Central Core Disease Research Articles

Congenital Myopathy Due to RyR1 Gene Mutation in a Newborn Masquerading as a Consequence of Hypoxic-ischemic Encephalopathy

Background: Congenital myopathies may be a cause of prolonged and persistent hypotonia and weakness in a newborn, which may be overlooked as a neurological consequence of hypoxic-ischemic encephalopathy. We report such a neonate which turned out to be a case of central core myopathy. Clinical Description: A female neonate born at 38 weeks of gestation suffered birth asphyxia and was referred at 25 days of life due to a persistent need for respiratory support. A review of history revealed that the mother had decreased fetal movements during the third trimester and polyhydramnios. On examination, the baby had a frog-like posture with a paucity of proximal movements with preserved distal movements. Considering the possibility of a congenital myopathy, investigations were carried out. The whole-exome sequencing revealed heterozygous nonsense variation in exon 20 of the RyR1 gene, thus diagnosing the infant as a case of congenital core myopathy. Management and Outcome: The baby was managed with supportive care including ventilatory and nutritional support. Baby remained ventilator dependent, but ultimately succumbed to complications by 2 months of age. Conclusions: A high index of suspicion is required in a newborn with birth asphyxia having unusually persistent hypotonia, thorough clinical examination and appropriate genetic testing can help in identifying an underlying congenital myopathy.

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.

Clinical features and genetic analysis of a child with Central core disease due to compound heterozygous variants of RYR1 gene

To explore the clinical features and genetic etiology of a child with Central core disease (CCD). A child with CCD who was treated at the Children's Hematology Department of the First Affiliated Hospital of Zhengzhou University in February 2022 was selected as the study subject. Muscle biopsy was performed. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA. The child was subjected to whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing. The child, a 12-year-old boy, had manifested motor retardation, facial weakness, ptosis, pectus carinatum, scoliosis, etc. Muscle biopsy showed that the central nucleus muscle fibers and atrophic muscle fibers were mainly type I. WES revealed that the child has harbored c.10561G>A (p.G3521S) and c.3448T>C (p.C1150R) compound heterozygous variants of the RYR1 gene. Sanger sequencing confirmed that they were inherited from his mother and father, respectively. Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were considered as likely pathogenic (PS4+PM1+PM2_Supporting+PP3；PM1+PM2_Supporting+PM3+PP3). By combining his clinical manifestation and results of muscle pathology and genetic testing, the child was diagnosed with CCD, which may be attributed to the c.10561G>A (p.G3521S) and c.3448T>C (p.C1150R) compound heterozygous variants of the RYR1 gene.

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.

Generation of two iPSC lines from patients with inherited central core disease and concurrent malignant hyperthermia caused by dominant missense variants in the RYR1 gene

RYR1 variants are the most common genetic cause of congenital myopathies, and typically cause central core disease (CCD) and/or malignant hyperthermia (MH). Here, we generated iPSC lines from two patients with CCD and MH caused by dominant RYR1 variants within the central region of the protein (p.Val2168Met and p.Arg2508Cys). Both lines displayed typical iPSC morphology, uniform expression of pluripotency markers, trilineage differentiation potential, and had normal karyotypes. These are the first RYR1 iPSC lines from patients with both CCD and MH. As these are common CCD/MH variants, these lines should be useful to study these conditions and test therapeutics.

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.

A brief history of the congenital myopathies – the myopathological perspective

Congenital myopathies are defined by early clinical onset, slow progression, hereditary nature and disease-specific myopathological lesions – however, with exceptions - demanding special techniques in regard to morphological diagnostic and research work-up. To identify an index disease in a family requires a muscle biopsy – and no congenital myopathy has ever been first described at autopsy. The nosographic history commenced when – in addition to special histopathological techniques in the earliest classical triad of central core disease, 1956, nemaline myopathy, 1963, and centronuclear myopathy, 1966/67, within a decade - electron microscopy and enzyme histochemistry were applied to unfixed frozen muscle tissue and, thus, revolutionized diagnostic and research myopathology. During the following years, the list of structure-defined congenital myopathies grew to some 40 conditions. Then, the introduction of immunohistochemistry allowed myopathological documentation of proteins and their abnormalities in individual congenital myopathies. Together with the diagnostic evolution of molecular genetics, many more congenital myopathies were described, without new disease-specific lesions or only already known ones. These were nosographically defined by individual mutations in hitherto congenital myopathies-unrelated genes. This latter development may also affect the nomenclature of congenital myopathies in that the mutant gene needs to be attached to the individually identified congenital myopathies with or without the disease-specific lesion, such as CCD-RYR1 or CM-RYR1. This principle is similar to that of the nomenclature of Congenital Disorders of Glycosylation. Retroactive molecular characterization of originally and first described congenital myopathies has only rarely been achieved.

Pancreatitis in RYR1-related disorders

Mutations in RYR1 encoding the ryanodine receptor (RyR) skeletal muscle isoform (RyR1) are a common cause of inherited neuromuscular disorders. Despite its expression in a wide range of tissues, non-skeletal muscle manifestations associated with RYR1 mutations have only been rarely reported. Here, we report three patients with a diagnosis of Central Core Disease (CCD), King-Denborough Syndrome (KDS) and Malignant Hyperthermia Susceptibility (MHS), respectively, who in addition to their (putative) RYR1-related disorder also developed symptoms and signs of acute pancreatitis. In two patients, episodes were recurrent, with severe multisystem involvement and sequelae. RyR1-mediated calcium signalling plays an important role in normal pancreatic function but has also been critically implicated in the pathophysiology of acute pancreatitis, particularly in bile acid- and ethanol-induced forms. Findings from relevant animal models indicate that pancreatic damage in these conditions may be ameliorated through administration of the specific RyR1 antagonist dantrolene and other compounds modifying pancreatic metabolism including calcium signalling. These observations suggest that patients with RYR1 gain-of-function variants may be at increased risk of developing acute pancreatitis, a condition which should therefore be considered in the health surveillance of such individuals.

RYR1 myopathy complicated by RSV bronchiolitis requiring intubation leading to posthypoxic leukoencephalopathy in a 4 year-old.

Central core disease due to RYR1 mutations is a rare heterogeneous myopathy characterized by skeletal muscle weakness. In light of both the rarity of presentation as well as the relatively broad spectrum of clinical phenotypes, there is a need to report treatment strategies for common complications of this condition. In this case, we outline the ICU management of a 4 year-old girl with central core disease caused by RYR1 mutation who was hospitalized due to respiratory syncytial virus (RSV) bronchiolitis leading to respiratory failure. Her hospital stay was complicated by multiple failed extubations, hospital infections, and post-anoxic leukoencephalopathy.

Challenging Neuromuscular Disease Cases.

The diagnosis of neuromuscular disorders requires a thorough history including family history and examination, with the next steps broadened now beyond electromyography and neuropathology to include genetic testing. The challenge in diagnosis can often be putting all the information together. With advances in genetic testing, some diagnoses that adult patients may have received as children deserve a second look and may result in diagnoses better defined or alternative diagnoses made. Clearly defining or redefining a diagnosis can result in understanding of potential other systems involved, prognosis, or potential treatments. This article presents several cases and approach to diagnosis as well as potential treatment and prognostic concerns, including seipinopathy, congenital myasthenic syndrome, central core myopathy, and myotonic dystrophy type 2.

Loss-of-rescue of Ryr1I4895T-related pathology by the genetic inhibition of the ER stress response mediator CHOP

RYR1 is the gene encoding the ryanodine receptor 1, a calcium release channel of the endo/sarcoplasmic reticulum. I4898T in RYR1 is one of the most common mutations that give rise to central core disease (CCD), with a variable phenotype ranging from mild to severe myopathy to lethal early-onset core-rod myopathy. Mice with the corresponding I4895T mutation in Ryr1 present mild myopathy when the mutation is heterozygous while I4895T homozygous is perinatal-lethal. Here we show that skeletal muscles of I4895T homozygous mice at birth present signs of stress of the endoplasmic reticulum (ER stress) and of the related unfolded protein response (UPR) with increased levels of the maladaptive mediators CHOP and ERO1. To gain information on the role of CHOP in the pathogenesis of RYR1I4895T-related myopathy, we generated compound Ryr1I4895T, Chop knock-out (-/-) mice. However, the genetic deletion of Chop, although it attenuates ER stress in the skeletal muscle of the newborns, does not rescue any phenotypic or functional features of Ryr1I4895T in mice: neither the perinatal-lethal phenotype nor the inability of Ryr1I4895T to respond to its agonist caffeine, but protects from ER stress-induced apoptosis. These findings suggest that genetic deletion of the ER stress response mediator CHOP is not sufficient to counteract the pathological Ryr1I4895T phenotype.

VP.04 Ryanodine receptor - related disorders

The ryanodine receptors (RyR1) is the main sarcoplasmic reticulum Ca²⁺ channel in skeletal muscle and acts as a connecting link between electrical stimulation and Ca²⁺-dependent muscle contraction. <i>RYR1</i> is expressed predominantly in fast and slow-twitch skeletal muscle and also in the esophagus and in cerebellar Purkinje cells in the brain. <i>RYR2</i> is the predominant isoform in cardiac muscle and brain. Its expression in the brain, brain stem, and spinal cord is widespread, but it is absent from the pituitary. <i>RYR3</i> is differentially expressed in the brain. Abnormal RyR1 activity compromises normal muscle function. RYR1-associated myopathies have been described as rare, histopathologically and clinically heterogeneous, and slowly progressive neuromuscular disorders. <i>RYR1</i> variants can lead to dysfunctional RyR1-mediated calcium release, malignant hyperthermia susceptibility, elevated oxidative stress, deleterious post-translational modifications, and decreased RyR1 expression. <i>RYR1</i> mutations cause disorders such as malignant hyperthermia, central core disease, congenital fibre type disproportion and centronuclear myopathy. However, <i>RYR1</i> is one of the largest genes of the human genome and accumulates numerous missense variants of uncertain significance (VUS), precluding an efficient molecular diagnosis for many patients and families. Here we describe several <i>RYR</i> mutations and correlate them to the diseases. We also validated large deletion in ryanodine receptor 1 (<i>RYR1</i>) using droplet digital PCR.

P.11 Clinical, pathological, imaging, and genetic characterization in a Taiwanese cohort with congenital myopathy

Congenital myopathy (CMP) is a genetically heterogeneous, hereditary disease entity characterized by clinically infantile hypotonia with facial weakness and histologically specific changes. The disease severity ranges from neonatal onset with respiratory failure to mild weakness with stationary course. This study aims to analyze the phenotypes and genotypes in Taiwanese patients with CMP in a referral center for neuromuscular diseases (NMDs). We enrolled 89 patients clinically suspected of having CMP who underwent muscle biopsy with subsequent genetic analysis in the past 15 years. Based on pathological features, we categorized the patients into nemaline myopathy, central core disease, multiminicore disease, congenital myopathy with uniform type 1, centronuclear myopathy, congenital fiber type disproportion, neuropathic change and others, respectively. Subsequent sequencing of target genes or panel for NMDs done in 65 patients and identified mutations in <i>ACTA1, NEB, KLHL40, RYR1, SEPN1, MEGF10, DNM2, MTM1, TPM2</i> and other genes unrelated to CMP. Our study revealed that RYR1-related myopathy counted for the largest proportion of CMP in Taiwan. As the causative genes of CMP are often big and benign polymorphism might be frequently identified, muscle pathology still plays an important role to support the final diagnosis. The domestic data could be useful by joining global registry for nature history study and future clinical trials.

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

Ryanodine receptors (RyRs) are main regulators of intracellular Ca2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca2+, increasing by ∼20-fold the potency of Ca2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

Malignant Hyperthermia and Absence of Ryanodine Receptors in a Child Presenting for Ptosis Repair

Introduction: Malignant hyperthermia (MH) is a hypermetabolic condition caused by a genetic cell membranes channel disruption leading to increased calcium release from the sarcoplasmic reticulum after exposure to triggering agents. Central Core Disease (CCD) is a rare inherited neuromuscular disorder with a wide spectrum of phenotypic presentations. Clinically, there is muscle weakness of variable degree and histopathologically there is evidence of core formation in the muscle fibers. Additional features may occur, including skeletal abnormalities such as foot deformities, scoliosis, or hip displacement. Association of CCD with a mutation of the ryanodine receptor RYR1 and MH is high. If not properly treated, MH could be fatal. Purpose: This study aims towards increasing the awareness of pediatricians, surgeons and anesthesiologists about possible occurrence of MH in patients presenting with only mild “clinical” signs of congenital myopathies such as CCD. Careful diagnostic investigations should be performed prior to surgical interventions and extreme caution during anesthesia. Material and Methods: We report the case of a 3 years old girl, with a history of ptosis, scoliosis, and good muscle tone, who developed unexpected MH during the surgical repair of her ptosis. The critically ill child was transferred to Pediatric Intensive Care Unit (PICU) for management of her MH. The management was done following the guidelines of the European Malignant Hyperthermia Group published in the British Journal of Anesthesia 2010. The patient responded well to treatment. She had a muscle biopsy done after recovery. Discussion: Early management and dantrolene administration are the most important factors to minimize MH morbidity and mortality. MH susceptibility is known in patients carrying mutations in the RYR1 receptors. In our case, RYR1 staining was negative on the muscle biopsy specimen suggesting abnormal or absent RYR1 function, with the presence of occasional cores. These findings, in addition to the ophthalmological and orthopedic history were diagnostic of CCD. In Vitro Contracture Testing (IVCT) was not done because it was not available. Genetic testing was refused by the parents due to financial reasons. Conclusion: In patients presenting with symptoms suggestive of muscle weakness, such as ptosis and scoliosis, and variable muscle tone; congenital myopathies should be considered. CCD and RYR1 receptors deficiency or mutation, if present, may lead to a possible risk of fatal MH, which can be prevented.

Clinical characteristics and genetic analysis of a pedigree affected with neonatal central core disease

To investigate the possible causative factors of central core disease(CCD), the clinical features of a neonatal case with CCD and five patients in the pedigree line were analyzed for RYR1 gene variant. Medical and family history inquiries and detailed clinical examinations were performed in the proband. High-throughput sequencing technology was applied to analyze the gene variant of the proband, and Sanger sequencing was applied to verify the pedigree distribution of the variant. The whole exon sequencing results showed that the proband has a missense variant of c. 14591A>C (p.Tyr4864Ser) in the RYR1 gene which was unreported previously; Sanger sequencing results showed that the father, grandfather, the eldest aunt and second aunt of the proband all carried the same variant. The c.14591 A>C variant of RYR1 gene was predicted to be a likely pathogenic (PM2+PM5+PP1+PP3) according to the American College of Medical Genetics and Genomics standards and guidelines. The RYR1 gene c.14591A>C (p.Tyr4864Ser) variant may be the genetic cause of the pedigree and genetic testing helps to clarify the diagnosis. Identification of this variant has enriched the variant spectrum of the RYR1 gene.

A review of major causative genes in congenital myopathies.

In this review, we focus on congenital myopathies, which are a genetically heterogeneous group of hereditary muscle diseases with slow or minimal progression. They are mainly defined and classified according to pathological features, with the major subtypes being core myopathy (central core disease), nemaline myopathy, myotubular/centronuclear myopathy, and congenital fiber-type disproportion myopathy. Recent advances in molecular genetics, especially next-generation sequencing technology, have rapidly increased the number of known causative genes for congenital myopathies; however, most of the diseases related to the novel causative genes are extremely rare. There remains no cure for congenital myopathies. However, there have been recent promising findings that could inform the development of therapy for several types of congenital myopathies, including myotubular myopathy, which indicates the importance of prompt and correct diagnosis. This review discusses the major causative genes (NEB, ACTA1, ADSSL1, RYR1, SELENON, MTM1, DNM2, and TPM3) for each subtype of congenital myopathies and the relevant latest findings.

Structural Insight Into Ryanodine Receptor Channelopathies.

The ryanodine receptors (RyRs) are large cation-selective ligand-gated channels that are expressed in the sarcoplasmic reticulum (SR) membrane. They mediate the controlled release of Ca2+ from SR and play an important role in many cellular processes. The mutations in RyRs are associated with several skeletal muscle and cardiac conditions, including malignant hyperthermia (MH), central core disease (CCD), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia (ARVD). Recent breakthroughs in structural biology including cryo-electron microscopy (EM) and X-ray crystallography allowed the determination of a number of near-atomic structures of RyRs, including wildtype and mutant structures as well as the structures in complex with different modulating molecules. This allows us to comprehend the physiological gating and regulatory mechanisms of RyRs and the underlying pathological mechanisms of the disease-causing mutations. In this review, based on the insights gained from the available high-resolution structures of RyRs, we address several questions: 1) what are the gating mechanisms of different RyR isoforms; 2) how RyRs are regulated by multiple channel modulators, including ions, small molecules, and regulatory proteins; 3) how do disease-causing mutations affect the structure and function of RyRs; 4) how can these structural information aid in the diagnosis of the related diseases and the development of pharmacological therapies.

Congenital myopathies: The current status.

Within the history of neuromuscular diseases (NMD), congenital myopathies (CM) represent a relatively new category introduced in the mid-nineteen hundreds upon advent and subsequent application of enzyme histochemistry and electron microscopy by establishing the three major CM, central core disease, nemaline myopathy, and centronuclear myopathy which later pluralized each when the molecular era began at the end of last century. Quickly, during the following 5 decades, many new CM entities were described, based on muscle biopsies and their CM-characteristic myopathology, the former a prerequisite to recognizing an individual CM, the latter of the nosological hallmark of the individual CM. When the molecular era ushered in immunohistochemistry the spectrum and nosography of CM altered in that some CM became allelic to other cohorts of NMD, e.g., congenital muscular dystrophies, other muscular dystrophies, distal myopathies based on different or identical mutations in the same gene. The nosological spectrum of a defective gene also enlarged by recognizing several entities with mutations in the same gene, and same or similar nosological conditions originated from mutations in different genes. Lately, however, CM were reported which lacked any individual myopathological hallmarks, but were clearly based on molecular defects, a fair number of them being newly identified ones. Few CM still remain without any molecular clarification. This nosographic development rendered the original definition of such new CM questionable and brought uncertainty to their classification and nomenclature.

Central Core Disease: Facial Weakness Differentiating Biallelic from Monoallelic Forms.

Central Core Disease (CCD) is a genetic neuromuscular disorder characterized by the presence of cores in muscle biopsy. The inheritance has been described as predominantly autosomal dominant (AD), and the disease may present as severe neonatal or mild adult forms. Here we report clinical and molecular data on a large cohort of Brazilian CCD patients, including a retrospective clinical analysis and molecular screening for RYR1 variants using Next-Generation Sequencing (NGS). We analyzed 27 patients from 19 unrelated families: four families (11 patients) with autosomal dominant inheritance (AD), two families (3 patients) with autosomal recessive (AR), and 13 sporadic cases. Biallelic RYR1 variants were found in six families (two AR and four sporadic cases) of the 14 molecularly analyzed families (~43%), suggesting a higher frequency of AR inheritance than expected. None of these cases presented a severe phenotype. Facial weakness was more common in biallelic than in monoallelic patients (p = 0.0043) and might be a marker for AR forms. NGS is highly effective for the identification of RYR1 variants in CCD patients, allowing the discovery of a higher proportion of AR cases with biallelic mutations. These data have important implications for the genetic counseling of the families.