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

OBJECTIVE: To explore the clinical features and genetic etiology of a child with Central core disease (CCD). METHODS: 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. RESULTS: 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). CONCLUSION: 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.

A case report of central core disease with repeated foaming at the mouth as the initial symptom.

BACKGROUND: Central core disease (CCD) is a congenital myopathy primarily observed in infants and children. It frequently manifests as limb weakness or delayed motor development, characterized by gradually progressing or non-worsening weakness and muscle atrophy primarily affecting the proximal limbs. Joint deformity is a prevalent clinical feature. Presently, there is no targeted treatment available for this condition. CASE DESCRIPTION: The infant, who was 42 days old, showed a repeated occurrence of foaming at the mouth for more than a month as the initial symptom. Initially, the local clinic misdiagnosed it as softening of the thyroid cartilage. However, when the infant underwent bronchoscopy at our hospital, it was discovered that the pharyngeal muscle was loose, and there was noticeable retraction of the base of the tongue. Additionally, the infant displayed evident hypotonia and an increase in creatine kinase levels. By conducting a thorough genetic examination, we confirmed that the infant had CCD. CONCLUSION: The onset of CCD may manifest as various symptoms. Medical practitioners need to be attentive in recognizing individuals who experience recurring pneumonia along with reduced muscle tone during the course of clinical diagnosis and treatment.

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.

A novel RYR1 variant in an infant with a unique fetal presentation of central core disease.

Ryanodine receptor type 1-related disorder (RYR1-RD) is the most common subgroup of congenital myopathies with a wide phenotypic spectrum ranging from mild hypotonia to lethal fetal akinesia. Genetic testing for myopathies is imperative as the diagnosis informs counseling regarding prognosis and recurrence risk, treatment options, monitoring, and clinical management. However, diagnostic challenges exist as current options are limited to clinical suspicion prompting testing including: single gene sequencing or familial variant testing, multi-gene panels, exome, genome sequencing, and invasive testing including muscle biopsy. The timing of diagnosis is of great importance due to the association of RYR1-RD with malignant hyperthermia (MH). MH is a hypermetabolic crisis that occurs secondary to excessive calcium release in muscles, leading to systemic effects that can progress to shock and death if unrecognized. Given the association of MH with pathogenic variants in RYR1, a diagnosis of RYR1-RD necessitates an awareness of medical team to avoid potentially triggering agents. We describe a case of a unique fetal presentation with bilateral diaphragmatic eventrations who had respiratory failure, dysmorphic facial features, and profound global hypotonia in the neonatal period. The diagnosis was made at several months of age, had direct implications on her clinical care related to anticipated need to long-term ventilator support, and ultimately death secondary an arrhythmia as a result of suspected MH. Our report reinforces the importance of having high suspicion for a genetic syndrome and pursuing early, rapid exome or genome sequencing as first line testing in critically ill neonatal intensive care unit patients and further evaluating the pathogenicity of a variant of uncertain significance in the setting of a myopathic phenotype.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

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.

Expanding the clinical-pathological and genetic spectrum of RYR1-related congenital myopathies with cores and minicores: an Italian population study.

Mutations in the RYR1 gene, encoding ryanodine receptor 1 (RyR1), are a well-known cause of Central Core Disease (CCD) and Multi-minicore Disease (MmD). We screened a cohort of 153 patients carrying an histopathological diagnosis of core myopathy (cores and minicores) for RYR1 mutation. At least one RYR1 mutation was identified in 69 of them and these patients were further studied. Clinical and histopathological features were collected. Clinical phenotype was highly heterogeneous ranging from asymptomatic or paucisymptomatic hyperCKemia to severe muscle weakness and skeletal deformity with loss of ambulation. Sixty-eight RYR1 mutations, generally missense, were identified, of which 16 were novel. The combined analysis of the clinical presentation, disease progression and the structural bioinformatic analyses of RYR1 allowed to associate some phenotypes to mutations in specific domains. In addition, this study highlighted the structural bioinformatics potential in the prediction of the pathogenicity of RYR1 mutations. Further improvement in the comprehension of genotype-phenotype relationship of core myopathies can be expected in the next future: the actual lack of the human RyR1 crystal structure paired with the presence of large intrinsically disordered regions in RyR1, and the frequent presence of more than one RYR1 mutation in core myopathy patients, require designing novel investigation strategies to completely address RyR1 mutation effect.

A review of core myopathy: central core disease, multiminicore disease, dusty core disease, and core-rod myopathy.

Core myopathies are clinically, pathologically, and genetically heterogeneous muscle diseases. Their onset and clinical severity are variable. Core myopathies are diagnosed by muscle biopsy showing focally reduced oxidative enzyme activity and can be pathologically divided into central core disease, multiminicore disease, dusty core disease, and core-rod myopathy. Although RYR1-related myopathy is the most common core myopathy, an increasing number of other causative genes have been reported, including SELENON, MYH2, MYH7, TTN, CCDC78, UNC45B, ACTN2, MEGF10, CFL2, KBTBD13, and TRIP4. Furthermore, the genes originally reported to cause nemaline myopathy, namely ACTA1, NEB, and TNNT1, have been recently associated with core-rod myopathy. Genetic analysis allows us to diagnose each core myopathy more accurately. In this review, we aim to provide up-to-date information about core myopathies.

A case of dermatomyositis in a patient with central core disease: unusual association with autoimmunity and genetic muscle disease.

BACKGROUND: Dermatomyositis is an inflammatory muscle disease caused by immune-mediated muscle injury, and central core disease (CCD) is a congenital myopathy associated with disturbed intracellular calcium homeostasis and excitation-contraction coupling. To date, CCD has not been reported to have autoantibodies or coexist with inflammatory myopathy. CASE PRESENTATION: Here, we described the case of a 25-year-old woman who had progressive proximal muscle weakness, myalgia, pruritic macular rash, skin ulcers, and calcinosis. Dermatomyositis was initially suspected based on the clinical symptoms accompanied by elevated muscle enzyme levels, electromyography abnormalities, and a positive antinuclear antibody test. However, the patient's muscle biopsy revealed the characteristic findings of both dermatomyositis and CCD, suggesting that dermatomyositis occurred in this patient with previously asymptomatic CCD. The patient did not have any pathogenic gene mutations associated with congenital myopathy, including RYR1 and SEPN1 in targeted next-generation sequencing. She received high-dose glucocorticoid therapy and azathioprine with a significant improvement in muscle strength. CONCLUSIONS: We present a case of rare coexistence of dermatomyositis and CCD. Clinicians should be aware that patients with CCD may have inflammatory myopathy that responds well to immunosuppressive therapy.

Clinical exome sequencing in the diagnosis of pediatric neuromuscular disease.

BACKGROUND: The diagnosis of uncommon pediatric neuromuscular disease (NMD) is challenging due to genetic and phenotypic heterogeneity, yet is important to guide treatment, prognosis, and recurrence risk. Patients with diagnostically challenging presentations typically undergo extensive testing with variable molecular diagnostic yield. Given the advancement in next generation sequencing (NGS), we investigated the value of clinical whole exome sequencing (ES) in uncommon pediatric NMD. METHODS: A retrospective cohort study of 106 pediatric NMD patients with a combination of ES, chromosomal microarray (CMA), and candidate gene testing was completed at a large tertiary referral center. RESULTS: A molecular diagnosis was achieved in 37/79 (46%) patients with ES, 4/44 (9%) patients with CMA, and 15/74 (20%) patients with candidate gene testing. In 2/79 (3%) patients, a dual molecular diagnosis explaining the neuromuscular disease process was identified. A total of 42 patients (53%) who received ES remained without a molecular diagnosis at the conclusion of the study. CONCLUSIONS: Due to NGS, molecular diagnostic yield of rare neurological diseases is at an all-time high. We show that ES has a higher diagnostic rate compared to other genetic tests in a complex pediatric neuromuscular disease cohort and should be considered early in the diagnostic journey for select NMD patients with challenging presentations in which a clinical diagnosis is not evident.

Evaluation of the Core Formation Process in Congenital Neuromuscular Disease With Uniform Type 1 Fiber and Central Core Disease.

Typical central core disease (CCD) is characterized pathologically by the presence of a core and is accompanied by type 1 fiber uniformity. Congenital neuromuscular disease with uniform type 1 fiber (CNMDU1) is characterized pathologically by the presence of type 1 fiber uniformity but without the abnormal structural changes in muscle fibers. Interestingly, typical CCD and 40% of CNMDU1 cases are caused by the same mutations in RYR1, and thus CNMDU1 has been considered an early precursor to CCD. To better understand the nature of CNMDU1, we re-evaluated muscle biopsies from 16 patients with CNMDU1 using immunohistochemistry to RYR1, triadin and TOM20, and compared this to muscle biopsies from 36 typical CCD patients. In CCD, RYR1, and triadin were present in the core regions, while TOM20 was absent in the core regions. Interestingly, in 5 CNMDU1 cases with the RYR1 mutation, RYR1, and triadin were similarly present in core-like areas, while TOM20 was absent in the subsarcolemmal region. Furthermore, there was a correlation between the core position and the disease duration or progression-the older patients in more advanced stages had more centralized cores. Our results indicate that CNMDU1 due to RYR1 mutation is a de facto core myopathy.

In vivo RyR1 reduction in muscle triggers a core-like myopathy.

Mutations in the RYR1 gene, encoding the skeletal muscle calcium channel RyR1, lead to congenital myopathies, through expression of a channel with abnormal permeability and/or in reduced amount, but the direct functional whole organism consequences of exclusive reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Tamoxifen-induced recombination in the RYR1 gene at adult age resulted in a progressive reduction in the protein amount reaching a stable level of 50% of the initial amount, and was associated with a progressive muscle weakness and atrophy. Measurement of calcium fluxes in isolated muscle fibers demonstrated a reduction in the amplitude of RyR1-related calcium release mirroring the reduction in the protein amount. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution and membrane remodeling. An increase in the expression level of many proteins was observed, as well as an inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Dusty Core Disease patients (a subtype of Central Core Disease), pointing to common pathophysiological mechanisms related to RyR1 reduction.

Core myopathies - a short review.

Congenital myopathies represent a clinically and genetically heterogeneous group of early-onset neuromuscular diseases with characteristic, but not always specific, histopathological features, often presenting with stable and/or slowly progressive truncal and proximal weakness. It is often not possible to have a diagnosis on clinical ground alone. Additional extraocular, respiratory, distal involvement, scoliosis, and distal laxity may provide clues. The "core myopathies" collectively represent the most common form of congenital myopathies, and the name pathologically corresponds to histochemical appearance of focally reduced oxidative enzyme activity and myofibrillar changes on ultrastructural studies. Because of the clinical, pathological, and molecular overlaps, central core disease and multiminicore disease will be discussed together.

Dominant or recessive mutations in the RYR1 gene causing central core myopathy in Brazilian patients.

Central Core Disease (CCD) is an inherited neuromuscular disorder characterized by the presence of cores in muscle biopsy. CCD is caused by mutations in the RYR1 gene. This gene encodes the ryanodine receptor 1, which is an intracellular calcium release channel from the sarcoplasmic reticulum to the cytosol in response to depolarization of the plasma membrane. Mutations in this gene are also associated with susceptibility to Malignant Hyperthermia (MHS). In this study, we evaluated 20 families with clinical and histological characteristics of CCD to identify primary mutations in patients, for diagnosis and genetic counseling of the families. We identified variants in the RYR1 gene in 19/20 families. The molecular pathogenicity was confirmed in 16 of them. Most of these variants (22/23) are missense and unique in the families. Two variants were recurrent in two different families. We identified six families with biallelic mutations, five compound heterozygotes with no consanguinity, and one homozygous, with consanguineous parents, resulting in 30% of cases with possible autosomal recessive inheritance. We identified seven novel variants, four of them classified as pathogenic. In one family, we identified two mutations in exon 102, segregating in cis, suggesting an additive effect of two mutations in the same allele. This work highlights the importance of using Next-Generation Sequencing technology for the molecular diagnosis of genetic diseases when a very large gene is involved, associated to a broad distribution of the mutations along it. These data also influence the prevention through adequate genetic counseling for the families and cautions against malignant hyperthermia susceptibility.

Cored in the act: the use of models to understand core myopathies.

The core myopathies are a group of congenital myopathies with variable clinical expression - ranging from early-onset skeletal-muscle weakness to later-onset disease of variable severity - that are identified by characteristic 'core-like' lesions in myofibers and the presence of hypothonia and slowly or rather non-progressive muscle weakness. The genetic causes are diverse; central core disease is most often caused by mutations in ryanodine receptor 1 (RYR1), whereas multi-minicore disease is linked to pathogenic variants of several genes, including selenoprotein N (SELENON), RYR1 and titin (TTN). Understanding the mechanisms that drive core development and muscle weakness remains challenging due to the diversity of the excitation-contraction coupling (ECC) proteins involved and the differential effects of mutations across proteins. Because of this, the use of representative models expressing a mature ECC apparatus is crucial. Animal models have facilitated the identification of disease progression mechanisms for some mutations and have provided evidence to help explain genotype-phenotype correlations. However, many unanswered questions remain about the common and divergent pathological mechanisms that drive disease progression, and these mechanisms need to be understood in order to identify therapeutic targets. Several new transgenic animals have been described recently, expanding the spectrum of core myopathy models, including mice with patient-specific mutations. Furthermore, recent developments in 3D tissue engineering are expected to enable the study of core myopathy disease progression and the effects of potential therapeutic interventions in the context of human cells. In this Review, we summarize the current landscape of core myopathy models, and assess the hurdles and opportunities of future modeling strategies.