Karen Gaudon

Identification of an Agrin Mutation that Causes Congenital Myasthenia and Affects Synapse Function

We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.

Phenotype genotype analysis in 15 patients presenting a congenital myasthenic syndrome due to mutations in DOK7

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of diseases caused by genetic defects affecting neuromuscular transmission. Mutations of DOK7 have recently been described in recessive forms of CMS. Dok-7 is a cytoplasmic post-synaptic protein co-activator of the muscle-specific receptor-tyrosine kinase (MuSK) involved in neuromuscular synaptogenesis and maintenance. We report clinical, morphological and molecular data on 15 patients with mutations in DOK7. Eleven different mutations (5 novel) were identified and all patients but one were found to carry at least the common c.1124_1127dupTGCC mutation. Patients with DOK7 mutations have a particular limb-girdle pattern, without tubular aggregates but a frequent lipidosis on the muscle biopsy. Changes in pre- and post-synaptic compartments of the neuromuscular junction were also observed in muscle biopsies: terminal axons showed defective branching which resulted in a unique terminal axon contacting en passant postsynaptic cups. Clinical features, muscle biopsy findings or response to therapy were confusing in several patients. Characterization of this distinct phenotype is essential to provide clues for targeted genetic screening and to predict the therapeutic response to anticholinesterase treatments or ephedrine as has been suggested.

A Mutation Causes MuSK Reduced Sensitivity to Agrin and Congenital Myasthenia

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of genetic disorders affecting neuromuscular transmission. The agrin/muscle-specific kinase (MuSK) pathway is critical for proper development and maintenance of the neuromuscular junction (NMJ). We report here an Iranian patient in whom CMS was diagnosed since he presented with congenital and fluctuating bilateral symmetric ptosis, upward gaze palsy and slowly progressive muscle weakness leading to loss of ambulation. Genetic analysis of the patient revealed a homozygous missense mutation c.2503A>G in the coding sequence of MUSK leading to the p.Met835Val substitution. The mutation was inherited from the two parents who were heterozygous according to the notion of consanguinity. Immunocytochemical and electron microscopy studies of biopsied deltoid muscle showed dramatic changes in pre- and post-synaptic elements of the NMJs. These changes induced a process of denervation/reinnervation in native NMJs and the formation, by an adaptive mechanism, of newly formed and ectopic NMJs. Aberrant axonal outgrowth, decreased nerve terminal ramification and nodal axonal sprouting were also noted. In vivo electroporation of the mutated MuSK in a mouse model showed disorganized NMJs and aberrant axonal growth reproducing a phenotype similar to that observed in the patient’s biopsy specimen. In vitro experiments showed that the mutation alters agrin-dependent acetylcholine receptor aggregation, causes a constitutive activation of MuSK and a decrease in its agrin- and Dok-7-dependent phosphorylation.

The CHRNE 1293insG founder mutation is a frequent cause of congenital myasthenia in North Africa.

Objective: Mutations in various genes of the neuromuscular junction cause congenital myasthenic syndrome (CMS). A single truncating mutation (ε1293insG) in the acetylcholine receptor epsilon subunit gene (CHRNE) was most often identified in CMS families originating from North Africa and was possibly a founder mutation. Methods: Twenty-three families were studied with an early onset form of CMS and originating from Tunisia, Algeria, Morocco, and Libya. Screening for the mutation ε1293insG was performed by direct sequencing. Haplotype analysis was done with 9 (CA)n repeat microsatellite markers and 6 SNPs flanking ε1293insG on chromosome 17p13-p12. Dating was calculated using the ESTIAGE method for rare genetic diseases. Results: The ε1293insG mutation was identified in 14 families (about 60% of the initial 23). The expression of the CMS in affected members of these families was relatively homogeneous, without fetal involvement or being life-threatening, with moderate hypotonia and oculobulbar involvement, mild and stable disease course, and good response to cholinesterase inhibitors. Haplotype analysis revealed a common conserved haplotype encompassing a distance of 63 kb. The estimated age of the founder event was at least 700 years. Conclusions: These results strongly support the hypothesis that ε1293insG derives from an ancient single founder event in the North African population. Identification of founder mutations in isolated or inbred populations may have important implications in the context of molecular diagnosis and genetic counseling of patients and families by detection of heterozygous carriers.

Multiexon deletions account for 15% of congenital myasthenic syndromes with RAPSN mutations after negative DNA sequencing

Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders that give rise to a defect in neuromuscular transmission. We described here three patients with a characteristic phenotype of recessive CMS and presenting mutation in the gene encoding rapsyn (RAPSN). Familial analysis showed that one allelic mutation failed to be detected by direct sequencing. An allelic quantification on patients DNA identified three novel multi-exon deletions of RAPSN. These three genomic rearrangements in RAPSN represent 15% of our CMS patients with RAPSN mutations and we emphasize that single-nucleotide polymorphism markers and a gene dosage method should be performed in addition to DNA direct sequencing analysis particularly when there is a genetic counselling issue.

Syndromes myasthéniques congénitaux dus à des mutations du gène de la rapsyne

Resume Debutant le plus souvent dans la periode neonatale, les syndromes myastheniques congenitaux (SMC) sont des affections genetiques a l’origine d’un dysfonctionnement de la transmission neuromusculaire. La majorite d’entre eux est due a des anomalies post-synaptiques, correspondant a des mutations du recepteur de l’acetylcholine (RACh). En 2002, le groupe d’A. Engel a rapporte les premiers cas de mutations du gene de la rapsyne qui est une autre molecule post-synaptique impliquee dans l’agregation du RACh. Depuis, plus d’une trentaine d’autres cas, dont 6 francais, ont ete rapportes. Trois categories de phenotypes cliniques ont ete trouvees : des formes neonatales severes ; 2) des formes plus legeres, debutant dans l’enfance ; 3) des cas affectant des patients Juifs originaires du Proche Orient, avec malformations faciales. Nos donnees sont confrontees aux autres observations publiees avec pour principales conclusions : la grande frequence de mutation N88K (homozygote dans 50 % des cas), une grande variete pour la seconde mutation (autre que N88K), la severite des cas heterozygotes (N88K + autre mutation), l’existence d’un effet fondateur dans la population europeenne. Il existe une variabilite phenotypique chez les patients homozygotes pour la mutation N88 K qui presentent un tableau clinique soit severe soit benin. Les 7 cas de SMC benins avec malformation faciale, precedemment decrits dans la population Juive d’Irak et d’Iran, ont ete rapportes par l’equipe d’A. Engel a une mutation homozygote de la region promotrice du gene.