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.

Physician-directed genetic screening to evaluate personal risk for medically actionable disorders: a large multi-center cohort study

Background The use of proactive genetic screening for disease prevention and early detection is not yet widespread. Professional practice guidelines from the American College of Medical Genetics and Genomics (ACMG) have encouraged reporting pathogenic variants that confer personal risk for actionable monogenic hereditary disorders, but only as secondary findings from exome or genome sequencing. The Centers for Disease Control and Prevention (CDC) recognizes the potential public health impact of three Tier 1 actionable disorders. Here, we report results of a large multi-center cohort study to determine the yield and potential value of screening healthy individuals for variants associated with a broad range of actionable monogenic disorders, outside the context of secondary findings. Methods Eligible adults were offered a proactive genetic screening test by health care providers in a variety of clinical settings. The screening panel based on next-generation sequencing contained up to 147 genes associated with monogenic disorders within cancer, cardiovascular, and other important clinical areas. Sequence and intragenic copy number variants classified as pathogenic, likely pathogenic, pathogenic (low penetrance), or increased risk allele were considered clinically significant and reported. Results were analyzed by clinical area and severity/burden of disease using chi-square tests without Yates’ correction. Results Among 10,478 unrelated adults screened, 1619 (15.5%) had results indicating personal risk for an actionable monogenic disorder. In contrast, only 3.1 to 5.2% had clinically reportable variants in genes suggested by the ACMG version 2 secondary findings list to be examined during exome or genome sequencing, and 2% had reportable variants related to CDC Tier 1 conditions. Among patients, 649 (6.2%) were positive for a genotype associated with a disease of high severity/burden, including hereditary cancer syndromes, cardiovascular disorders, or malignant hyperthermia susceptibility. Conclusions This is one of the first real-world examples of specialists and primary care providers using genetic screening with a multi-gene panel to identify health risks in their patients. Nearly one in six individuals screened for variants associated with actionable monogenic disorders had clinically significant results. These findings provide a foundation for further studies to assess the role of genetic screening as part of regular medical care.

Marked Facial Weakness, Ptosis, and Hanging Jaw: A Case with RYR1-Related Congenital Centronuclear Myopathy

Congenital myopathies are an expanding spectrum of neuromuscular disorders with early infantile or childhood onset hypotonia and slowly or nonprogressive skeletal muscle weakness. RYR1-related myopathies are the most common and frequently diagnosed class of congenital myopathies. Malignant hyperthermia susceptibility and central core disease are autosomal dominant or de novo RYR1 disorder, whereas multiminicore, congenital fiber type disproportion and centronuclear myopathy are autosomal recessive RYR1 disorders. The presence of ptosis, ophthalmoparesis, facial, and proximal muscles weakness, with the presence of dusty cores and multiple internal nuclei on muscle biopsy are clues to the diagnosis. We describe an 18-year-old male, who presented with early infantile onset ptosis, ophthalmoplegia, myopathic facies, hanging lower jaw, and proximal muscle weakness confirmed as an RYR1-related congenital centronuclear myopathy on genetic analysis and muscle biopsy.

Spinal Anesthesia

Spinal anesthesia is the injection of local anesthetics and additives into the subarachnoid space, providing analgesia by creating a sensory block and providing suitable surgical conditions by creating a motor block. In the pediatric population, spinal “intrathecal” anesthesia can be used as an alternative to general anesthesia for lower extremity and abdominal surgery in high-risk patients (e.g., premature or former premature infants, teenagers with malignant hyperthermia susceptibility or chronic respiratory disease). Preservative-free morphine injected into the intrathecal space can also be used for postoperative pain control, for example, after scoliosis repair.

Variant Curation Expert Panel Recommendations for RYR1 Pathogenicity Assertions in Malignant Hyperthermia Susceptibility

ABSTRACTPurposePrevention of malignant hyperthermia (MH) requires an understanding of RYR1 variant pathogenicity to assess the risk of exposure to triggering agents. Personalized medicine, especially secondary findings and eventually genomic screening, will contribute toward this goal.MethodsWe specified ACMG/AMP criteria for variant interpretation for RYR1 and MH. Proposed rules were piloted on 84 variants. We applied quantitative evidence calibration for several criteria using likelihood ratios based on the Bayesian framework.ResultsSeven ACMG/AMP criteria were adopted without changes, ten were adopted with RYR1-specific modifications, and nine were dropped. The in silico (PP3 and BP4) and hot spot criteria (PM1) were evaluated quantitatively. REVEL gave an OR of 23:1 for PP3 and 16:1 for BP4 using trichotomized cut-offs of >0.85 (pathogenic) and <0.5 (benign). The PM1 hotspot criterion had an OR of 24:1. PP3 and PM1 were implemented at moderate strength. Applying the revised ACMG criteria to 44 recognized MH variants, 30 were assessed as pathogenic, 12 as likely pathogenic, and two as VUS.ConclusionCuration of these variants will facilitate interpretation of RYR1/MH genomic testing results, which is especially important for secondary findings analyses. Our approach to quantitatively calibrating criteria are generalizable to other variant curation expert panels.

Ryanodine receptor 1-related disorders: an historical perspective and proposal for a unified nomenclature

The RYR1 gene, which encodes the sarcoplasmic reticulum calcium release channel or type 1 ryanodine receptor (RyR1) of skeletal muscle, was sequenced in 1988 and RYR1 variations that impair calcium homeostasis and increase susceptibility to malignant hyperthermia were first identified in 1991. Since then, RYR1-related myopathies (RYR1-RM) have been described as rare, histopathologically and clinically heterogeneous, and slowly progressive neuromuscular disorders. RYR1 variants can lead to dysfunctional RyR1-mediated calcium release, malignant hyperthermia susceptibility, elevated oxidative stress, deleterious post-translational modifications, and decreased RyR1 expression. RYR1-RM-affected individuals can present with delayed motor milestones, contractures, scoliosis, ophthalmoplegia, and respiratory insufficiency.Historically, RYR1-RM-affected individuals were diagnosed based on morphologic features observed in muscle biopsies including central cores, cores and rods, central nuclei, fiber type disproportion, and multi-minicores. However, these histopathologic features are not always specific to RYR1-RM and often change over time. As additional phenotypes were associated with RYR1 variations (including King-Denborough syndrome, exercise-induced rhabdomyolysis, lethal multiple pterygium syndrome, adult-onset distal myopathy, atypical periodic paralysis with or without myalgia, mild calf-predominant myopathy, and dusty core disease) the overlap among diagnostic categories is ever increasing. With the continuing emergence of new clinical subtypes along the RYR1 disease spectrum and reports of adult-onset phenotypes, nuanced nomenclatures have been reported (RYR1- [related, related congenital, congenital] myopathies). In this narrative review, we provide historical highlights of RYR1 research, accounts of the main diagnostic disease subtypes and propose RYR1-related disorders (RYR1-RD) as a unified nomenclature to describe this complex and evolving disease spectrum.

Malignant hyperthermia susceptibility: utilization of genetic results in an electronic medical record to increase safety

Aim: This manuscript describes implementation of clinical decision support for providers concerned with perioperative complications of malignant hyperthermia susceptibility. Materials & methods: Clinical decision support for malignant hyperthermia susceptibility was implemented in 2018 based around our pre-emptive genotyping platform. We completed a brief descriptive review of patients who underwent pre-emptive testing, focused particularly on RYR1 and CACNA1S genes. Results: To date, we have completed pre-emptive genetic testing on more than 10,000 patients; 13 patients having been identified as a carrier of a pathogenic or likely pathogenic variant of RYR1 or CACNA1S. Conclusion: An alert system for malignant hyperthermia susceptibility – as an extension of our pre-emptive genomics platform – was implemented successfully. Implementation strategies and lessons learned are discussed herein.

Adaptive thermogenesis enhances the life-threatening response to heat in mice with an Ryr1 mutation

Mutations in the skeletal muscle Ca2+ release channel, the type 1 ryanodine receptor (RYR1), cause malignant hyperthermia susceptibility (MHS) and a life-threatening sensitivity to heat, which is most severe in children. Mice with an MHS-associated mutation in Ryr1 (Y524S, YS) display lethal muscle contractures in response to heat. Here we show that the heat response in the YS mice is exacerbated by brown fat adaptive thermogenesis. In addition, the YS mice have more brown adipose tissue thermogenic capacity than their littermate controls. Blood lactate levels are elevated in both heat-sensitive MHS patients with RYR1 mutations and YS mice due to Ca2+ driven increases in muscle metabolism. Lactate increases brown adipogenesis in both mouse and human brown preadipocytes. This study suggests that simple lifestyle modifications such as avoiding extreme temperatures and maintaining thermoneutrality could decrease the risk of life-threatening responses to heat and exercise in individuals with RYR1 pathogenic variants.