RYR1-related Myopathies Research Articles

Compound heterozygous RYR1-RM mouse model reveals disease pathomechanisms and muscle adaptations to promote postnatal survival.

Pathogenic variants in the type I ryanodine receptor (RYR1) result in a wide range of muscle disorders referred to as RYR1-related myopathies (RYR1-RM). We developed the first RYR1-RM mouse model resulting from co-inheritance of two different RYR1 missense alleles (Ryr1TM/SC-ΔL mice). Ryr1TM/SC-ΔL mice exhibit a severe, early onset myopathy characterized by decreased body/muscle mass, muscle weakness, hypotrophy, reduced RYR1 expression, and unexpectedly, incomplete postnatal lethality with a plateau survival of ~50% at 12 weeks of age. Ryr1TM/SC-ΔL mice display reduced respiratory function, locomotor activity, and invivo muscle strength. Extensor digitorum longus muscles from Ryr1TM/SC-ΔL mice exhibit decreased cross-sectional area of type IIb and type IIx fibers, as well as a reduction in number of type IIb fibers. Exvivo functional analyses revealed reduced Ca2+ release and specific force production during electrically-evoked twitch stimulation. In spite of a ~threefold reduction in RYR1 expression in single muscle fibers from Ryr1TM/SC-ΔL mice at 4 weeks and 12 weeks of age, RYR1 Ca2+ leak was not different from that of fibers from control mice at either age. Proteomic analyses revealed alterations in protein synthesis, folding, and degradation pathways in the muscle of 4- and 12-week-old Ryr1TM/SC-ΔL mice, while proteins involved in the extracellular matrix, dystrophin-associated glycoprotein complex, and fatty acid metabolism were upregulated in Ryr1TM/SC-ΔL mice that survive to 12 weeks of age. These findings suggest that adaptations that optimize RYR1 expression/Ca2+ leak balance, sarcolemmal stability, and fatty acid biosynthesis provide Ryr1TM/SC-ΔL mice with an increased survival advantage during postnatal development.

04P Characterising patient iPSC-derived models of RYR1-related myopathies

04P Characterising patient iPSC-derived models of RYR1-related myopathies

Structural changes and contractility in muscle assessed by magnetic resonance imaging in individuals with ryanodine receptor 1-related rhabdomyolysis or myalgia.

Ryanodine receptor 1 (RYR1)-related myopathies associated with variants in the RYR1 gene present with a wide range of symptoms and severity. Two of the milder phenotypes associated with dominant pathogenic variants in RYR1 are rhabdomyolysis and myalgia. Only a few studies have investigated the muscle function and structure of individuals with RYR1-related rhabdomyolysis/myalgia objectively, showing inconsistent results. This study aimed to describe structural changes and contractility of muscles in individuals with RYR1-related rhabdomyolysis/myalgia. We investigated 15 individuals with dominant variants in the RYR1-gene and compared them with 15 age-, sex-, and body mass index (BMI)-matched controls using MRI, stationary isokinetic dynamometry, and comprehensive clinical evaluation. No significant differences were found between individuals with RYR1-related rhabdomyolysis/myalgia and healthy controls in peak torque, fat fraction, cross-sectional area, contractile cross-sectional area, or contractility (p > .05) in muscles of the lower back (MRI data only), thigh, or calf. On clinical examination, three individuals exhibited weakness in hip or back extension on the Medical Research Council (MRC) test and eight had muscle hypertrophy. Individuals with weakness were not hypertrophic. Most individuals with RYR1-related rhabdomyolysis/myalgia have close to normal strength, and normal fat fraction and contractility of muscles, and therefore constitute a mild phenotype of RYR1-related myopathies.

Update on RYR1-related myopathies.

RYR1-related myopathy (RYR1-RM) is a group of myopathies caused by mutations in the RYR1 gene, which encodes the ryanodine receptor 1 (RYR1). This review discusses recent advances in the clinical features, pathology, pathogenesis, and therapeutics of RYR1-RM. Although treatments such as salbutamol, pyridostigmine, and N-acetylcysteine have been explored as potential therapies for RYR1-RM, none have been conclusively proven to be effective. However, recent clinical trials of Rycal ARM210 in patients with RYR1-RM have shown promising results, including reduced fatigue and improved proximal muscle strength.Recent advances in three-dimensional structural analysis of RYR1 channels, facilitated by cryo-electron microscopy (cryo-EM), have elucidated the distinct molecular mechanisms underlying RYR1 functionality. Additionally, high-throughput screening methods, including FRET-based and endoplasmic reticulum Ca 2+ -based assays, have been successful in identifying potential candidates for the treatment of RYR1-RM. Recent advances in clinical and pathological understanding have provided new insights into RYR1-RM. Novel pathomechanisms elucidated by cryo-EM and rapid screening methods have led to the identification of several promising drug candidates. We are hopeful about the potential of Rycal, other new drugs, and gene therapy, offering a promising outlook for the future.

Functional benefit of CRISPR-Cas9-induced allele deletion for RYR1 dominant mutation

More than 700 pathogenic or probably pathogenic variations have been identified in the RYR1 gene causing various myopathies collectively known as "RYR1-related myopathies”. There is no treatment for these myopathies, and gene therapy stands out as one of the most promising approaches. In the context of a dominant form of Central Core Disease due to a RYR1 mutation, we aimed at showing the functional benefit of inactivating specifically the mutated RYR1 allele by guiding CRISPR/Cas9 cleavages onto frequent single nucleotide polymorphisms (SNPs) segregating on the same chromosome. Whole-genome sequencing was used to pinpoint SNPs localized on the mutant RYR1 allele and identified specific CRISPR/Cas9 guide-RNAs. Lentiviruses encoding these guide-RNAs and the SpCas9 nuclease were used to transduce immortalized patient myoblasts, inducing the specific deletion of the mutant RYR1 allele. The efficiency of the deletion was assessed at DNA and RNA levels, and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study provides in-cellulo proof of concept regarding the benefits of mutant RYR1 allele deletion, in the case of a dominant RYR1 mutation, from both a molecular and functional perspective, and could apply potentially to 20% of all patients with a RYR1 mutation.

Unusual cause of muscle weakness, type II respiratory failure and pulmonary hypertension: a case report of ryanodine receptor type 1(RYR1)-related myopathy

BackgroundPatients with congenital myopathies may experience respiratory involvement, resulting in restrictive ventilatory dysfunction and respiratory failure. Pulmonary hypertension (PH) associated with this condition has never been reported in congenital ryanodine receptor type 1(RYR1)-related myopathy.Case presentation A 47-year-old woman was admitted with progressively exacerbated chest tightness and difficulty in neck flexion. She was born prematurely at week 28. Her bilateral lower extremities were edematous and muscle strength was grade IV−. Arterial blood gas analysis revealed hypoventilation syndrome and type II respiratory failure, while lung function test showed restrictive ventilation dysfunction, which were both worse in the supine position. PH was confirmed by right heart catheterization (RHC), without evidence of left heart disease, congenital heart disease, or pulmonary artery obstruction. Polysomnography indicated nocturnal hypoventilation. The ultrasound revealed reduced mobility of bilateral diaphragm. The level of creatine kinase was mildly elevated. Magnetic resonance imaging showed myositis of bilateral thigh muscle. Muscle biopsy of the left biceps brachii suggested muscle malnutrition and congenital muscle disease. Gene testing revealed a missense mutation in the RYR1 gene (exon33 c.C4816T). Finally, she was diagnosed with RYR1-related myopathy and received long-term non-invasive ventilation (NIV) treatment. Her symptoms and cardiopulmonary function have been greatly improved after 10 months.ConclusionsWe report a case of RYR1-related myopathy exhibiting hypoventilation syndrome, type II respiratory failure and PH associated with restrictive ventilator dysfunction. Pulmonologists should keep congenital myopathies in mind in the differential diagnosis of type II respiratory failure, especially in patients with short stature and muscle weakness.

Generation of two iPSC lines from adult central core disease patients with dominant missense variants in the RYR1 gene

RYR1 variants are a common cause of congenital myopathies, including multi-minicore disease (MmD) and central core disease (CCD). Here, we generated iPSC lines from two CCD patients with dominant RYR1 missense variants that affect the transmembrane (pore) and SPRY3 protein domains (p.His4813Tyr and p.Asn1346Lys, respectively). Both lines had typical iPSC morphology, expressed canonical pluripotency markers, exhibited trilineage differentiation potential, and had normal karyotypes. Together with existing RYR1 iPSC lines, these represent important tools to study and develop treatments for RYR1-related myopathies.

Successful Correction by Prime Editing of a Mutation in the RYR1 Gene Responsible for a Myopathy.

We report the first correction from prime editing a mutation in the RYR1 gene, paving the way to gene therapies for RYR1-related myopathies. The RYR1 gene codes for a calcium channel named Ryanodine receptor 1, which is expressed in skeletal muscle fibers. The failure of this channel causes muscle weakness in patients, which leads to motor disabilities. Currently, there are no effective treatments for these diseases, which are mainly caused by point mutations. Prime editing allows for the modification of precise nucleotides in the DNA. Our results showed a 59% correction rate of the T4709M mutation in the RYR1 gene in human myoblasts by RNA delivery of the prime editing components. It is to be noted that T4709M is recessive and, thus, persons having a heterozygous mutation are healthy. These results are the first demonstration that correcting mutations in the RYR1 gene is possible.

Generation of two induced pluripotent stem cell lines from a 33-year-old central core disease patient with a heterozygous dominant c.14145_14156delCTACTGGGACA (p.Asn4715_Asp4718del) deletion in the RYR1 gene

Central core disease (CCD) is a congenital disorder that results in hypotonia, delayed motor development, and areas of reduced oxidative activity in the muscle fibre. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 33-year-old male with CCD, caused by a previously unreported dominant c.14145_14156delCTACTGGGACA (p.Asn4715_Asp4718del) deletion in the RYR1 gene. Both lines demonstrated typical morphology, pluripotency, trilineage differentiation, and had a normal karyotype. As the first published iPSC model of CCD caused by an RYR1 variant these lines are a potential resource for further investigation of RYR1-related myopathies in a human context.

Ryanodine receptor type 1 content decrease-induced endoplasmic reticulum stress is a hallmark of myopathies.

Decreased ryanodine receptor type 1 (RyR1) protein levels are a well-described feature of recessive RYR1-related myopathies. The aim of the present study was twofold: (1) to determine whether RyR1 content is also decreased in other myopathies and (2) to investigate the mechanisms by which decreased RyR1 protein triggers muscular disorders. We used publicly available datasets, muscles from human inflammatory and mitochondrial myopathies, an inducible muscle-specific RYR1 recessive mouse model and RyR1 knockdown in C2C12 muscle cells to measure RyR1 content and endoplasmic reticulum (ER) stress markers. Proteomics, lipidomics, molecular biology and transmission electron microscopy approaches were used to decipher the alterations associated with the reduction of RyR1 protein levels. RYR1 transcripts were reduced in muscle samples of patients suffering from necrotizing myopathy (P=0.026), inclusion body myopathy (P=0.003), polymyositis (P<0.001) and juvenile dermatomyositis (P<0.001) and in muscle samples of myotonic dystrophy type 2 (P<0.001), presymptomatic (P<0.001) and symptomatic (P<0.001) Duchenne muscular dystrophy, Becker muscular dystrophy (P=0.004) and limb-girdle muscular dystrophy type 2A (P=0.004). RyR1 protein content was also significantly decreased in inflammatory myopathy (-75%, P<0.001) and mitochondrial myopathy (-71%, P<0.001) muscles. Proteomics data showed that depletion of RyR1 protein in C2C12 myoblasts leads to myotubes recapitulating the common molecular alterations observed in myopathies. Mechanistically, RyR1 protein depletion reduces ER-mitochondria contact length (-26%, P<0.001), Ca2+ transfer to mitochondria (-48%, P=0.002) and the mitophagy gene Parkinson protein 2 transcripts (P=0.037) and induces mitochondrial accumulation (+99%, P=0.005) and dysfunction (P<0.001). This was associated to the accumulation of deleterious sphingolipid species. Our data showed increased levels of the ER stress marker chaperone-binding protein/glucose regulated protein 78, GRP78-Bip, in RyR1 knockdown myotubes (+45%, P=0.046), in mouse RyR1 recessive muscles (+58%, P=0.001) and in human inflammatory (+96%, P=0.006) and mitochondrial (+64%, P=0.049) myopathy muscles. This was accompanied by increased protein levels of the pro-apoptotic protein CCAAT-enhancer-binding protein homologous protein, CHOP-DDIT3, in RyR1 knockdown myotubes (+27%, P<0.001), mouse RyR1 recessive muscles (+63%, P=0.009), human inflammatory (+50%, P=0.038) and mitochondrial (+51%, P=0.035) myopathy muscles. In publicly available datasets, the decrease in RYR1 content in myopathies was also associated to increased ER stress markers and RYR1 transcript levels are inversely correlated with ER stress markers in the control population. Decreased RyR1 is commonly observed in myopathies and associated to ER stress in vitro, in mouse muscle and in human myopathy muscles, suggesting a potent role of RyR1 depletion-induced ER stress in the pathogenesis of myopathies.

Orthognathic surgery in RYR1-related congenital myopathy: a patient report

Orthognathic surgery in RYR1-related congenital myopathy: a patient report

P416 Systemic NAD+ deficiency reveals a potential therapeutic target for RYR1-related myopathies

P416 Systemic NAD+ deficiency reveals a potential therapeutic target for RYR1-related myopathies

P415 Mitoquinol Mesylate and PUFA: an alternative therapeutic approach for RYR1-related myopathies

P415 Mitoquinol Mesylate and PUFA: an alternative therapeutic approach for RYR1-related myopathies

Long-term Natural History of Pediatric Dominant and Recessive RYR1-Related Myopathy.

RYR1-related myopathies are the most common congenital myopathies, but long-term natural history data are still scarce. We aim to describe the natural history of dominant and recessive RYR1-related myopathies. A cross-sectional and longitudinal retrospective data analysis of pediatric cases with RYR1-related myopathies seen between 1992-2019 in 2 large UK centers. Patients were identified, and data were collected from individual medical records. Sixty-nine patients were included in the study, 63 in both cross-sectional and longitudinal studies and 6 in the cross-sectional analysis only. Onset ranged from birth to 7 years. Twenty-nine patients had an autosomal dominant RYR1-related myopathy, 31 recessive, 6 de novo dominant, and 3 uncertain inheritance. Median age at the first and last appointment was 4.0 and 10.8 years, respectively. Fifteen% of patients older than 2 years never walked (5 recessive, 4 de novo dominant, and 1 dominant patient) and 7% lost ambulation during follow-up. Scoliosis and spinal rigidity were present in 30% and 17% of patients, respectively. Respiratory involvement was observed in 22% of patients, and 12% needed ventilatory support from a median age of 7 years. Feeding difficulties were present in 30% of patients, and 57% of those needed gastrostomy or tube feeding. There were no anesthetic-induced malignant hyperthermia episodes reported in this cohort. We observed a higher prevalence of prenatal/neonatal features in recessive patients, in particular hypotonia and respiratory difficulties. Clinical presentation, respiratory outcomes, and feeding outcomes were consistently more severe at presentation and in the recessive group. Conversely, longitudinal analysis suggested a less progressive course for motor and respiratory function in recessive patients. Annual change in forced vital capacity was -0.2%/year in recessive vs -1.4%/year in dominant patients. This clinical study provides long-term data on disease progression in RYR1-related myopathies that may inform management and provide essential milestones for future therapeutic interventions.

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

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

Early diagnosis of congenital muscular pathologies using next-generation sequencing: experiences from a tertiary center in Morocco

BackgroundCongenital muscular dystrophies (CMD) and congenital myopathies (CM) are clinically and genetically heterogeneous groups of neuromuscular disorders resulting in prenatal or early-onset hypotonia, muscle weakness, myogenic pattern, and dystrophic or myopathic features on muscle biopsy. In this study, we provide a genetic and molecular characterization of CMD and CM in Moroccan patients.Patients and methodsIn this cohort, we investigated 23 Moroccan patients from 21 families who consented to genetic testing. Firstly, genetic analysis in the probands was conducted by next-generation sequencing (NGS) technology using two approaches: targeted NGS gene panel and clinical exome sequencing to study the mutational spectrum and to achieve an accurate diagnosis of these hereditary myopathies in Morocco.ResultsNGS data analysis revealed 16 pathogenic variants harbored in 17 unrelated patients that were genetically resolved. The phenotypic forms identified were in order: LAMA2-related CMD (52.94%), LMNA-CMD (23.53%), and RYR1-related congenital myopathy (17.65%). The congenital titinopathy group was less frequent (5.88%). Here, we identified two novel recessive variants in LAMA2 gene: c.2164G > A (p.Glu722Lys), and c.(6992 + 1_6993-1)_(7300 + 1_7301-1)del p.(Pro2332Glnfs*10). Additionally, we expanded the phenotypic spectrum of a known heterozygous LMNA c.1718C > T p.(Ser573Leu) variant, and we report it for the first time to a form of CMD.ConclusionsThe introduction of the NGS tool in clinical practice allowed us to improve the diagnosis and the management of these neuromuscular diseases and to highlight the importance of molecular genetic diagnosis of these disorders that are underestimated in the Moroccan population.

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.

Gene therapy strategies using CRISPR/Cas9 for RyR1-related myopathies.

Type 1 ryanodine receptor (RyR1) is the intracellular calcium channel responsible for skeletal muscle contraction. Mutations in the RYR1 gene can lead to RyR1-related myopathies (RyR1-RM), a group of genetic disorders. They are characterised by abnormal calcium homeostasis and muscle weakness of variable severity. Currently, there is no treatment for these pathologies. This study aims to present proof of concept for gene therapy approaches. Immortalized skeletal muscle cells from patients affected by RyR1-Related myopathies have been edited using Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9). CRISPR guides targeting different part of the gene were design in order to adapt the strategy depending on the patient's mutation and its localisation. The consequences of the expected editing will be verified at the DNA, RNA and functional level. The objective is to determine if these therapeutic approaches could improve calcium release in patient's cells and skeletal muscle contraction.

Using Cluster Analysis to Overcome the Limits of Traditional Phenotype-Genotype Correlations: The Example of RYR1-Related Myopathies.

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype-phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype-phenotype correlations, we found clustering to overcome the limits of the "single-dimension" paradigm traditionally used to describe genotype-phenotype relationships.

Oral N-acetylcysteine Trial for RYR1-related Myopathies

Investigators from NIH, Hyperion Biotechnology Inc., and Hospital for Sick Children studied the effect of oral N-acetylcysteine (NAC) on decreasing oxidative stress and increasing physical endurance in individuals with ryanodine receptor 1-related myopathies (RYR1-RM).