Caveolinopathy: Clinical, histological, and muscle imaging features and follow-up in a multicenter retrospective cohort.

BACKGROUND AND PURPOSE: CAV3 gene mutations, mostly inherited as an autosomal dominant trait, cause various skeletal muscle diseases. Clinical presentations encompass proximal myopathy, distal myopathy, or isolated persistent high creatine kinase (CK) with a major overlapping phenotype. METHODS: Twenty-three patients with CAV3 symptomatic mutations, from 16 different families, were included in a retrospective cohort. Mean follow-up duration was 24.2 ± 15.0 years. Clinical and functional data were collected during the follow-up. The results of muscle imaging, electroneuromyography, muscle histopathology, immunohistochemistry, and caveolin-3 Western blot analysis were also compiled. RESULTS: Exercise intolerance was the most common phenotype (52%). Eighty percent of patients had calf hypertrophy, and only 65% of patients presented rippling. One patient presented initially with camptocormia. A walking aid was required in only two patients. Electroneuromyography was mostly normal. CK level was elevated in all patients. No patient had cardiac or respiratory impairment. Muscle imaging showed fatty involvement of semimembranosus, semitendinosus, rectus femoris, biceps brachialis, and spinal muscles. Almost all (87%) of the biopsies were abnormal but without any specific pattern. Whereas a quarter of patients had normal caveolin-3 immunohistochemistry results, Western blots disclosed a reduced amount of the protein. We report nine mutations, including four not previously described. No phenotype-genotype correlation was evidenced. CONCLUSIONS: Caveolinopathy has diverse clinical, muscle imaging, and histological presentations but often has limited functional impact. Mild forms of the disease, an atypical phenotype, and normal caveolin-3 immunostaining are pitfalls leading to misdiagnosis.

Comprehensive morphometric assessment of deltoid muscle development in children: A cross-sectional study.

BACKGROUND: Normative values for different morphometric parameters of muscle fibres during paediatric development, i.e. from 0 to 18 years, are currently unavailable. They would be of major importance to accurately evaluate pathological changes and could be used as reference biomarkers for evaluating treatment response in clinical trials, or physiological adjustments in sports or ageing. METHODS: Data were derived from 482 images with a total of 33 094 fibres from 10 µm cross-sections of snap-frozen muscle from 83 deltoid muscle biopsies from patients, 0-18 years, without neuromuscular pathology stained with ATPase 9.4. Data was acquired and analysed with patented image analysis algorithms from "CARPACCIO.cloud". Several parameters were extracted or calculated, including cross-sectional area (CSA), fibre type, circularity, as well as the Minimum diameter of Feret (MinFeret). FINDINGS: This study illustrates changes in quantitative parameters for muscle morphology over the course of paediatric development and the pivotal changes occurring around puberty. Only fibre size parameters (MinFeret, CSA) are dependent on gender, and only after puberty. All other parameters vary in a similar manner for females and males. The proportion of type 1 fibres is essentially constant from birth to age 10, decreasing to ≈40% by age 18. Circularity decreases with age, to plateau after age 10 for both fibre types. INTERPRETATION: Normative values and reference charts for muscle fibre types in this age range have been generated to allow comparison of data from patients in pathology laboratories working on neuromuscular diseases. FUNDING: BPI FRANCE, PULSALYS, Association de l'Institut de Myologie, French National Research Agency (ANR), LABEX CORTEX of Université de Lyon.

FYCO1 Increase and Effect of Arimoclomol-Treatment in Human VCP-Pathology.

Dominant VCP-mutations cause a variety of neurological manifestations including inclusion body myopathy with early-onset Paget disease and frontotemporal dementia 1 (IBMPFD). VCP encodes a ubiquitously expressed multifunctional protein that is a member of the AAA+ protein family, implicated in multiple cellular functions ranging from organelle biogenesis to ubiquitin-dependent protein degradation. The latter function accords with the presence of protein aggregates in muscle biopsy specimens derived from VCP-patients. Studying the proteomic signature of VCP-mutant fibroblasts, we identified a (pathophysiological) increase of FYCO1, a protein involved in autophagosome transport. We confirmed this finding applying immunostaining also in muscle biopsies derived from VCP-patients. Treatment of fibroblasts with arimoclomol, an orphan drug thought to restore physiologic cellular protein repair pathways, ameliorated cellular cytotoxicity in VCP-patient derived cells. This finding was accompanied by increased abundance of proteins involved in immune response with a direct impact on protein clearaqnce as well as by elevation of pro-survival proteins as unravelled by untargeted proteomic profiling. Hence, the combined results of our study reveal a dysregulation of FYCO1 in the context of VCP-etiopathology, highlight arimoclomol as a potential drug and introduce proteins targeted by the pre-clinical testing of this drug in fibroblasts.

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies.

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

FDX2 and ISCU Gene Variations Lead to Rhabdomyolysis With Distinct Severity and Iron Regulation.

BACKGROUND AND OBJECTIVES: To determine common clinical and biological traits in 2 individuals with variants in ISCU and FDX2, displaying severe and recurrent rhabdomyolyses and lactic acidosis. METHODS: We performed a clinical characterization of 2 distinct individuals with biallelic ISCU or FDX2 variants from 2 separate families and a biological characterization with muscle and cells from those patients. RESULTS: The individual with FDX2 variants was clinically more affected than the individual with ISCU variants. Affected FDX2 individual fibroblasts and myoblasts showed reduced oxygen consumption rates and mitochondrial complex I and PDHc activities, associated with high levels of blood FGF21. ISCU individual fibroblasts showed no oxidative phosphorylation deficiency and moderate increase of blood FGF21 levels relative to controls. The severity of the FDX2 individual was not due to dysfunctional autophagy. Iron was excessively accumulated in ISCU-deficient skeletal muscle, which was accompanied by a downregulation of IRP1 and mitoferrin2 genes and an upregulation of frataxin (FXN) gene expression. This excessive iron accumulation was absent from FDX2 affected muscle and could not be correlated with variable gene expression in muscle cells. DISCUSSION: We conclude that FDX2 and ISCU variants result in a similar muscle phenotype, that differ in severity and skeletal muscle iron accumulation. ISCU and FDX2 are not involved in mitochondrial iron influx contrary to frataxin.

New insights about myofibrillar myopathies: the role of metalloproteinases 2 and 9 in the pathogenesis

Backgroung: There are few reports suggesting that gene expression and activation of various matrix metalloproteinases (MMPs) are deregulated. MMP-2 and MMP-9 represent the two MMPs, which degrade type IV collagen, the component of basement membrane. Methods: We analysed the involvement of gelatinases, MMP-2 and MMP-9, in the pathogenesis of myofibrillar myopathy (MFM). Muscle specimens from 23 patients well diagnosed with MFM, were immunostained by MMP-2 and MMP-9. We analysed qualitatively the immunoexpression in three compartments: subsarcolemmal (SSC), intracytoplasmic (ICC) and perinuclear (PNC).Results: 95,7% and 100% samples showed MMP-2 and MMP-9 upregulation ICC, respectively. PNC showed MMP-2 (82,6%) and MMP-9 (8,7%) regulation (p<0.001). SSC and ICC did not present statistical significance. There was no correlation between mutated gene and immunohistochemical pattern distribution.Conclusion: Our results suggest that MMP-2 and/or MMP-9 could participate in the pathomechanism of MFM, causing damage of sarcomere and deposition of protein aggregates.

Introdução: Existem poucos relatos sugerindo que a expressão gênica e a ativação de várias metaloproteinases de matriz (MMPs) estão desreguladas. MMP-2 e MMP-9 representam as duas MMPs, que degradam o colágeno tipo IV, o componente da membrana basal.Método: Analisamos o envolvimento das gelatinases, MMP-2 e MMP-9, na patogênese da miopatia miofibrilar (MFM). Amostras de músculos de 23 pacientes bem diagnosticados com MFM foram imunocoradas por MMP-2 e MMP-9. Analisamos qualitativamente a imunoexpressão em três compartimentos: subsarcolemal (SSC), intracitoplasmático (ICC) e perinuclear (PNC).Resultados: 95,7% e 100% das amostras apresentaram ICC de regulação positiva de MMP-2 e MMP-9, respectivamente. PNC mostrou regulação MMP-2 (82,6%) e MMP-9 (8,7%) (p <0,001). SSC e ICC não apresentaram significância estatística. Não houve correlação entre o gene mutado e a distribuição do padrão imunohistoquímico.Conclusão: Nossos resultados sugerem que MMP-2 e / ou MMP-9 podem participar do patomecanismo da MFM, causando dano ao sarcômero e deposição de agregados proteicos.

Metformin rescues muscle function in BAG3 myofibrillar myopathy models.

Dominant de novo mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation. To elucidate the mechanism of disease in, and identify therapies for, BAG3 myofibrillar myopathy, we generated two zebrafish models, one conditionally expressing BAG3P209L and one with a nonsense mutation in bag3. While transgenic BAG3P209L-expressing fish display protein aggregation, modeling the early phase of the disease, bag3-/- fish exhibit exercise dependent fiber disintegration, and reduced swimming activity, consistent with later stages of the disease. Detailed characterization of the bag3-/- fish, revealed an impairment in macroautophagic/autophagic activity, a defect we confirmed in BAG3 patient samples. Taken together, our data highlights that while BAG3P209L expression is sufficient to promote protein aggregation, it is the loss of BAG3 due to its sequestration within aggregates, which results in impaired autophagic activity, and subsequent muscle weakness. We therefore screened autophagy-promoting compounds for their effectiveness at removing protein aggregates, identifying nine including metformin. Further evaluation demonstrated metformin is not only able to bring about the removal of protein aggregates in zebrafish and human myoblasts but is also able to rescue the fiber disintegration and swimming deficit observed in the bag3-/- fish. Therefore, repurposing metformin provides a promising therapy for BAG3 myopathy.Abbreviations:ACTN: actinin, alpha; BAG3: BAG cochaperone 3; CRYAB: crystallin alpha B; DES: desmin; DMSO: dimethyl sulfoxide; DNAJB6: DnaJ heat shock protein family (Hsp40) member B6; dpf: days post fertilization; eGFP: enhanced green fluorescent protein; FDA: Food and Drug Administration; FHL1: four and a half LIM domains 1; FLNC: filamin C; hpf: hours post-fertilization; HSPB8: heat shock protein family B [small] member 8; LDB3/ZASP: LIM domain binding 3; MYOT: myotilin; TTN: titin; WT: wild-type.

Clinical phenotype and loss of the slow skeletal muscle troponin T in three new patients with recessive TNNT1 nemaline myopathy.

BACKGROUND: Congenital nemaline myopathies are rare pathologies characterised by muscle weakness and rod-shaped inclusions in the muscle fibres. METHODS: Using next-generation sequencing, we identified three patients with pathogenic variants in the Troponin T type 1 (TNNT1) gene, coding for the troponin T (TNT) skeletal muscle isoform. RESULTS: The clinical phenotype was similar in all patients, associating hypotonia, orthopaedic deformities and progressive chronic respiratory failure, leading to early death. The anatomopathological phenotype was characterised by a disproportion in the muscle fibre size, endomysial fibrosis and nemaline rods. Molecular analyses of TNNT1 revealed a homozygous deletion of exons 8 and 9 in patient 1; a heterozygous nonsense mutation in exon 9 and retention of part of intron 4 in muscle transcripts in patient 2; and a homozygous, very early nonsense mutation in patient 3.Western blot analyses confirmed the absence of the TNT protein resulting from these mutations. DISCUSSION: The clinical and anatomopathological presentations of our patients reinforce the homogeneous character of the phenotype associated with recessive TNNT1 mutations. Previous studies revealed an impact of recessive variants on the tropomyosin-binding affinity of TNT. We report in our patients a complete loss of TNT protein due to open reading frame disruption or to post-translational degradation of TNT.

A Heterozygous Mutation in the Filamin C Gene Causes an Unusual Nemaline Myopathy With Ring Fibers.

Autosomal dominant pathogenic variants in the filamin C gene (FLNC) have been associated with myofibrillar myopathies, distal myopathies, and isolated cardiomyopathies. Mutations in different functional domains of FLNC can cause various clinical phenotypes. A novel heterozygous missense variant c.608G>A, p.(Cys203Tyr) in the actin binding domain of FLCN was found to cause an upper limb distal myopathy (MIM #614065). The muscle MRI findings are similar to those observed in FLNC-myofibrillar myopathy (MIM #609524). However, the muscle biopsy revealed >20% of muscle fibers with nemaline bodies, in addition to numerous ring fibers and a predominance of type 1 fibers. Overall, this case shows some unique and rare aspects of FLNC-myopathy constituting a new morphologic phenotype of FLNC-related myopathies.

Lamin-Related Congenital Muscular Dystrophy Alters Mechanical Signaling and Skeletal Muscle Growth.

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in the LMNA gene, which encodes the nuclear envelope proteins lamins A and C. The most frequent diseases associated with LMNA mutations are characterized by skeletal and cardiac involvement, and include autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B, and LMNA-related congenital muscular dystrophy (LMNA-CMD). Although the exact pathophysiological mechanisms responsible for LMNA-CMD are not yet understood, severe contracture and muscle atrophy suggest that mutations may impair skeletal muscle growth. Using human muscle stem cells (MuSCs) carrying LMNA-CMD mutations, we observe impaired myogenic fusion with disorganized cadherin/ß catenin adhesion complexes. We show that skeletal muscle from Lmna-CMD mice is unable to hypertrophy in response to functional overload, due to defective fusion of activated MuSCs, defective protein synthesis and defective remodeling of the neuromuscular junction. Moreover, stretched myotubes and overloaded muscle fibers with LMNA-CMD mutations display aberrant mechanical regulation of the yes-associated protein (YAP). We also observe defects in MuSC activation and YAP signaling in muscle biopsies from LMNA-CMD patients. These phenotypes are not recapitulated in closely related but less severe EDMD models. In conclusion, combining studies in vitro, in vivo, and patient samples, we find that LMNA-CMD mutations interfere with mechanosignaling pathways in skeletal muscle, implicating A-type lamins in the regulation of skeletal muscle growth.

Clathrin plaques and associated actin anchor intermediate filaments in skeletal muscle.

Clathrin plaques are stable features of the plasma membrane observed in several cell types. They are abundant in muscle, where they localize at costameres that link the contractile apparatus to the sarcolemma and connect the sarcolemma to the basal lamina. Here, we show that clathrin plaques and surrounding branched actin filaments form microdomains that anchor a three-dimensional desmin intermediate filament (IF) web. Depletion of clathrin plaque and branched actin components causes accumulation of desmin tangles in the cytoplasm. We show that dynamin 2, whose mutations cause centronuclear myopathy (CNM), regulates both clathrin plaques and surrounding branched actin filaments, while CNM-causing mutations lead to desmin disorganization in a CNM mouse model and patient biopsies. Our results suggest a novel paradigm in cell biology, wherein clathrin plaques act as platforms capable of recruiting branched cortical actin, which in turn anchors IFs, both essential for striated muscle formation and function.

'Dusty core disease' (DuCD): expanding morphological spectrum of RYR1 recessive myopathies.

Several morphological phenotypes have been associated to RYR1-recessive myopathies. We recharacterized the RYR1-recessive morphological spectrum by a large monocentric study performed on 54 muscle biopsies from a large cohort of 48 genetically confirmed patients, using histoenzymology, immunohistochemistry, and ultrastructural studies. We also analysed the level of RyR1 expression in patients' muscle biopsies. We defined "dusty cores" the irregular areas of myofibrillar disorganisation characterised by a reddish-purple granular material deposition with uneven oxidative stain and devoid of ATPase activity, which represent the characteristic lesion in muscle biopsy in 54% of patients. We named Dusty Core Disease (DuCD) the corresponding entity of congenital myopathy. Dusty cores had peculiar histological and ultrastructural characteristics compared to the other core diseases. DuCD muscle biopsies also showed nuclear centralization and type1 fibre predominance. Dusty cores were not observed in other core myopathies and centronuclear myopathies. The other morphological groups in our cohort of patients were: Central Core (CCD: 21%), Core-Rod (C&R:15%) and Type1 predominance "plus" (T1P+:10%). DuCD group was associated to an earlier disease onset, a more severe clinical phenotype and a lowest level of RyR1 expression in muscle, compared to the other groups. Variants located in the bridge solenoid and the pore domains were more frequent in DuCD patients. In conclusion, DuCD is the most frequent histopathological presentation of RYR1-recessive myopathies. Dusty cores represent the unifying morphological lesion among the DuCD pathology spectrum and are the morphological hallmark for the recessive form of disease.

Loss of Sarcomeric Scaffolding as a Common Baseline Histopathologic Lesion in Titin-Related Myopathies.

Titin-related myopathies are heterogeneous clinical conditions associated with mutations in TTN. To define their histopathologic boundaries and try to overcome the difficulty in assessing the pathogenic role of TTN variants, we performed a thorough morphological skeletal muscle analysis including light and electron microscopy in 23 patients with different clinical phenotypes presenting pathogenic autosomal dominant or autosomal recessive (AR) mutations located in different TTN domains. We identified a consistent pattern characterized by diverse defects in oxidative staining with prominent nuclear internalization in congenital phenotypes (AR-CM) (n = 10), ± necrotic/regenerative fibers, associated with endomysial fibrosis and rimmed vacuoles (RVs) in AR early-onset Emery-Dreifuss-like (AR-ED) (n = 4) and AR adult-onset distal myopathies (n = 4), and cytoplasmic bodies (CBs) as predominant finding in hereditary myopathy with early respiratory failure (HMERF) patients (n = 5). Ultrastructurally, the most significant abnormalities, particularly in AR-CM, were multiple narrow core lesions and/or clear small areas of disorganizations affecting one or a few sarcomeres with M-band and sometimes A-band disruption and loss of thick filaments. CBs were noted in some AR-CM and associated with RVs in HMERF and some AR-ED cases. As a whole, we described recognizable histopathological patterns and structural alterations that could point toward considering the pathogenicity of TTN mutations.

Mutations in GFPT1-related congenital myasthenic syndromes are associated with synaptic morphological defects and underlie a tubular aggregate myopathy with synaptopathy.

Mutations in GFPT1 (glutamine-fructose-6-phosphate transaminase 1), a gene encoding an enzyme involved in glycosylation of ubiquitous proteins, cause a limb-girdle congenital myasthenic syndrome (LG-CMS) with tubular aggregates (TAs) characterized predominantly by affection of the proximal skeletal muscles and presence of highly organized and remodeled sarcoplasmic tubules in patients' muscle biopsies. We report here the first long-term clinical follow-up of 11 French individuals suffering from LG-CMS with TAs due to GFPT1 mutations, of which nine are new. Our retrospective clinical evaluation stresses an evolution toward a myopathic weakness that occurs concomitantly to ineffectiveness of usual CMS treatments. Analysis of neuromuscular biopsies from three unrelated individuals demonstrates that the maintenance of neuromuscular junctions (NMJs) is dramatically impaired with loss of post-synaptic junctional folds and evidence of denervation-reinnervation processes affecting the three main NMJ components. Moreover, molecular analyses of the human muscle biopsies confirm glycosylation defects of proteins with reduced O-glycosylation and show reduced sialylation of transmembrane proteins in extra-junctional area. Altogether, these results pave the way for understanding the etiology of this rare neuromuscular disorder that may be considered as a "tubular aggregates myopathy with synaptopathy".

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy.

Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

QIL1 mutation causes MICOS disassembly and early onset fatal mitochondrial encephalopathy with liver disease.

Previously, we identified QIL1 as a subunit of mitochondrial contact site (MICOS) complex and demonstrated a role for QIL1 in MICOS assembly, mitochondrial respiration, and cristae formation critical for mitochondrial architecture (Guarani et al., 2015). Here, we identify QIL1 null alleles in two siblings displaying multiple clinical symptoms of early-onset fatal mitochondrial encephalopathy with liver disease, including defects in respiratory chain function in patient muscle. QIL1 absence in patients' fibroblasts was associated with MICOS disassembly, abnormal cristae, mild cytochrome c oxidase defect, and sensitivity to glucose withdrawal. QIL1 expression rescued cristae defects, and promoted re-accumulation of MICOS subunits to facilitate MICOS assembly. MICOS assembly and cristae morphology were not efficiently rescued by over-expression of other MICOS subunits in patient fibroblasts. Taken together, these data provide the first evidence of altered MICOS assembly linked with a human mitochondrial disease and confirm a central role for QIL1 in stable MICOS complex formation.

Impaired Presynaptic High-Affinity Choline Transporter Causes a Congenital Myasthenic Syndrome with Episodic Apnea.

The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.