Impaired gating of γ- and ε-AChR respectively causes Escobar syndrome and fast-channel myasthenia.

OBJECTIVE: To dissect the kinetic defects of acetylcholine receptor (AChR) γ subunit variant in an incomplete form of the Escobar syndrome without pterygium and compare it with those of a variant of corresponding residue in the AChR ε subunit in a congenital myasthenic syndrome (CMS). METHODS: Whole exome sequencing, α-bungarotoxin binding assay, single channel patch-clamp recordings, and maximum likelihood analysis of channel kinetics. RESULTS: We identified compound heterozygous variants in AChR γ and ε subunits in three Escobar syndrome (1-3) and three CMS patients (4-6), respectively. Each Escobar syndrome patient carries γP121R along with γV221Afs*44 in patients 1 and 2, and γY63* in patient 3. Three CMS patients share εP121T along with εR20W, εG-8R, and εY15H in patients 4, 5, and 6, respectively. Surface expressions of γP121R- and εP121T-AChR were 80% and 138% of the corresponding wild-type AChR, whereas εR20W, εG-8R, and εY15H reduced receptor expression to 27%, 35%, and 30% of wild-type εAChR, respectively. γV221Afs*44 and γY63* are null variants. Thus, γP121R and εP121T determine the phenotype. γP121R and εP121T shorten channel opening burst duration to 28% and 18% of corresponding wild-type AChR by reducing the channel gating equilibrium constant 44- and 63-fold, respectively. INTERPRETATION: Similar impairment of channel gating efficiency of a corresponding P121 residue in the acetylcholine-binding site of the AChR γ and ε subunits causes Escobar syndrome without pterygium and fast-channel CMS, respectively, suggesting that therapy for the fast-channel CMS will benefit Escobar syndrome.

Clinical and Pathologic Features of Congenital Myasthenic Syndromes Caused by 35 Genes-A Comprehensive Review.

Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders characterized by impaired neuromuscular signal transmission due to germline pathogenic variants in genes expressed at the neuromuscular junction (NMJ). A total of 35 genes have been reported in CMS (AGRN, ALG14, ALG2, CHAT, CHD8, CHRNA1, CHRNB1, CHRND, CHRNE, CHRNG, COL13A1, COLQ, DOK7, DPAGT1, GFPT1, GMPPB, LAMA5, LAMB2, LRP4, MUSK, MYO9A, PLEC, PREPL, PURA, RAPSN, RPH3A, SCN4A, SLC18A3, SLC25A1, SLC5A7, SNAP25, SYT2, TOR1AIP1, UNC13A, VAMP1). The 35 genes can be classified into 14 groups according to the pathomechanical, clinical, and therapeutic features of CMS patients. Measurement of compound muscle action potentials elicited by repetitive nerve stimulation is required to diagnose CMS. Clinical and electrophysiological features are not sufficient to identify a defective molecule, and genetic studies are always required for accurate diagnosis. From a pharmacological point of view, cholinesterase inhibitors are effective in most groups of CMS, but are contraindicated in some groups of CMS. Similarly, ephedrine, salbutamol (albuterol), amifampridine are effective in most but not all groups of CMS. This review extensively covers pathomechanical and clinical features of CMS by citing 442 relevant articles.

Proteomic profiling of sporadic late-onset nemaline myopathy.

OBJECTIVE: To define the proteomic profile of sporadic late-onset nemaline myopathy (SLONM) and explore its pathogenesis. METHODS: We performed mass spectrometry on laser-dissected frozen muscle samples from five patients with SLONM, three of whom with an associated monoclonal protein (MP), and four controls, to determine the proteomic profile of SLONM. Furthermore, we assessed the role of the MP by evaluating the expression of the immunoglobulin light chain variable regions (IGVL). RESULTS: There were 294 differentially expressed proteins: 272 upregulated and 22 downregulated. Among the top 100 upregulated proteins, the most common categories were: nuclear or nucleic acid metabolism (24%), extracellular matrix and basal lamina (17%), immune response (13%), and actin dynamics (8%). Downregulated proteins consisted mostly of contractile proteins. Among upregulated proteins, there were 65 with a role related to the immune system, including eight proteins involved in major histocompatibility complex 1 (MHC1) and antigen processing, 15 in MHCII complex and phagocytosis, and 23 in B and/or T-cell function. Among nine upregulated immunoglobulin proteins, there were two IGVL genes. However, these were also detected in SLONM cases without an MP, with no evidence of clonally dominant immunoglobulin deposition. In muscle sections from SLONM patients, nemaline rods tended to accumulate in atrophic fibers with marked rarefaction of the myofibrils. Increased MHC1 reactivity was present in fibers containing nemaline rods as well as adjacent nonatrophic fibers. CONCLUSION: Our findings suggest that aberrant immune activation is present in SLONM, but do not support a direct causal relationship between the MP and SLONM.

Expanding Spectrum of Desmin-Related Myopathy, Long-term Follow-up, and Cardiac Transplantation.

BACKGROUND AND OBJECTIVES: We aimed to determine the genetic and clinical phenotypes of patients with desmin-related myopathy and long-term outcomes after cardiac transplantation. METHODS: We performed a retrospective review of cardiac and neurologic manifestations of patients with genetically confirmed desmin-related myopathy (January 1, 1999-January 1, 2020). RESULTS: Twenty-five patients in 20 different families were recognized. Median age at onset of symptoms was 20 (range 4-50) years; median follow-up time was 36 (range 1-156) months. Twelve patients initially presented with skeletal muscle involvement, and 13 presented with cardiac disease. Sixteen patients had both cardiac and skeletal muscle involvement. Clinically muscle weakness distribution was distal (n = 11), proximal (n = 4), or both (n = 7) in 22 patients. Skeletal muscle biopsy from patients with missense and splice site variants (n = 12) showed abnormal fibers containing amorphous material in Gomori trichrome-stained sections. Patients with cardiac involvement had atrioventricular conduction abnormalities or cardiomyopathy. The most common ECG abnormality was complete atrioventricular block in 11 patients, all of whom required a permanent pacemaker at a median age of 25 (range 16-48) years. Sudden cardiac death resulting in implantable cardioverter-defibrillator (ICD) shocks or resuscitation was reported in 3 patients; a total of 5 patients had ICDs. Orthotopic cardiac transplantation was performed in 3 patients at 20, 35, and 39 years of age. DISCUSSION: Pathogenic variants in desmin can lead to varied neurologic and cardiac phenotypes beginning at a young age. Two-thirds of the patients have both neurologic and cardiac symptoms, usually starting in the third decade. Heart transplantation was tolerated with improved cardiac function and quality of life.

Sleep and Breathing After Nusinersen Therapy in a Child With Spinal Muscular Atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) type 3 is an autosomal recessive neurological disorder associated with a deletion/mutation in the survival motor neuron gene, with gradually progressive degeneration of the motor neurons of the spinal cord and brainstem, which causes muscle weakness responsible for impairment of swallowing, breathing, and mobility. REPORT OF CASE: We report an 11-year-old girl with SMA type 3 with moderate to severe obstructive sleep apnea (OSA) syndrome refractory to adenotonsillectomy and noninvasive ventilatory support. She was started on nusinersen, which is a novel disease modifying therapy for SMA. This new treatment led to improvement of the OSA in a short period, likely from better respiratory muscle function. CONCLUSIONS: The improvement in OSA supports the role of nusinersen in sleep-related upper respiratory muscle function in SMA type 3.

Determinants of the repetitive-CMAP occurrence and therapy efficacy in slow-channel myasthenia.

OBJECTIVE: To find determinants of the occurrence of repetitive compound muscle action potential (R-CMAP) and to assess the efficacy of channel blocker therapy in slow-channel congenital myasthenic syndrome (SCCMS). METHODS: Neurologic examination, EMG study, laboratory test, muscle biopsy, and next-generation and Sanger sequencing; literature review of reported patients with SCCMS, including EMG, kinetics of mutant acetylcholine receptors (AChRs), and response to therapy; and simulation of the decay phase of endplate potential (EPP) were performed. RESULTS: Three newly characterized and 57 reported patients with SCCMS with mutations of AChR subunits were included. In patients with R-CMAP, the length of channel opening bursts of mutant AChR was increased 8.68 ± 2.82 (mean ± SD)-fold compared to wild-type; in patients without R-CMAP, the length was increased 3.84 ± 0.65-fold (95% confidence interval 3.18-6.50, p = 0.000014). The EPP amplitude after refractory period of action potential in muscle fiber is above the threshold in patients with R-CMAP but below the threshold in patients without R-CMAP. In patients with good results from channel blocker therapy, treatment was initiated 11.60 ± 5.17 years after onset of symptoms; in patients with no to moderate benefit from channel blocker therapy, treatment was initiated 30.70 ± 12.72 years after onset (95% confidence interval -28.57 to -9.63, p = 0.00089). CONCLUSIONS: In SCCMS, the R-CMAP occurrence is related to the extent of prolongation of the opening episodes of mutant AChR channel. Channel blocker treatment is more effective the sooner it is started after the onset of symptoms. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that channel blocker therapy in patients with SCCMS improves symptoms.

A novel fast-channel myasthenia caused by mutation in ß subunit of AChR reveals subunit-specific contribution of the intracellular M1-M2 linker to channel gating.

Genetic variants causing the fast-channel congenital myasthenic syndrome (CMS) have been identified in the α, δ, and ε but not the ß subunit of acetylcholine receptor (AChR). A 16-year-old girl with severe myasthenia had low-amplitude and fast-decaying miniature endplate potentials. Mutation analysis revealed two heteroallelic variants in CHRNB1 encoding the AChR ß subunit: a novel c.812C>T (p.P248L) variant in M1-M2 linker (p.P271L in HGVS nomenclature), and a ~430 bp deletion causing loss of exon 8 leading to frame-shift and a premature stop codon (p.G251Dfs*21). P248 is conserved in all ß subunits of different species, but not in other AChR subunits. Measurements of radio-labeled α-bungarotoxin binding show that ßP248L reduces AChR expression to 60% of wild-type. Patch clamp recordings of ACh-elicited single channel currents demonstrate that ßP248L shortens channel opening bursts from 3.3 ms to 1.2 ms, and kinetic analyses predict that the decay of the synaptic response is accelerated 2.4-fold due to reduced probability of channel reopening. Substituting ßP248 with threonine, alanine or glycine reduces the burst duration to 2.3, 1.7, and 1.5 ms, respectively. In non-ß subunits, substituting leucine for residues corresponding to ßP248 prolongs the burst duration to 4.5 ms in the α subunit, shortens it to 2.2 ms in the δ subunit, and has no effect in the ε subunit. Conversely, substituting proline for residues corresponding to ßP248 prolongs the burst duration to 8.7 ms in the α subunit, to 4.6 ms in the δ subunit, but has no effect in the ε subunit. Thus, this fast channel CMS is caused by the dual defects of ßP248L in reducing expression of the mutant receptor and accelerating the decay of the synaptic response. The results also reveal subunit-specific contributions of the M1-M2 linker to the durations of channel opening bursts.

A second cohort of CHD3 patients expands the molecular mechanisms known to cause Snijders Blok-Campeau syndrome.

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

Developmental brain abnormalities and acute encephalopathy in a patient with myopathy with extrapyramidal signs secondary to pathogenic variants in MICU1.

Mitochondria play a variety of roles in the cell, far beyond their widely recognized role in ATP generation. One such role is the regulation and sequestration of calcium, which is done with the help of the mitochondrial calcium uniporter (MCU) and its regulators, MICU1 and MICU2. Genetic variations in MICU1 and MICU2 have been reported to cause myopathy, developmental disability and neurological symptoms typical of mitochondrial disorders. The symptoms of MICU1/2 deficiency have generally been attributed to calcium regulation in the metabolic and biochemical roles of mitochondria. Here, we report a female child with heterozygous MICU1 variants and multiple congenital brain malformations on MRI. Specifically, she shows anterior perisylvian polymicrogyria, dysmorphic basal ganglia, and cerebellar dysplasia in addition to white matter abnormalities. These novel findings suggest that MICU1 is necessary for proper neurodevelopment through a variety of potential mechanisms, including calcium-mediated regulation of the neuronal cytoskeleton, Miro1-MCU complex-mediated mitochondrial movement, or enhancing ATP production. This case provides new insight into the molecular pathogenesis of MCU dysfunction and may represent a novel diagnostic feature of calcium-based mitochondrial disease.

Congenital myasthenic syndrome-associated agrin variants affect clustering of acetylcholine receptors in a domain-specific manner.

Congenital myasthenic syndromes (CMS) are caused by mutations in molecules expressed at the neuromuscular junction. We report clinical, structural, ultrastructural, and electrophysiologic features of 4 CMS patients with 6 heteroallelic variants in AGRN, encoding agrin. One was a 7.9-kb deletion involving the N-terminal laminin-binding domain. Another, c.4744G>A - at the last nucleotide of exon 26 - caused skipping of exon 26. Four missense mutations (p.S1180L, p.R1509W, p.G1675S, and p.Y1877D) expressed in conditioned media decreased AChR clusters in C2C12 myotubes. The agrin-enhanced phosphorylation of MuSK was markedly attenuated by p.Y1877D in the LG3 domain and moderately attenuated by p.R1509W in the LG1 domain but not by the other 2 mutations. The p.S1180L mutation in the SEA domain facilitated degradation of secreted agrin. The p.G1675S mutation in the LG2 domain attenuated anchoring of agrin to the sarcolemma by compromising its binding to heparin. Anchoring of agrin with p.R1509W in the LG1 domain was similarly attenuated. Mutations of agrin affect AChR clustering by enhancing agrin degradation or by suppressing MuSK phosphorylation and/or by compromising anchoring of agrin to the sarcolemma of the neuromuscular junction.

Slow-channel myasthenia due to novel mutation in M2 domain of AChR delta subunit.

OBJECTIVE: To characterize the molecular and phenotypic basis of a severe slow-channel congenital myasthenic syndrome (SCCMS). METHODS: Intracellular and single-channel recordings from patient endplates; alpha-bungarotoxin binding studies; direct sequencing of AChR genes; microsatellite analysis; kinetic analysis of AChR activation; homology modeling of adult human AChR structure. RESULTS: Among 24 variants reported to cause SCCMS only two appear in the AChR δ-subunit. We here report a 16-year-old patient harboring a novel δL273F mutation (δL294F in HGVS nomenclature) in the second transmembrane domain (M2) of the AChR δ subunit. Kinetic analyses with ACh and the weak agonist choline indicate that δL273F prolongs the channel opening bursts 9.4-fold due to a 75-fold increase in channel gating efficiency, whereas a previously identified εL269F mutation (εL289F in HGVS nomenclature) at an equivalent location in the AChR ε-subunit prolongs channel opening bursts 4.4-fold due to a 30-fold increase in gating efficiency. Structural modeling of AChR predicts that inter-helical hydrophobic interactions between the mutant residue in the δ and ε subunit and nearby M2 domain residues in neighboring α subunits contribute to structural stability of the open relative to the closed channel states. INTERPRETATION: The greater increase in gating efficiency by δL273F than by εL269F explains why δL273F has more severe clinical effects. Both δL273F and εL269F impair channel gating by disrupting hydrophobic interactions with neighboring α-subunits. Differences in the extent of impairment of channel gating in δ and ε mutant receptors suggest unequal contributions of ε/α and δ/α subunit pairs to gating efficiency.

A homozygous mutation in GMPPB leads to centronuclear myopathy with combined pre- and postsynaptic defects of neuromuscular transmission.

Mutations in GMPPB cause a wide spectrum of neuromuscular syndromes, including muscular dystrophies and congenital myasthenic syndrome. The mechanisms by which GMPPB mutations impair neuromuscular transmission however remain incompletely understood. We expand here upon a previous report of one such patient presenting with a myopathy-congenital myasthenic syndrome overlap phenotype. Fatigable proximal muscle weakness developed gradually between 13 and 25 years of age, with subsequent stabilization. Low-frequency repetitive nerve stimulation showed a decrement, while a muscle biopsy demonstrated the presence of a centronuclear myopathy. Genetic testing identified a homozygous c.458C > T (p.Thr153Ile) variant in GMPPB. In-vitro microelectrode recordings and ultrastructural studies showed impairment of both pre- and postsynaptic neuromuscular transmission, thus demonstrating the presence of not only postsynaptic, but also presynaptic pathology in GMPPB-related disorders.

Idiopathic inflammatory myopathy: Interrater variability in muscle biopsy reading.

OBJECTIVE: To determine interrater variability in diagnosing individual muscle biopsy abnormalities and diagnosis. METHODS: We developed a scoring tool to analyze consensus in muscle biopsy reading of an ad hoc workgroup of international experts. Twenty-four samples from patients with suspected idiopathic inflammatory myopathy (IIM) were randomly selected, providing sections that were stained with standard histologic and immunohistochemical methods. Sections were made available on an online platform, and experts were queried about myopathologic features within 4 pathologic domains: muscle fibers, inflammation, connective tissue, and vasculature. A short clinical presentation of cases was included, and experts were asked to give a tentative diagnosis of polymyositis, dermatomyositis, inclusion-body myositis, antisynthetase syndrome-related myositis, immune-mediated necrotizing myopathy, nonspecific myositis, or other disease. Fleiss κ values, scoring interrater variability, showed the highest agreement within the muscle fiber and connective tissue domains. RESULTS: Despite overall low κ values, moderate agreement was achieved for tentative diagnosis, supporting the idea of using holistic muscle biopsy interpretation rather than adding up individual features. CONCLUSION: The assessment of individual pathologic features needs to be standardized and harmonized and should be measured for sensitivity and specificity for subgroup classification. Standardizing the process of diagnostic muscle biopsy reading would allow identification of more homogeneous patient cohorts for upcoming treatment trials.

Congenital myasthenic syndromes in adult neurology clinic: A long road to diagnosis and therapy.

OBJECTIVE: To investigate the diagnostic challenges of congenital myasthenic syndromes (CMS) in adult neuromuscular practice. METHODS: We searched the Mayo Clinic database for patients with CMS diagnosed in adulthood in the neuromuscular clinic between 2000 and 2016. Clinical, laboratory, and electrodiagnostic data were reviewed. RESULTS: We identified 34 patients with CMS, 30 of whom had a molecular diagnosis (14 DOK7, 6 RAPSN, 2 LRP4, 2 COLQ, 2 slow-channel syndrome, 1 primary acetylcholine receptor deficiency, 1 AGRN, 1 GFPT1, and 1 SCN4A). Ophthalmoparesis was often mild and present in 13 patients. Predominant limb-girdle weakness occurred in 19 patients. Two patients had only ptosis. Age at onset ranged from birth to 39 years (median 5 years). The median time from onset to diagnosis was 26 years (range 4-56 years). Thirteen patients had affected family members. Fatigable weakness was present when examined. Creatine kinase was elevated in 4 of 23 patients (range 1.2-4.2 times the upper limit of normal). Repetitive nerve stimulation revealed a decrement in 30 patients. Thirty-two patients were previously misdiagnosed with seronegative myasthenia gravis (n = 16), muscle diseases (n = 15), weakness of undetermined cause (n = 8), and others (n = 4). Fifteen patients received immunotherapy or thymectomy without benefits. Fourteen of the 25 patients receiving pyridostigmine did not improve or worsen. CONCLUSION: Misdiagnosis occurred in 94% of the adult patients with CMS and causes a median diagnostic delay of nearly 3 decades from symptom onset. Seronegative myasthenia gravis and muscle diseases were the 2 most common misdiagnoses, which led to treatment delay and unnecessary exposure to immunotherapy, thymectomy, or muscle biopsy.

Novel Homozygous Variant in TTC19 Causing Mitochondrial Complex III Deficiency with Recurrent Stroke-Like Episodes: Expanding the Phenotype.

A 7-year-old boy with family history of consanguinity presented with developmental delay and recurrent hemiplegia involving both sides of the body, with variable facial and ocular involvement. Brain MRI showed bilateral striatal necrosis with cystic degeneration and lactate peaks on spectroscopy. Biochemical testing demonstrated mildly elevated lactate and pyruvate. Whole-exome sequencing revealed a novel homozygous pathogenic frameshift mutation in gene TTC19, diagnostic of mitochondrial complex III deficiency.

The unfolding landscape of the congenital myasthenic syndromes.

Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is impaired by one or more specific mechanisms. Since the advent of next-generation sequencing methods, the discovery of novel CMS targets and phenotypes has proceeded at an accelerated rate. Here, we review the current classification of CMS and describe our findings in five of these targets identified and investigated in our laboratory in the past 5 years. Defects in LRP4 hinder synaptic development and maintenance; the defects in PREPL are predicted to diminish filling of the synaptic vesicle with acetylcholine; and defects in SNAP25, Munc13-1, and synaptotbrevin-1 impede synaptic vesicle exocytosis.

Novel de novo variant in EBF3 is likely to impact DNA binding in a patient with a neurodevelopmental disorder and expanded phenotypes: patient report, in silico functional assessment, and review of published cases.

Pathogenic variants in EBF3 were recently described in three back-to-back publications in association with a novel neurodevelopmental disorder characterized by intellectual disability, speech delay, ataxia, and facial dysmorphisms. In this report, we describe an additional patient carrying a de novo missense variant in EBF3 (c.487C>T, p.(Arg163Trp)) that falls within a conserved residue in the zinc knuckle motif of the DNA binding domain. Without a solved structure of the DNA binding domain, we generated a homology-based atomic model and performed molecular dynamics simulations for EBF3, which predicted decreased DNA affinity for p.(Arg163Trp) compared with wild-type protein and control variants. These data are in agreement with previous experimental studies of EBF1 showing the paralogous residue is essential for DNA binding. The conservation and experimental evidence existing for EBF1 and in silico modeling and dynamics simulations to validate comparable behavior of multiple variants in EBF3 demonstrates strong support for the pathogenicity of p.(Arg163Trp). We show that our patient presents with phenotypes consistent with previously reported patients harboring EBF3 variants and expands the phenotypic spectrum of this newly identified disorder with the additional feature of a bicornuate uterus.

Whole exome sequencing of a patient with suspected mitochondrial myopathy reveals novel compound heterozygous variants in RYR1.

BACKGROUND: Pathogenic variants in ryanodine receptor 1 (RYR1, MIM# 180901) are the cause of congenital myopathy with fiber-type disproportion, malignant hyperthermia susceptibility type 1, central core disease of muscle, multiminicore disease and other congenital myopathies. METHODS: We present a patient with global developmental delay, hypotonia, myopathy, joint hypermobility, and multiple other systemic complaints that were noted early in life. Later she was found to have multiple bone deformities involving her spine, with severe scoliosis that was corrected surgically. She was also diagnosed with ophthalmoplegia, chronic hypercapnic respiratory failure, and hypertension. At 22 years of age she presented to the genetics clinic with a diagnosis of mitochondrial myopathy and underwent whole exome sequencing (WES). RESULTS: Whole exome sequencing revealed two novel compound heterozygous variants in RYR1 (c.7060_7062del, p.Val2354del and c.4485_4500del, p.Tyr1495X). CONCLUSION: Review of her clinical, pathologic, and genetic findings pointed to a diagnosis of a congenital myopathy with fiber-type disproportion.