Expanding the spectrum of LAMB2: Pierson syndrome associated with neuromuscular junction disorder in two patients.

LAMB2 gene disorders present with different phenotypes. Pierson syndrome (PS) is a common phenotype associated with LAMB2 variants. Neuromuscular phenotype has been reported including hypotonia and developmental delay. However, neuromuscular junction abnormalities represented as congenital myasthenic syndrome (CMS) was reported in one adult patient only. Here, in this paper, we present two pediatric cases with a severe presentation of PS and have CMS so expanding the knowledge of LAMB2 related phenotypes. The first patient had hypotonia and global developmental delay. Targeted genetic testing panel demonstrated homozygous pathogenic variant in the LAMB2 gene (c.5182C>T, pGln1728*) which was reported by Maselli et al. 2009. Repetitive nerve stimulation (RNS) showed a decremental response at low frequency of 3 Hz. On the other hand, the second patient had profound weakness since birth. Tri-Whole exome sequencing showed homozygous pathogenic variant in the LAMB2 gene c.2890C>T, pArg964*. A trial of salbutamol did not improve the symptoms. Both patients passed away from sequala of PS. The spectrum of phenotypic changes associated with LAMB2 mutations is still expanding, and further investigation into the various clinical and morphologic presentations associated with these mutations is important to better identify and manage affected individuals.

Similar metabolic pathways are affected in both Congenital Myasthenic Syndrome-22 and Prader-Willi Syndrome.

Loss of prolyl endopeptidase-like (PREPL) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome-22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC-p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl-/- and PWS-IC-p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular parallels between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders.

[Prenatal diagnosis of a case with Congenital myasthenic syndrome due to compound heterozygous variants of SCN4A gene].

OBJECTIVE: To explore the clinical and genetic characteristics of a fetus diagnosed with Congenital myasthenic syndrome type 16 (CMS16). METHODS: A couple who had visited Tianjin Medical University General Hospital in February 2018 due to "adverse outcome of two pregnancies" was selected as the study subject. Clinical data was gathered. Peripheral blood and amniotic fluid samples were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. Low-depth whole-genome sequencing was carried out to detect copy number variation (CNV) in the fetus. RESULTS: The couple's first pregnancy had resulted in a miscarriage at 27+5 weeks, when ultrasound had revealed pleural effusion and polyhydramnios in the fetus. Their second pregnancy was terminated at 30+5 weeks due to fetal hand malformations, polyhydramnios and pleural fluid. Both couple had denied family history of genetic conditions. For their third pregnancy, no CNV abnormality was detected, whilst a compound heterozygous variants, including a maternally derived c.3172C>T (p.R1058W) and paternal c.1431delG (p.K477fs*89) in the SCN4A gene were detected. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.3172C>T (p.R1058W) was predicted as a likely pathogenic variant (PM1+PM2_supporting+PP3+PP4), whilst the c.1431delG (p.K477fs*89) was predicted as a pathogenic variant (PVS1+PM2_supporting+PP4). CONCLUSION: The c.3172C>T (p.R1058W) and c.1431delG (p.K477fs*89) compound heterozygous variants of the SCN4A gene probably underlay the CMS16 in the third fetus.

Preimplantation genetic testing as a means of preventing hereditary congenital myasthenic syndrome caused by RAPSN.

BACKGROUND: Congenital myasthenic syndrome is a heterogeneous group of inherited neuromuscular transmission disorders. Variants in RAPSN are a common cause of CMS, accounting for approximately 14%-27% of all CMS cases. Whether preimplantation genetic testing for monogenic disease (PGT-M) could be used to prevent the potential birth of CMS-affected children is unclear. METHODS: Application of WES (whole-exome sequencing) for carrier testing and guidance for the PGT-M in the absence of a genetically characterized index patient as well as assisted reproductive technology were employed to prevent the occurrence of birth defects in subsequent pregnancy. The clinical phenotypes of stillborn fetuses were also assessed. RESULTS: The family carried two likely pathogenic variants in RAPSN(NM_005055.5): c.133G>A (p.V45M) and c.280G>A (p.E94K). And the potential birth of CMS-affected child was successfully prevented, allowing the family to have offspring devoid of disease-associated variants and exhibiting a normal phenotype. CONCLUSION: This report constitutes the first documented case of achieving a CMS-free offspring through PGT-M in a CMS-affected family. By broadening the known variant spectrum of RAPSN in the Chinese population, our findings underscore the feasibility and effectiveness of PGT-M for preventing CMS, offering valuable insights for similarly affected families.

COLQ-Congenital myasthenic syndrome in an Iranian cohort: the clinical and genetics spectrum.

BACKGROUND: Congenital myasthenic syndrome (CMS) is a group of neuromuscular disorders caused by abnormal signal transmission at the motor endplate. Mutations in the collagen-like tail subunit gene (COLQ) of acetylcholinesterase are responsible for recessive forms of synaptic congenital myasthenic syndromes with end plate acetylcholinesterase deficiency. Clinical presentation includes ptosis, ophthalmoparesis, and progressive weakness with onset at birth or early infancy. METHODS: We followed 26 patients with COLQ-CMS over a mean period of 9 years (ranging from 3 to 213 months) and reported their clinical features, electrophysiologic findings, genetic characteristics, and therapeutic management. RESULTS: In our population, the onset of symptoms ranged from birth to 15 years. Delayed developmental motor milestones were detected in 13 patients (∼ 52%), and the most common presenting signs were ptosis, ophthalmoparesis, and limb weakness. Sluggish pupils were seen in 8 (∼ 30%) patients. All patients who underwent electrophysiologic study showed a significant decremental response (> 10%) following low-frequency repetitive nerve stimulation. Moreover, double compound muscle action potential was evident in 18 patients (∼ 75%). We detected 14 variants (eight novel variants), including six missense, three frameshift, three nonsense, one synonymous and one copy number variation (CNV), in the COLQ gene. There was no benefit from esterase inhibitor treatment, while treatment with ephedrine and salbutamol was objectively efficient in all cases. CONCLUSION: Despite the rarity of the disease, our findings provide valuable information for understanding the clinical and electrophysiological features as well as the genetic characterization and response to the treatment of COLQ-CMS.

VAMP1-Related Congenital Myasthenic Syndrome: A Case Report and Literature Review.

Congenital myasthenic syndrome-25 (CMS-25) is an autosomal recessive neuromuscular disorder caused by a homozygous mutation in VAMP1 gene. To date, only eight types of allelic variants in VAMP1 gene have been reported in 12 cases of CMS-25. Here, we report on an 8-year-old boy with motor developmental delay, axial hypotonia, myopathic face, muscle weakness, strabismus, ptosis, pectus carinatum, kyphoscoliosis, joint contractures, joint laxity, seizures, and recurrent nephrolithiasis. He also had feeding difficulties and recurrent aspiration pneumonia. Brain magnetic resonance imaging at 20 months of age showed left focal cerebellar hypoplasia. Genetic analysis revealed a homozygous missense variant of c.202C > T (p.Arg68Ter) in the VAMP1 gene. Treatment with oral pyridostigmine was started, which resulted in mild improvement in muscle strength. Salbutamol syrup was added a few months later, but no significant improvement was observed. This case report presents novel findings such as focal cerebellar hypoplasia and nephrolithiasis in VAMP1-related CMS-25. Consequently, this case report extends the clinical spectrum. Further studies are needed to expand the genotype-phenotype correlations in VAMP1-related CMS-25.

Rare disease research workflow using multilayer networks elucidates the molecular determinants of severity in Congenital Myasthenic Syndromes.

Exploring the molecular basis of disease severity in rare disease scenarios is a challenging task provided the limitations on data availability. Causative genes have been described for Congenital Myasthenic Syndromes (CMS), a group of diverse minority neuromuscular junction (NMJ) disorders; yet a molecular explanation for the phenotypic severity differences remains unclear. Here, we present a workflow to explore the functional relationships between CMS causal genes and altered genes from each patient, based on multilayer network community detection analysis of complementary biomedical information provided by relevant data sources, namely protein-protein interactions, pathways and metabolomics. Our results show that CMS severity can be ascribed to the personalized impairment of extracellular matrix components and postsynaptic modulators of acetylcholine receptor (AChR) clustering. This work showcases how coupling multilayer network analysis with personalized -omics information provides molecular explanations to the varying severity of rare diseases; paving the way for sorting out similar cases in other rare diseases.

Expanding the genetic and phenotypic spectrum of congenital myasthenic syndrome: new homozygous VAMP1 splicing variants in 2 novel individuals.

We report the cases of two Spanish pediatric patients with hypotonia, muscle weakness and feeding difficulties at birth. Whole-exome sequencing (WES) uncovered two new homozygous VAMP1 (Vesicle Associated Membrane Protein 1) splicing variants, NM_014231.5:c.129+5 G > A in the boy patient (P1) and c.341-24_341-16delinsAGAAAA in the girl patient (P2). This gene encodes the vesicle-associated membrane protein 1 (VAMP1) that is a component of a protein complex involved in the fusion of synaptic vesicles with the presynaptic membrane. VAMP1 has a highly variable C-terminus generated by alternative splicing that gives rise to three main isoforms (A, B and D), being VAMP1A the only isoform expressed in the nervous system. In order to assess the pathogenicity of these variants, expression experiments of RNA for VAMP1 were carried out. The c.129+5 G > A and c.341-24_341-16delinsAGAAAA variants induced aberrant splicing events resulting in the deletion of exon 2 (r.5_131del; p.Ser2TrpfsTer7) in the three isoforms in the first case, and the retention of the last 14 nucleotides of the 3' of intron 4 (r.340_341ins341-14_341-1; p.Ile114AsnfsTer77) in the VAMP1A isoform in the second case. Pathogenic VAMP1 variants have been associated with autosomal dominant spastic ataxia 1 (SPAX1) and with autosomal recessive presynaptic congenital myasthenic syndrome (CMS). Our patients share the clinical manifestations of CMS patients with two important differences: they do not show the typical electrophysiological pattern that suggests pathology of pre-synaptic neuromuscular junction, and their muscular biopsies present hypertrophic fibers type 1. In conclusion, our data expand both genetic and phenotypic spectrum associated with VAMP1 variants.

[Congenital Myasthenic Syndromes].

Congenital myasthenic syndromes (CMS) are characterized by congenital defects in the neuromuscular signal transmission and are caused by pathogenic variants in 36 genes. Recently identified forms of CMS include TOR1AIP1-CMS, CHD8-CMS, PURA-CMS, and TEFM-CMS. Most forms of CMS are caused by autosomal recessive variants, whereas four forms of CMS are caused by autosomal dominant variants, in which adult-onset cases are not rare. As myasthenic features are not always observed and muscle hypotrophy is sometimes observed, CMS should be considered in differential diagnosis of congenital myopathies and other neuromuscular diseases. Low- and high-frequency repetitive nerve stimulation is essential to diagnose CMS for patients who develop muscle weakness at less than 2 years of age. Tubular aggregates are observed in muscle biopsy in four forms of CMS, and serum CK levels are elevated in some forms of CMS. As rational therapies are available for most forms of CMS, identification of causative gene variants by genetic analysis is required.

Alteration of actin cytoskeletal organisation in fetal akinesia deformation sequence.

Fetal akinesia deformation sequence (FADS) represents the severest form of congenital myasthenic syndrome (CMS), a diverse group of inherited disorders characterised by impaired neuromuscular transmission. Most CMS originate from defects in the muscle nicotinic acetylcholine receptor, but the underlying molecular pathogenesis is only poorly understood. Here we show that RNAi-mediated silencing of FADS-related proteins rapsyn and NUP88 in foetal fibroblasts alters organisation of the actin cytoskeleton. We show that fibroblasts from two independent FADS individuals have enhanced and shorter actin stress fibre bundles, alongside with an increased number and size of focal adhesions, with an otherwise normal overall connectivity and integrity of the actin-myosin cytoskeleton network. By proximity ligation assays and bimolecular fluorescence complementation, we show that rapsyn and NUP88 localise nearby adhesion plaques and that they interact with the focal adhesion protein paxillin. Based on these findings we propose that a respective deficiency in rapsyn and NUP88 in FADS alters the regulation of actin dynamics at focal adhesions, and thereby may also plausibly dictate myofibril contraction in skeletal muscle of FADS individuals.

Calcitriol ameliorates motor deficits and prolongs survival of Chrne-deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2.

Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including congenital myasthenic syndromes (CMS). Germline mutations in CHRNE encoding the acetylcholine receptor (AChR) ε subunit are the most common cause of CMS. An active form of vitamin D, calcitriol, binds to vitamin D receptor (VDR) and regulates gene expressions. We found that calcitriol enhanced MuSK phosphorylation, AChR clustering, and myotube twitching in co-cultured C2C12 myotubes and NSC34 motor neurons. RNA-seq analysis of co-cultured cells showed that calcitriol increased the expressions of Rspo2, Rapsn, and Dusp6. ChIP-seq of VDR revealed that VDR binds to a region approximately 15 â€‹kbp upstream to Rspo2. Biallelic deletion of the VDR-binding site of Rspo2 by CRISPR/Cas9 in C2C12 myoblasts/myotubes nullified the calcitriol-mediated induction of Rspo2 expression and MuSK phosphorylation. We generated Chrne knockout (Chrne KO) mouse by CRISPR/Cas9. Intraperitoneal administration of calcitriol markedly increased the number of AChR clusters, as well as the area, the intensity, and the number of synaptophysin-positive synaptic vesicles, in Chrne KO mice. In addition, calcitriol ameliorated motor deficits and prolonged survival of Chrne KO mice. In the skeletal muscle, calcitriol increased the gene expressions of Rspo2, Rapsn, and Dusp6. We propose that calcitriol is a potential therapeutic agent for CMS and other diseases with defective neuromuscular signal transmission.

Mutations in PTPN11 could lead to a congenital myasthenic syndrome phenotype: a Noonan syndrome case series.

The RASopathies are a group of genetic rare diseases caused by mutations affecting genes involved in the RAS/MAPK (RAS-mitogen activated protein kinase) pathway. Among them, PTPN11 pathogenic variants are responsible for approximately 50% of Noonan syndrome (NS) cases and, albeit to a lesser extent, of Leopard syndrome (LPRD1), which present a few overlapping clinical features, such as facial dysmorphism, developmental delay, cardiac defects, and skeletal deformities. Motor impairment and decreased muscle strength have been recently reported. The etiology of the muscle involvement in these disorders is still not clear but probably multifactorial, considering the role of the RAS/MAPK pathway in skeletal muscle development and Acetylcholine Receptors (AChR) clustering at the neuromuscular junction (NMJ). We report, herein, four unrelated children carrying three different heterozygous mutations in the PTPN11 gene. Intriguingly, their phenotypic features first led to a clinical suspicion of congenital myasthenic syndrome (CMS), due to exercise-induced fatigability with a variable degree of muscle weakness, and serum proteomic profiling compatible with a NMJ defect. Moreover, muscle fatigue improved after treatment with CMS-specific medication. Although the link between PTPN11 gene and neuromuscular transmission is unconfirmed, an increasing number of patients with RASopathies are affected by muscle weakness and fatigability. Hence, NS or LPDR1 should be considered in children with suspected CMS but negative genetic workup for known CMS genes or additional symptoms indicative of NS, such as facial dysmorphism or intellectual disability.

Clinical and genetic characterisation of a large Indian congenital myasthenic syndrome cohort.

Congenital myasthenic syndromes (CMS) are a rare group of inherited disorders caused by gene defects associated with the neuromuscular junction and potentially treatable with commonly available medications such as acetylcholinesterase inhibitors and ß2 adrenergic receptor agonists. In this study, we identified and genetically characterized the largest cohort of CMS patients from India to date. Genetic testing of clinically suspected patients evaluated in a South Indian hospital during the period 2014-19 was carried out by standard diagnostic gene panel testing or using a two-step method that included hotspot screening followed by whole-exome sequencing. In total, 156 genetically diagnosed patients (141 families) were characterized and the mutational spectrum and genotype-phenotype correlation described. Overall, 87 males and 69 females were evaluated, with the age of onset ranging from congenital to fourth decade (mean 6.6 ± 9.8 years). The mean age at diagnosis was 19 ± 12.8 (1-56 years), with a mean diagnostic delay of 12.5 ± 9.9 (0-49 years). Disease-causing variants in 17 CMS-associated genes were identified in 132 families (93.6%), while in nine families (6.4%), variants in genes not associated with CMS were found. Overall, postsynaptic defects were most common (62.4%), followed by glycosylation defects (21.3%), synaptic basal lamina genes (4.3%) and presynaptic defects (2.8%). Other genes found to cause neuromuscular junction defects (DES, TEFM) in our cohort accounted for 2.8%. Among the individual CMS genes, the most commonly affected gene was CHRNE (39.4%), followed by DOK7 (14.4%), DPAGT1 (9.8%), GFPT1 (7.6%), MUSK (6.1%), GMPPB (5.3%) and COLQ (4.5%). We identified 22 recurrent variants in this study, out of which eight were found to be geographically specific to the Indian subcontinent. Apart from the known common CHRNE variants p.E443Kfs*64 (11.4%) and DOK7 p.A378Sfs*30 (9.3%), we identified seven novel recurrent variants specific to this cohort, including DPAGT1 p.T380I and DES c.1023+5G>A, for which founder haplotypes are suspected. This study highlights the geographic differences in the frequencies of various causative CMS genes and underlines the increasing significance of glycosylation genes (DPAGT1, GFPT1 and GMPPB) as a cause of neuromuscular junction defects. Myopathy and muscular dystrophy genes such as GMPPB and DES, presenting as gradually progressive limb girdle CMS, expand the phenotypic spectrum. The novel genes MACF1 and TEFM identified in this cohort add to the expanding list of genes with new mechanisms causing neuromuscular junction defects.

LRP4 site-specific variants in the third ß-propeller domain causes congenital myasthenic syndrome type 17.

LRP4 is expressed in many organs. It mediates SOST-dependent inhibition of bone formation and acts as an inhibitor of WNT signaling. It is also a postsynaptic end plate cell surface receptor at the neuromuscular junction and is central to its development, maintenance, and function. Pathogenic variants of LRP4 that specifically affect the canonical WNT signaling pathway are known to be associated with Cenani-Lenz syndactyly syndrome or the overlapping condition sclerosteosis. However, site-specific pathogenic variants of LRP4 have been associated with the congenital myasthenic syndrome (CMS) type 17 with no abnormal bone phenotype. Only two studies reported biallelic variants of LRP4 associated with CMS17 that presented during childhood. All three reported variants (NM_002334.4: p.Glu1233Ala, p.Glu1233Lys, or p.Arg1277His) are located within the 3'-edge of the third ß-propeller domain of LRP4. We report on a patient with a biallelic variant of the LRP4 gene presenting with a severe and neonatal lethal phenotype; we also provide a literature review of the previously reported patients. A female neonate, born to healthy consanguineous parents, presented with severe hypotonia, congenital diaphragmatic hernia, pulmonary hypertension, and progressive hypoxemia. Two of her siblings presented with a similar condition in the past, and all three died shortly after birth. Clinical exome sequencing revealed homozygosity for the pathogenic variant NM_002334.4:c.3698A > C (p.[Glu1233Ala]).

Electrophysiological evaluation of the neuromuscular junction: a brief review.

The nerve terminal and muscle membrane compose the neuromuscular junction. After opening the voltage-gated calcium channels, action potentials from the motor axons provoke a cascade for the acetylcholine release from synaptic vesicles to the synaptic cleft, where it binds to its receptor at the muscle membrane for depolarization. Low amplitude compound muscle action potential typically presents in presynaptic disorders, increasing by more than 100% after a 10-second effort in the Lambert-Eaton myasthenic syndrome and less in botulism. Needle electromyography may show myopathic motor unit action potentials and morphological instability ("jiggle") due to impulse blocking. Low-frequency repetitive nerve stimulation (RNS) is helpful in postsynaptic disorders, such as myasthenia gravis and most congenital myasthenic syndromes, where the number of functioning acetylcholine receptors is reduced. Low-frequency RNS with a decrement >10% is abnormal when comparing the 4th to the first compound muscle action potential amplitude. High-frequency RNS is helpful in presynaptic disorders like Lambert-Eaton myasthenic syndrome, botulism, and some rare congenital myasthenic syndromes. The high-frequency RNS releases more calcium, increasing the acetylcholine with a compound muscle action potential increment. Concentric needle records apparent single-fiber action potentials (spikes). A voluntary activation measures the jitter between spikes from two endplates. An electrical activation measures the jitter of one spike (one endplate). The jitter is the most sensitive test for detecting a neuromuscular junction dysfunction. Most neuromuscular junction disorders are responsive to treatment.

O nervo terminal e a membrana muscular compõem a junção neuromuscular. Após a abertura dos canais de cálcio dependentes de voltagem, os potenciais de ação do axônio motor provocam uma cascata de eventos que libera acetilcolina das vesículas para a fenda sináptica, ligando-se ao receptor na membrana muscular para despolarização. O potencial de ação muscular composto de baixa amplitude ocorre nas desordens pré-sinápticas, aumentando em mais de 100% após esforço de 10 segundos na síndrome miastênica de Lambert-Eaton e menos no botulismo. A eletromiografia pode mostrar potenciais de ação da unidade motora miopáticos e instabilidade morfológica ("jiggle") devido ao bloqueio do impulso. Estimulação nervosa repetitiva (ENR) de baixa frequência é útil nos distúrbios pós-sinápticos, como miastenia gravis e a maioria das síndromes miastênicas congênitas, quando há número reduzido de receptores de acetilcolina funcionantes. ENR de baixa frequência com decremento >10% é anormal comparando-se à amplitude do quarto com o primeiro potencial de ação muscular composto. ENR de alta frequência é útil nas doenças pré-sinápticas, como síndrome miastênica de Lambert-Eaton, botulismo e algumas síndromes miastênicas congênitas raras. ENR de alta frequência libera mais cálcio, aumenta acetilcolina, resultando em incremento do potencial de ação muscular composto. O eletrodo de agulha concêntrico registra potenciais de ação aparente de fibra única (PAAFU). Ativação voluntária mede jitter entre dois PAAFUs (duas junções neuromusculares). Ativação elétrica mede jitter de um PAAFU (uma junção neuromuscular). Jitter é o teste mais sensível para detectar disfunção de junção neuromuscular. A maioria dos distúrbios juncionais é responsiva ao tratamento.