New mutation in the beta 1 propeller domain of LRP4 responsible for congenital myasthenic syndrome associated with Cenani-Lenz syndrome

International audienceCongenital myasthenic syndromes are a clinically and genetically heterogeneous group of inherited disorders caused by defective synaptic transmission at the neuromuscular junction, characterized by muscle weakness and fatigability. Until now, many mutations encoding postsynaptic proteins as Agrin, MuSK and LRP4 have been identified as responsible for increasingly complex CMS phenotypes. The majority of mutations identified in LRP4 gene causes bone diseases including CLS and sclerosteosis-2 and in rare cases of CMS with mutations in LRP4 gene has been described so far.In the French cohort of CMS patients, we identified a novel LRP4 homozygous missense mutation (c.1820A>G; p.Thy607Cys) within the 1 propeller domain in a patient presenting CMS symptoms, including muscle weakness, fluctuating fatigability and a decrement in compound muscle action potential (CMAP) in spinal nerves, associated with congenital agenesis of the hands and feet and renal malformation. Expression studies revealed the decrease of agrin and Wnt11 binding to mutated β1 propeller domain leading to a disruption of LRP4/MuSK signaling associated with a lack of AChR aggregation in patient myotubes. Little improvement in the patient's symptoms were observed with the treatments usually used for CMS.To date, mutations in the LRP4 gene have been reported in several different phenotypes including CLS and CMS but never associated together in the same patient. Besides, this study appears useful for the future diagnosis and treatment of patients with co-existing CLS and CMS and highlights the importance of structural and functional studies in the diagnosis and possible treatment of these atypical forms

Identification of a new splice site mutation in synaptotagmin-2 responsible for a severe and early presynaptic form of congenital myasthenic syndrome

International audienceCongenital myasthenic syndromes (CMS) are a clinically and genetically heterogeneous group of inherited disorders caused by defective synaptic transmission at the neuromuscular junction (NMJ) and characterized by fluctuation of muscle weakness and fatigability. Recently, many mutations encoding presynaptic and ubiquitous proteins have been identified as responsible for increasingly complex CMS phenotypes of CMS. Among them, this is the case of autosomal dominant mutations in Synaptotagmin2 (SYT2) C2B domain that have been linked to described as responsible for presynaptic CMS combined to Lambert-Eaton myasthenic syndromes and motor neuropathy forms. SYT2 is the major synaptotagmin isoform expressed at the NMJ and acts as a calcium sensor that is mediated by the presence of two tandem C2 domains. In the French cohort of CMS patients, we recently identified in a consanguineous family a new homozygote recessive intronic mutation in SYT2 causing an early and severe presynaptic CMS. Using a minigene construct we demonstrated that this intronic mutation in the donor splice site of SYT2 intron 4 leads to a SYT2 in-frame exon 4 skipping suppressing the N-terminal part of C2A domain. Morphological and functional studies revealed that defects in SYT2 C2A domain affects NMJs maintenance, synaptic transmission and triggers a decrease of SYT2 expression partially compensated by the upregulation of SYT1 expression at the NMJ. This study reports the identification of a new severe presynaptic CMS form associated to a recessive intronic mutation in SYT2 and completes the previously reported data on the dominant SYT2-related motor neuropathy and Lambert-Eaton myasthenic syndrome

Identification of a new splice site mutation in synaptotagmin-2 responsible for a severe and early presynaptic form of congenital myasthenic syndrome

International audienceCongenital myasthenic syndromes (CMS) are a clinically and genetically heterogeneous group of inherited disorders caused by defective synaptic transmission at the neuromuscular junction (NMJ) and characterized by fluctuation of muscle weakness and fatigability. Recently, many mutations encoding presynaptic and ubiquitous proteins have been identified as responsible for increasingly complex CMS phenotypes of CMS. Among them, this is the case of autosomal dominant mutations in Synaptotagmin2 (SYT2) C2B domain that have been linked to described as responsible for presynaptic CMS combined to Lambert-Eaton myasthenic syndromes and motor neuropathy forms. SYT2 is the major synaptotagmin isoform expressed at the NMJ and acts as a calcium sensor that is mediated by the presence of two tandem C2 domains. In the French cohort of CMS patients, we recently identified in a consanguineous family a new homozygote recessive intronic mutation in SYT2 causing an early and severe presynaptic CMS. Using a minigene construct we demonstrated that this intronic mutation in the donor splice site of SYT2 intron 4 leads to a SYT2 in-frame exon 4 skipping suppressing the N-terminal part of C2A domain. Morphological and functional studies revealed that defects in SYT2 C2A domain affects NMJs maintenance, synaptic transmission and triggers a decrease of SYT2 expression partially compensated by the upregulation of SYT1 expression at the NMJ. This study reports the identification of a new severe presynaptic CMS form associated to a recessive intronic mutation in SYT2 and completes the previously reported data on the dominant SYT2-related motor neuropathy and Lambert-Eaton myasthenic syndrome

The cell polarity protein Vangl2 in the muscle shapes the neuromuscular synapse by binding to and regulating the tyrosine kinase MuSK

International audienceThe development of the neuromuscular junction (NMJ) requires dynamic trans-synaptic coordination orchestrated by secreted factors, including Wnt family morphogens. To investigate how these synaptic cues in NMJ development are transduced, particularly in the regulation of acetylcholine receptor (AChR) accumulation in the postsynaptic membrane, we explored the function of Van Gogh-like protein 2 (Vangl2), a core component of Wnt planar cell polarity signaling. We found that conditional, muscle-specific ablation of Vangl2 in mice reproduced the NMJ differentiation defects seen in mice with global Vangl2 deletion. These alterations persisted into adulthood and led to NMJ disassembly, impaired neurotransmission, and deficits in motor function. Vangl2 and the musclespecific receptor tyrosine kinase MuSK were functionally associated in Wnt signaling in the muscle. Vangl2 bound to and promoted the signaling activity of MuSK in response to Wnt11. The loss of Vangl2 impaired RhoA activation in cultured mouse myotubes and caused dispersed, rather than clustered, organization of AChRs at the postsynaptic or muscle cell side of NMJs in vivo. Our results identify Vangl2 as a key player of the core complex of molecules shaping neuromuscular synapses and thus shed light on the molecular mechanisms underlying NMJ assembly

New mutation in the β1 propeller domain of LRP4 responsible for congenital myasthenic syndrome associated with Cenani–Lenz syndrome

International audienceCongenital myasthenic syndromes (CMS) are a clinically and genetically heterogeneous group of rare diseases due to mutations in neuromuscular junction (NMJ) protein-coding genes. Until now, many mutations encoding postsynaptic proteins as Agrin, MuSK and LRP4 have been identified as responsible for increasingly complex CMS phenotypes. The majority of mutations identified in LRP4 gene causes bone diseases including CLS and sclerosteosis-2 and rare cases of CMS with mutations in LRP4 gene has been described so far. In the French cohort of CMS patients, we identified a novel LRP4 homozygous missense mutation (c.1820A > G; p.Thy607Cys) within the β1 propeller domain in a patient presenting CMS symptoms, including muscle weakness, fluctuating fatigability and a decrement in compound muscle action potential in spinal accessory nerves, associated with congenital agenesis of the hands and feet and renal malformation. Mechanistic expression studies show a significant decrease of AChR aggregation in cultured patient myotubes, as well as altered in vitro binding of agrin and Wnt11 ligands to the mutated β1 propeller domain of LRP4 explaining the dual phenotype characterized clinically and electoneuromyographically in the patient. These results expand the LRP4 mutations spectrum associated with a previously undescribed clinical association involving impaired neuromuscular transmission and limb deformities and highlighting the critical role of a yet poorly described domain of LRP4 at the NMJ. This study raises the question of the frequency of this rare neuromuscular form and the future diagnosis and management of these cases

New recessive mutations in SYT2 causing severe presynaptic congenital myasthenic syndromes

International audienceObjective To report the identification of 2 new homozygous recessive mutations in the synaptotagmin 2 ( SYT2 ) gene as the genetic cause of severe and early presynaptic forms of congenital myasthenic syndromes (CMSs). Methods Next-generation sequencing identified new homozygous intronic and frameshift mutations in the SYT2 gene as a likely cause of presynaptic CMS. We describe the clinical and electromyographic patient phenotypes, perform ex vivo splicing analyses to characterize the effect of the intronic mutation on exon splicing, and analyze the functional impact of this variation at the neuromuscular junction (NMJ). Results The 2 infants presented a similar clinical phenotype evoking first a congenital myopathy characterized by muscle weakness and hypotonia. Next-generation sequencing allowed to the identification of 1 homozygous intronic mutation c.465+1G>A in patient 1 and another homozygous frameshift mutation c.328_331dup in patient 2, located respectively in the 5′ splice donor site of SYT2 intron 4 and in exon 3. Functional studies of the intronic mutation validated the abolition of the splice donor site of exon 4 leading to its skipping. In-frame skipping of exon 4 that encodes part of the C2A calcium-binding domain of SYT2 is associated with a loss-of-function effect resulting in a decrease of neurotransmitter release and severe pre- and postsynaptic NMJ defects. Conclusions This study identifies new homozygous recessive SYT2 mutations as the underlying cause of severe and early presynaptic form of CMS expanding the genetic spectrum of recessive SYT2 -related CMS associated with defects in neurotransmitter release

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

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

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

International audienceThe neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMS5), 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