Recessive RYR1 Mutations Research Articles

Quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying recessive Ryr1 mutations linked to congenital myopathies.

Skeletal muscles are a highly structured tissue responsible for movement and metabolic regulation, which can be broadly subdivided into fast and slow twitch muscles with each type expressing common as well as specific sets of proteins. Congenital myopathies are a group of muscle diseases leading to a weak muscle phenotype caused by mutations in a number of genes including RYR1. Patients carrying recessive RYR1 mutations usually present from birth and are generally more severely affected, showing preferential involvement of fast twitch muscles as well as extraocular and facial muscles. In order to gain more insight into the pathophysiology of recessive RYR1-congential myopathies, we performed relative and absolute quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying p.Q1970fsX16 and p.A4329D RyR1 mutations which were identified in a child with a severe congenital myopathy. Our in-depth proteomic analysis shows that recessive RYR1 mutations not only decrease the content of RyR1 protein in muscle, but change the expression of 1130, 753, and 967 proteins EDL, soleus and extraocular muscles, respectively. Specifically, recessive RYR1 mutations affect the expression level of proteins involved in calcium signaling, extracellular matrix, metabolism and ER protein quality control. This study also reveals the stoichiometry of major proteins involved in excitation contraction coupling and identifies novel potential pharmacological targets to treat RyR1-related congenital myopathies.

Molecular basis of impaired muscle function in a mouse model of congenital myopathy due to compound heterozygous RYR1 mutations

Congenital myopathies (CM) are a group of early-onset, genetically diverse muscle disorders of variable severity with characteristic muscle biopsy findings. Mutations in RYR1, the gene encoding the RYR1, are the most common genetic cause, responsible for ∼30% of all human CM. They are linked to the pharmacogenetic disorder malignant hyperthermia susceptibility and to various disease phenotypes, including central core disease (which is primarily dominantly inherited), multiminicore disease (which is predominantly recessively inherited), some forms of centronuclear myopathy and congenital fiber-type disproportion (which can be either dominantly or recessively inherited), and King-Denborough syndrome (a CM characterized by skeletal abnormalities, dysmorphic features, and malignant hyperthermia susceptibility). The recessive forms of RYR1-linked CM are more severe, affecting children at birth and, in addition to profound muscle weakness, may also affect facial and extraocular muscles and cause skeletal deformities and feeding difficulties. To study the mechanism leading to the profound muscle weakness characterized by recessive RYR1-CM, we created transgenic mice knocked in for the compound heterozygous RYR1 p.Q1970fsX16+p.A4329D mutations (double knock-in mouse, or DKI) identified in a severely affected child. The in vivo and ex vivo physiological functions of fast twitch, slow twitch, and extraocular muscles were severely impaired in DKI mice; in addition, the mutations were accompanied by a >50% decrease in RYR1 protein in all muscles examined, as well as changes in the expression of many proteins important for muscle function and chromatin structure. Muscle ultrastructure was disorganized, with fewer CRU and mitochondria and presence of cores. MyHC-EO, the superfast and ocular-muscle-specific myosin heavy isoform, was almost undetectable in EOMs from DKI mutant mice. Thus, the DKI mouse model faithfully recapitulates the human disease and could be exploited for preclinical studies aimed at developing therapeutic strategies to treat neuromuscular disorders linked to recessive RYR1 mutations.

CONGENITAL MYOPATHIES: EP.51 Treatment of an animal model carrying recessive RYR1 mutations with inhibitors targeting epigenetic enzymes

CONGENITAL MYOPATHIES: EP.51 Treatment of an animal model carrying recessive RYR1 mutations with inhibitors targeting epigenetic enzymes

Characterization of an Animal Model for Congenital Myopathies Linked to Recessive RyR1 Mutations

Characterization of an Animal Model for Congenital Myopathies Linked to Recessive RyR1 Mutations

Congenital ptosis, scoliosis, and malignant hyperthermia susceptibility in siblings with recessive RYR1 mutations

Malignant hyperthermia susceptibility is a rare pharmacogenic disorder of skeletal muscle calcium regulation caused by mutations in the skeletal muscle ryanodine receptor 1 gene (RYR1). It is important to identify children who are candidates for ophthalmic surgery who might harbor RYR1 mutations because intraoperative malignant hyperthermia is potentially lethal. We report 2 siblings with congenital ptosis and scoliosis who were considered for ptosis surgery but were found to harbor underlying recessive RYR1 mutations.

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies.

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.

Excitation-Contraction Coupling in Human Extraocular Muscles:There is more than Meets the Eye

Excitation-contraction coupling is the physiological mechanism whereby an electrical signal detected by the dihydropyridine receptor, is converted into an increase in [Ca2+], via activation of ryanodine receptors. Mutations in RYR1, the gene encoding ryanodine receptor 1, are the underlying cause of various congenital myopathies including Central Core Disease, Multiminicore disease, some forms of Centronucleal myopathy and Congenital Fiber Type disproportion. Interestingly, patients with recessive but not dominant RYR1 mutations show a significant reduction of ryanodine receptor protein in muscle biopsies as well as ophthalmoplegia. In order to understand why the patients with recessive RYR1 mutations specifically showed involvement of the extraocular muscles, we investigated the excitation-contraction coupling machinery in biopsies obtained from “normal” patients undergoing resection eye surgery. Our results show that the major proteins involved in skeletal muscle excitation-contraction coupling are expressed differently in human extraocular muscles compared to leg muscles. In particular the transcripts encoding ryanodine receptor 3, cardiac calsequestrin and the alfa 1 subunit of the cardiac dihydropyridine receptor were upregulated by at least 100 fold, whereas ryanodine receptor 1 and the alfa 1 subunit of the skeletal dihydropyridine receptor were reduced 10 fold. Myotubes obtained from extraocular muscle biopsies also exhibited changes in their calcium homeostasis, and particularly the resting [Ca2+] was lower and the depolarization induced Ca2+ influx was 3 fold higher compared to that observed in leg muscle-derived myotubes. These results indicate that extraocular muscles have a different mode of calcium handling; moreover, the presence of ophthalmoplegia in patients with recessive RYR1 mutations is most likely due to the lower endogenous levels of RyR1 expressed by the extraocular muscles.

P.9.15 Centronuclear myopathies: The experience of Italian Network for congenital myopathies

Centronuclear myopathies (CNMs) are clinically and genetically heterogeneous diseases with common histological findings of centrally located nuclei in myofibers with predominance and hypotrophy of type 1 fibers. CNMs can be classified in 3 main forms: the X-linked myotubular myopathy (XLMTM), caused by mutations in the myotubularin gene (MTM1); the autosomal-dominant form caused by mutations in the dynamin 2 gene (DNM2); the autosomal-recessive form related to mutations in the amphiphysin 2 gene (BIN1). More recently mutations in ryanodine receptor 1 gene (RYR1) have also been associated to CNMs. We report the genetic findings of a cohort of 50 patients with clinical and histological diagnosis of CNM who have been referred in the last 2 decade to the main tertiary Italian centers for neuromuscular diseases. Most patients were screened for the 4 candidate genes although the flow-chart™ of genetic testing was addressed by gender, pattern of inheritance and age of onset and/or disease severity. The genetic defect was identified in 29 patients (58%): 14 were mutated in MTM1 (48%), 12 in DNM2 (42%), and 3 in RYR1(10%). No mutations in BIN1 were detected. A total of 21 different mutations were identified, 6 of which were not previously reported, including 5 in MTM1 and 1 in RYR1. All MTM1 patients had neonatal onset and all but 2 showed severe disease progression. Patients with mutations in DNM2 had variable age of onset and different degree of muscle weakness. The patient carrying recessive RYR1 mutations had severe congenital hypotonia and ophthalmoparesis, in contrast to heterozygous patients who had a milder phenotype. In conclusion our data confirm that (1) XLMTM represent the most severe phenotype (2) DNM2-related CNMs are associated to a wide spectrum of phenotypes (3) CNMs related to recessive RYR1 mutations can mimic XLMTM 4) 40% of our cohort is genetically uncharacterized further confirming the genetic heterogeneity of CNMs.

C.P.2 In silico analysis of recessive RYR1 mutations identifies novel potential disease mechanisms

Mutations in RYR1 are responsible for malignant hyperthermia (MH) and several forms of congenital myopathy. Dominant mutations that give rise to central core disease (CCD) and MH cluster in three hotspot regions and the disease mechanisms associated with these diseases are well studied. In comparison, little is known about how recessive mutations, which occur throughout the gene, lead to disease. To address this issue, we reviewed the literature to map potentially functional domains within the ryanodine receptor type 1 (RyR1) and then analysed the nature and position of RYR1 mutations in 74 individuals with recessive disease, together with their clinical and histological phenotypes. Hypomorphic mutations, which abolish or markedly reduce RyR1 protein expression, were associated with more severe clinical phenotypes and with histological phenotypes other than CCD, such as multi-minicore disease, centronuclear myopathy and congenital fibre type disproportion. For non-hypomorphic mutations, CCD mutations were enriched in MH/CCD hotspot 3 and non-CCD core mutations in hotspot 2. Clustering of non-hypomorphic mutations in the triadin-binding domain (which lies within hotspot region 3) highlights abnormal triadin-binding as a potentially important disease mechanism. Non-hypomorphic mutations associated with ophthalmoplegia clustered in regions involved in ‘interdomain interactions’ which play roles in stabilising the closed conformation of the RyR1 channel. These results suggest that the variability seen in recessive RYR1 histological phenotypes may stem from differences in disease mechanism, determined in part by the location of non-hypomorphic mutations. Reduced overall RyR1 function, reduced stability of the closed channel conformation and abnormal triadin-RyR1 interactions are highlighted as potentially important disease mechanisms in various recessive RYR1-myopathies.

Core-Rod Myopathy Is a Genetically Heterogeneous Condition: Clinical and Morphological Analysis of a Cohort of Patients without Molecular Characterization (P07.203)

Objective: To better define clinical and morphological features of a cohort of 8 core-rod myopathy patients without a molecular diagnosis and possibly orientate new genes discovery. Background Congenital myopathies (CM) are a group of rare inherited muscle disorders characterized by particular histopathological alterations on muscle biopsy. Core-rod myopathy is a CM which presents with cores and rods as distinctive morphological picture. Up to now, this entity has been frequently associated with dominant or recessive RYR1 mutations. Recessive mutations of the NEB gene, dominant mutation in KBTBD13 gene and CFL2 recessive mutations have also been described. Nevertheless the molecular defect underlying several cases is still unknown. Design/Methods: A cohort of 8 patients, 4 adults and 4 paediatric patients (mean age: 31y) from 8 families, underwent neurological evaluation and muscle biopsy. Molecular studies for , RYR1, KBTBD13, NEB, and CFL2 were performed. Results: 4 patients (2 adults and 2 children) presented a homogeneous clinical phenotype characterised by axial weakness with bilateral foot–drop. The other patients, showed a marked clinical variability. Cardiac involvement was reported in 1 patient and respiratory involvement was present in another one. Hyper laxity and/or retractions were reported. Morphology is highly homogeneous, cores and rods lesions were clearly observed in different areas in the same or in distinct muscle fibres. RYR1 mutations were excluded in transcript for 4 patients and in the C-terminal regions for the other 4. ACTA1 and KBTBD13 were excluded in all patients, CFL2 in 4 and NEB mutations were excluded in 2 patients. Conclusions: We identified two different clinical groups. The first one presents an homogeneous phenotype with axial weakness and bilateral foot-drop. The second one exhibits a large clinical variability. By contrast, muscular morphology is invariably homogeneous. Our work confirms the genetic heterogeneity of this rare congenital myopathy and paves the way for the identification of new genes. Disclosure: Dr. Malfatti has nothing to disclose. Dr. Taratuto has nothing to disclose. Dr. Monges has nothing to disclose. Dr. Lubienecki has nothing to disclose. Dr. Stojkovic has received personal compensation for activities with Biogen Idec, Bayer Schering Pharma, Teva, Merck, Sanofi, and LFB. Dr. Lacene has nothing to disclose. Dr. Guycheney has nothing to disclose. Dr. Monnier has nothing to disclose. Dr. Romero has nothing to disclose.

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.

Core Myopathies

The core myopathies, Central Core Disease and Multiminicore Disease, are heterogeneous congenital myopathies with the common defining histopathological feature of focally reduced oxidative enzyme activity (central cores, multiminicores). Mutations in the gene encoding for the skeletal muscle ryanodine (RyR1) receptor are the most common cause. Mutations in the selenoprotein N (SEPN1) gene cause a less common variant. Pathogenic mechanisms underlying dominant RYR1 mutations have been extensively characterized, whereas those associated with recessive RYR1 and SEPN1 mutations are emerging. Identifying a specific genetic defect from the histopathological diagnosis of a core myopathy is complex and ought to be informed by a combined appraisal of histopathological, clinical, and, increasingly, muscle magnetic resonance imaging data. The present review aims at giving an overview of the main genetic and clinicopathological findings, with a major emphasis on features likely to inform the diagnostic process, as well as current treatments and perspectives for future research.

Dominant and recessive RYR1 mutations in adults with core lesions and mild muscle symptoms

Ryanodine receptor gene (RYR1) mutations have been associated with central core disease (CCD), multiminicore/minicore/multicore disease (MmD), and susceptibility to malignant hyperthermia (MH). Patients with muscle symptoms in adulthood, who had features compatible with CCD/MmD, underwent clinical, histological, and genetic (RYR1 and SEPN1 genes) evaluations. Published cases of CCD and MmD with adult onset were also reviewed. Eight patients fulfilled the criteria for further analysis. Five RYR1 mutations, 4 of them unreported, were detected in 3 patients. Compound heterozygosity was proven in 1 case. To our knowledge, this is the only report of adult onset associated with recessive RYR1 mutations and central core/multiminicores on muscle biopsy. Although adult patients with CCD, MmD, and minimally symptomatic MH with abnormal muscle biopsy findings usually have a mild clinical course, differential diagnosis and carrier screening is crucial for prevention of potentially life-threatening reactions to general anesthesia.

Recessive RYR1 mutations cause unusual congenital myopathy with prominent nuclear internalization and large areas of myofibrillar disorganization

To report the clinical, pathological and genetic findings in a group of patients with a previously not described phenotype of congenital myopathy due to recessive mutations in the gene encoding the type 1 muscle ryanodine receptor channel (RYR1). Seven unrelated patients shared a predominant axial and proximal weakness of varying severity, with onset during the neonatal period, associated with bilateral ptosis and ophthalmoparesis, and unusual muscle biopsy features at light and electron microscopic levels. Muscle biopsy histochemistry revealed a peculiar morphological pattern characterized by numerous internalized myonuclei in up to 51% of fibres and large areas of myofibrillar disorganization with undefined borders. Ultrastructurally, such areas frequently occupied the whole myofibre cross section and extended to a moderate number of sarcomeres in length. Molecular genetic investigations identified recessive mutations in the ryanodine receptor (RYR1) gene in six compound heterozygous patients and one homozygous patient. Nine mutations are novel and four have already been reported either as pathogenic recessive mutations or as changes affecting a residue associated with dominant malignant hyperthermia susceptibility. Only two mutations were located in the C-terminal transmembrane domain whereas the others were distributed throughout the cytoplasmic region of RyR1. Our data enlarge the spectrum of RYR1 mutations and highlight their clinical and morphological heterogeneity. A congenital myopathy featuring ptosis and external ophthalmoplegia, concomitant with the novel histopathological phenotype showing fibres with large, poorly delimited areas of myofibrillar disorganization and internal nuclei, is highly suggestive of an RYR1-related congenital myopathy.

G.P.1.02 Phenotypic spectrum of core-rod myopathy caused by dominant or recessive RYR1 mutations

G.P.1.01 Phenotypic variations of central core disease O. Paciello , A. Tammaro , L. Passamano , M. Scutifero , E. Picillo , A. Di Martino , S. Papparella , L. Politano University of Naples Federico II, Department of Veterinary Pathology, Naples, Italy, Cardarelli Hospital, Biotechnology Center for Malignant Hyperthermia, Naples, Italy, 3 Second University of Naples, Exp. Medicine – Cardiomyology and Medical Genetics, Naples, Italy, University of Naples Federico II, Department of Veterinary Myology, Naples, Italy

Molecular mechanisms and phenotypic variation in RYR1-related congenital myopathies

Dominant mutations in the skeletal muscle ryanodine receptor (RYR1) gene are well-recognized causes of both malignant hyperthermia susceptibility (MHS) and central core disease (CCD). More recently, recessive RYR1 mutations have been described in few congenital myopathy patients with variable pathology, including multi-minicores. Although a clinical overlap between patients with dominant and recessive RYR1 mutations exists, in most cases with recessive mutations the pattern of muscle weakness is remarkably different from that observed in dominant CCD. In order to characterize the spectrum of congenital myopathies associated with RYR1 mutations, we have investigated a cohort of 44 patients from 28 families with clinical and/or histopathological features suggestive of RYR1 involvement. We have identified 25 RYR1 mutations, 9 of them novel, including 12 dominant and 13 recessive mutations. With only one exception, dominant mutations were associated with a CCD phenotype, prominent cores and predominantly occurred in the RYR1 C-terminal exons 101 and 102. In contrast, the 13 recessive RYR1 mutations were distributed evenly along the entire RYR1 gene and were associated with a wide range of clinico-pathological phenotypes. Protein expression studies in nine cases suggested a correlation between specific mutations, RyR1 protein levels and resulting phenotype: in particular, whilst patients with dominant or recessive mutations associated with typical CCD phenotypes appeared to have normal RyR1 expression, individuals with more generalized weakness, multi-minicores and external ophthalmoplegia had a pronounced depletion of the RyR1 protein. The phenomenon of protein depletion was observed in some patients compound heterozygous for recessive mutations at the genomic level and silenced another allele in skeletal muscle, providing additional information on the mechanism of disease in these patients. Our data represent the most extensive study of RYR1-related myopathies and indicate complex genotype-phenotype correlations associated with mutations differentially affecting assembly and function of the RyR1 calcium release channel.

Characterization of recessive RYR1 mutations in core myopathies

Characterization of recessive RYR1 mutations in core myopathies

Epigenetic Allele Silencing Unveils Recessive RYR1 Mutations in Core Myopathies

Epigenetic regulation of gene expression is a source of genetic variation, which can mimic recessive mutations by creating transcriptional haploinsufficiency. Germline epimutations and genomic imprinting are typical examples, although their existence can be difficult to reveal. Genomic imprinting can be tissue specific, with biallelic expression in some tissues and monoallelic expression in others or with polymorphic expression in the general population. Mutations in the skeletal-muscle ryanodine-receptor gene (RYR1) are associated with malignant hyperthermia susceptibility and the congenital myopathies central core disease and multiminicore disease. RYR1 has never been thought to be affected by epigenetic regulation. However, during the RYR1-mutation analysis of a cohort of patients with recessive core myopathies, we discovered that 6 (55%) of 11 patients had monoallelic RYR1 transcription in skeletal muscle, despite being heterozygous at the genomic level. In families for which parental DNA was available, segregation studies showed that the nonexpressed allele was maternally inherited. Transcription analysis in patients' fibroblasts and lymphoblastoid cell lines indicated biallelic expression, which suggests tissue-specific silencing. Transcription analysis of normal human fetal tissues showed that RYR1 was monoallelically expressed in skeletal and smooth muscles, brain, and eye in 10% of cases. In contrast, 25 normal adult human skeletal-muscle samples displayed only biallelic expression. Finally, the administration of the DNA methyltransferase inhibitor 5-aza-deoxycytidine to cultured patient skeletal-muscle myoblasts reactivated the transcription of the silenced allele, which suggests hypermethylation as a mechanism for RYR1 silencing. Our data indicate that RYR1 undergoes polymorphic, tissue-specific, and developmentally regulated allele silencing and that this unveils recessive mutations in patients with core myopathies. Furthermore, our data suggest that imprinting is a likely mechanism for this phenomenon and that similar mechanisms could play a role in human phenotypic heterogeneity.

Characterization of recessive RYR1 mutations in core myopathies

We have characterized at the molecular level, three families with core myopathies carrying apparent recessive mutations in their RYR1 gene and studied the pharmacological properties of myotubes carrying endogenous mutations as well as the properties of mutant channels expressed in HEK293 cells. The proband of family 1 carried p.Ala1577Thr+p.Gly2060Cys in trans, having inherited a mutation from each parent. Immunoblot analysis of proteins from the patient's skeletal muscle revealed low levels of ryanodine receptor (RyR1) but neither substitution alone or in combination affected the functional properties of RyR1 channels in a discernable way. Two affected siblings in family 2 carried p.Arg109Trp+p.Met485Val substitutions in cis, inherited from the unaffected father. Interestingly, both affected siblings only transcribed the mutated paternal allele in skeletal muscle, whereas the maternal allele was silent. Single-channel measurements showed that recombinant, mutant RyR1 channels carrying both substitutions lost the ability to conduct Ca2+. In this case as well, low levels of RyR1 were present in skeletal muscle extracts. The proband of family 3 carried p.Ser71Tyr+p.Asn2283His substitutions in trans. Recombinant channels with Asn2283His substitution showed an increased activity, whereas recombinant channels with p.Ser71Tyr+p.Asn2283His substitution lost activity upon isolation. Taken together, our data suggest major differences in the ways RYR1 mutations may affect patients with core myopathies, by compromising RyR1 protein expression, stability and/or activity.

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands