyperthermia, central core disease, and severe skeletal malformation

Malignant hyperthermia susceptibility (MHS) and central core disease (CCD) have been shown to result from missense mutations in the ryanodine receptor gene of the skeletal muscle (RYR1). A 15-year-old patient who had spondylocostal dysostosis (SCD) developed an MH crisis during general anesthesia. The patient was characterized phenotypically by block vertebrae, vertebral fusion, short neck and thorax, fused ribs, craniofacial abnormalities, spina bifida occulta, and a diaphragmatic defect closed surgically in early infancy. The diagnosis MH susceptible (MHS) was confirmed by the in vitro contracture test (IVCT) on a muscle biopsy. Surprisingly, the histopathological investigation revealed the presence of CCD too. Molecular genetic investigation of the RYR1 gene was performed to search for known MH-related mutations. Cluster regions of the RYR1 gene, in which mutations have already been found, were examined by direct automated sequencing. In addition to the diagnosis MHS and CCD we were able to identify a novel RYR1 mutation in exon 46: 7358ATC > ACC, resulting in an Ile2453Thr substitution. This mutation was also present in the mother, in whom MH disposition and CCD were determined by muscle investigations. We suggest that the newly identified RYR1 mutation is closely associated with MH and CCD. A probable causative role of the RYR1 gene in SCD patients should be assessed by further genetic investigations.     

Abnormal myosin post-translational modifications and ATP turnover time associated with human congenital myopathy-related RYR1 mutations

Aim

Conditions related to mutations in the gene encoding the skeletal muscle ryanodine receptor 1 (RYR1) are genetic muscle disorders and include congenital myopathies with permanent weakness, as well as episodic phenotypes such as rhabdomyolysis/myalgia. Although RYR1 dysfunction is the primary mechanism in RYR1-related disorders, other downstream pathogenic events are less well understood and may include a secondary remodeling of major contractile proteins. Hence, in the present study, we aimed to investigate whether congenital myopathy-related RYR1 mutations alter the regulation of the most abundant contractile protein, myosin.

Methods

We used skeletal muscle tissues from five patients with RYR1-related congenital myopathy and compared those with five controls and five patients with RYR1-related rhabdomyolysis/myalgia. We then defined post-translational modifications on myosin heavy chains (MyHCs) using LC/MS. In parallel, we determined myosin relaxed states using Mant-ATP chase experiments and performed molecular dynamics (MD) simulations.

Results

LC/MS revealed two additional phosphorylations (Thr1309-P and Ser1362-P) and one acetylation (Lys1410-Ac) on the β/slow MyHC of patients with congenital myopathy. This method also identified six acetylations that were lacking on MyHC type IIa of these patients (Lys35-Ac, Lys663-Ac, Lys763-Ac, Lys1171-Ac, Lys1360-Ac, and Lys1733-Ac). MD simulations suggest that modifying myosin Ser1362 impacts the protein structure and dynamics. Finally, Mant-ATP chase experiments showed a faster ATP turnover time of myosin heads in the disordered–relaxed conformation.

Conclusions

Altogether, our results suggest that RYR1 mutations have secondary negative consequences on myosin structure and function, likely contributing to the congenital myopathic phenotype.     

Mutations and polymorphisms of the skeletal muscle α‐actin gene (ACTA1)

The ACTA1 gene encodes skeletal muscle α‐actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease‐causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease‐causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core‐like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype–phenotype correlations, we have developed a locus‐specific database for ACTA1 variations ( http://waimr.uwa.edu.au ). Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Identification and characterization of three novel mutations in the CASQ1 gene in four patients with tubular aggregate myopathy

Here, we report the identification of three novel missense mutations in the calsequestrin‐1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca²⁺ concentrations, showed a reduced Ca²⁺‐dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca²⁺‐dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca²⁺ in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca²⁺ and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca²⁺ entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca²⁺ handling in skeletal muscle fibers.     

A truncation in the RYR1 gene associated with central core lesions in skeletal muscle fibres

A novel single-nucleotide deletion in exon 100 of the RYR1 gene, corresponding to deletion of nucleotide 14,510 in the human RyR1 mRNA (c14510delA), was identified in a man with malignant hyperthermia and in his two daughters who were normal for malignant hyperthermia. This deletion results in a RyR1 protein lacking the last 202 amino acid residues. All three subjects heterozygotic for the mutated allele presented with a prevalence of type 1 fibres with central cores, although none experienced clinical signs of myopathy. Expression of the truncated protein resulted in non-functional RYR1 calcium release channels. Expression of wild-type and RyR1(R4836fsX4838) proteins resulted in heterozygotic release channels with overall functional properties similar to those of wild-type RyR1 channels. Nevertheless, small differences in sensitivity to calcium and caffeine were observed in heterotetrameric channels, which also presented an altered assembly/stability in sucrose-gradient centrifugation analysis. Altogether, these data suggest that altered RYR1 tetramer assembly/stability coupled with subtle chronic changes in Ca2+ homoeostasis over the long term may contribute to the development of core lesions and incomplete malignant hyperthermia susceptibility penetrance in individuals carrying this novel RYR1 mutation.     

Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India

We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH‐cytochrome b5 reductase (NADH‐CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice‐site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three‐dimensional (3D) structure of human erythrocyte NADH‐CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype–phenotype correlation in NADH‐CYB5R deficiency.     

Search for three known mutations in the RYR1 gene in 48 Danish families with malignant hyperthermia

We have examined 48 Danish families in which malignant hyperthermia reactions have occurred, with respect to three of six published mutations in the gene for the calcium release channel of sarcoplasmic reticulum (the RYR1 gene) believed to cause malignant hyperthermia susceptibility in man. The mutations are Arg614Cys, also known as the "pig mutation"; Arg163Cys; and Ile403Met. The only mutation found was Arg163Cys, which was detected in only one family. The results of this study indicate that other mutations must underlie the disorder in most Danish malignant hyperthermia-susceptible families, and that the "pig mutation" is not a frequent cause of malignant hyperthermia susceptibility in Denmark.     

Screening of the ryanodine receptor gene in 105 malignant hyperthermia families: novel mutations and concordance with the in vitro contracture test.

Malignant hyperthermia (MH) in man is an autosomal dominant disorder of skeletal muscle Ca(2+)-regulation. During anesthesia in predisposed individuals, it is triggered by volatile anesthetics and depolarizing muscle relaxants. In >50% of the families, MH susceptibility is linked to the gene encoding the skeletal muscle ryanodine receptor (RYR1), the calcium release channel of the sarcoplasmic reticulum, on chromosome 19q12-13.2. To date, 21 RYR1 mutations have been identified in a number of pedigrees. Four of them are also associated with central core disease (CCD), a congenital myopathy. Screening for these 21 mutations in 105 MH families including 10 CCD families phenotyped by the in vitro contracture test (IVCT) according to the European protocol revealed the following approximate distribution: 9% Arg-614-Cys, 1% Arg-614-Leu, 1% Arg-2163-Cys, 1% Val-2168-Met, 3% Thr-2206-Met and 7% Gly-2434-Arg. In one CCD family, the disease was caused by a recently reported MH mutation, Arg-2454-His. Two novel mutations, Thr-2206-Arg and Arg-2454-Cys were detected, each in a single pedigree. In the 109 individuals of the 25 families with RYR1 mutations cosegregation between genetic result and IVCT was almost perfect, only three genotypes were discordant with the IVCT phenotypes, suggesting a true sensitivity of 98.5% and a specificity of minimally 81.8% for this test. Screening of the transmembraneous region of RYR1 did not yield a new mutation confirming the cytosolic portion of the protein to be of main functional importance for disease pathogenesis.     

Null mutations causing depletion of the type 1 ryanodine receptor (RYR1) are commonly associated with recessive structural congenital myopathies with cores

Mutations of the ryanodine receptor cause dominant and recessive forms of congenital myopathies with cores. Quantitative defects of RYR1 have been reported in families presenting with recessive forms of the disease and epigenic regulation has been recently proposed to explain potential maternal monoallelic silencing of the RYR1 gene. We investigated nine families presenting with a recessive form of the disease and showing a quantitative defect of RYR1 expression. Genetic analysis allowed the identification of a mutation on both alleles of the RYR1 gene for all patients, 15 being novel variants. We evidenced for all patients an alteration of the expression of the RYR1 gene caused by amorphic mutations responsible either for mRNA or protein instability. In seven families the variant present on the second allele was a missense mutation. In the remaining two families the second variant led to a hypomorphic expression of the RYR1 gene and was associated with a severe neonatal phenotype, pointing out the minimal amount of RYR1 needed for skeletal muscle function. Noticeably, a novel additional exon 3b was characterized in the most severely affected cases. This study showed that all cases presenting with a quantitative defect of RYR1 expression in our panel of patients affected by recessive core myopathies were caused by the presence of one recessive null allele and that variability of the phenotype depended on the nature of the mutation present on the second allele. Our study also indicated that presence of a second mutation must be investigated in sporadic cases or in dominant cases presenting with a familial clinical variability.     

Recessive ACTA1 variant causes congenital muscular dystrophy with rigid spine

Variants in ACTA1, which encodes α-skeletal actin, cause several congenital myopathies, most commonly nemaline myopathy. Autosomal recessive variants comprise approximately 10% of ACTA1 myopathy. All recessive variants reported to date have resulted in loss of skeletal α-actin expression from muscle and severe weakness from birth. Targeted next-generation sequencing in two brothers with congenital muscular dystrophy with rigid spine revealed homozygous missense variants in ACTA1. Skeletal α-actin expression was preserved in these patients. This report expands the clinical and histological phenotype of ACTA1 disease to include congenital muscular dystrophy with rigid spine and dystrophic features on muscle biopsy. This represents a new class of recessive ACTA1 variants, which do not abolish protein expression.     

Genetic risk for malignant hyperthermia in non-anesthesia-induced myopathies

Malignant hyperthermia (MH) is a pharmacogenetic, autosomal dominantly inherited disorder of skeletal muscle triggered by volatile anesthetics and infrequently by extreme exertion and heat exposure. MH has variable penetrance with an incidence ranging from 1 in 5000 to 1 in 50,000-100,000 anesthesias. Mutations in the ryanodine receptor gene, RYR1, are found in 50-70% of cases. We hypothesized that a portion of patients with drug-induced muscle diseases, unrelated to anesthesia, such as severe statin myopathy, have underlying genetic liability that may include RYR1 gene mutations. DNA samples were collected from 885 patients in 4 groups: severe statin myopathy (n=197), mild statin myopathy (n=163), statin-tolerant controls (n=133), and non-drug-induced myopathies of unknown etiology characterized by exercise-induced muscle pain and weakness (n=392). Samples were screened for 105 mutations and variants in 26 genes associated with 7 categories of muscle disease including 34 mutations and variants in the RYR1 gene. Disease-causing mutations or variants in RYR1 were present in 3 severe statin myopathy cases, 1 mild statin myopathy case, 8 patients with non-drug-induced myopathy, and none in controls. These results suggest that disease-causing mutations and certain variants in the RYR1 gene may contribute to underlying genetic risk for non-anesthesia-induced myopathies and should be included in genetic susceptibility screening in patients with severe statin myopathy and in patients with non-statin-induced myopathies of unknown etiology.     

A search for three known RYR1 gene mutations in 41 Swedish families with predisposition to malignant hyperthermia

Eight mutations in the gene (the RYR1 gene) encoding the calcium release channel of sarcoplasmic reticulum (SR) in skeletal muscle are so far known to be very closely linked to malignant hyperthermia susceptibility in man and are regarded to be causative. We have examined 41 Swedish families where malignant hyperthermia had occurred in at least one member during anaesthesia, with respect to three of the known mutations. The mutations were Arg163Cys; Ile403Met and Arg614Cys (also known as the "pig mutation"). In three (i.e. 7%) of the families we detected the Arg614Cys mutation, and this was the only one of the mutations searched for that was observed. This indicates that other mutations than those searched for in this study must cause malignant hyperthermia susceptibility in most Swedish malignant hyperthermia susceptible families.     

Correlations between genotype and pharmacological, histological, functional, and clinical phenotypes in malignant hyperthermia susceptibility

Malignant hyperthermia susceptibility (MHS) is a subclinical pharmacogenetic disorder caused by an impairment of skeletal muscle calcium homeostasis in response to triggering agents. While in vitro contracture testing (IVCT) is the gold standard for defining MHS, molecular analysis is increasingly used to diagnosis MHS. Mutations associated with MHS have been reported in two genes: RYR1 and CACNA1S. Mutations in RYR1 are also responsible for central core disease (CCD), a myopathy that can be associated with a positive IVCT response. We report here the results of correlation studies performed with molecular, pharmacological, histological, and functional data obtained in 175 families (referred to as confirmed (129) or potential (46) MHS families). Extensive molecular analysis allowed us to identify a variant in 60% of the confirmed MHS families, and resulted in the characterization of 11 new variants in the RYR1 gene. Most mutations clustered to MH1 and MH2 domains of RYR1. Functional analysis allowed us to assign a causative role for seven MHS mutations that we propose to add to the panel of MHS mutations used for genetic testing. The use of genetic data to determine MHS status led to a 99.5% sensitivity for IVCT. IVCT-positive/mutation-negative diagnoses were analyzed not only in terms of specificity for IVCT, but also to assess the presence of a second MHS trait in families, and the genetic heterogeneity of the disease. Histological analyses revealed the presence of cores in more than 20% of muscle biopsies originating from 242 genotyped and tested MHS patients who did not present with clinical symptoms. This indicates that these patients must be considered as MHS patients with cores, and are clearly differentiated from CCD patients who have been tested positive for MHS.