Electrophysiological and morphological characterization of a case of autosomal recessive congenital myasthenic syndrome with acetylcholine receptor deficiency due to a N88K rapsyn homozygous mutation
Introduction
Congenital myasthenic syndromes (CMSs) are rare hereditary diseases that cause dysfunction of neuromuscular transmission resulting in fatigable muscle weakness. CMSs form a heterogeneous group of disorders, which are classified as originating from pre-synaptic, synaptic or post-synaptic defects (for a review see Ref. [1]). Within the class of post-synaptic CMSs, genes coding for the different subunits of the acetylcholine receptor (AChR) have been identified as causing kinetic abnormalities of the AChR or low expression of the AChR [1]. In patients with low AChR expression where the genes coding for the AChR subunits have been excluded, three mutations in the rapsyn gene (RAPSN) have been reported for the first time [2]. Since then several novel rapsyn mutations were described by our group and others [3], [4], [5], [6].
Rapsyn is a 43 kDa peripheral protein [7], [8], which is required for AChR clustering (for a review see Ref. [9]). Rapsyn consists of a myristoylated amino terminus, seven tetratricopeptide repeats (TPRs), a coiled-coil domain and a RING-H2 domain. The role of each part has been elucidated and it was shown that the TPR domains are sufficient for self-association, the coiled-coil domain is necessary for nicotinic AChR clustering [10] and the RING-H2 domain interacts with the β-dystroglycan cytoplasmic domain [11].
During neuromuscular junction (NMJ) development, the agrin–MuSK–rapsyn pathway plays a key role in clustering nicotinic AChRs. The nerve releases a protein called agrin, which binds to a muscle-specific tyrosine kinase known as MuSK. MuSK then acts downstream through a cytoplasmic effector protein, rapsyn, which is phosphorylated and acts as a linker to promote AChR clustering [9], [12]. The role of these interacting molecules in this pathway is not completely understood. Rapsyn knockout mice lack AChR clusters and die at birth from respiratory failure. Their cultured myotubes fail to respond to agrin [13]. Recently, it was shown that some of the molecules implicated in this pathway are related to human diseases. Rapsyn and MuSK antibodies were identified in myasthenia gravis patients without AChR antibodies [14], [15] and RAPSN mutations were found in some CMS patients. Patients who have mutations in RAPSN have diminished nicotinic AChRs and rapsyn at NMJs in their muscle biopsy. Similarly, co-expression of rapsyn mutations with AChR subunits in cultured HEK cells diminishes co-clustering of rapsyn and AChRs [2].
In this study, we excluded mutations in the genes coding for the different subunits of the AChR and identified the rapsyn N88K mutation in a patient presenting a CMS with AChR deficiency. We demonstrated that both rapsyn and AChR were drastically reduced at the NMJ whose subneural structure became unfolded. Finally, microelectrophysiology suggested that in addition to the reduction in subsynaptic receptor density, interference with rapsyn–AChR interactions led to pronounced frequency-dependent synaptic depression.
Section snippets
Patient
The propositus was a 28-year-old French man. His parents were from two different but close French regions: the Corrèze (mother) and the Dordogne (father). His birth was uneventful and he developed normally during infancy. From the age of 5 years, he complained of easy fatigability during exercise, had a waddling gait and was unable to run fast. Fluctuations were very clear with walking distance from 800 m at best to 10 m at worst. His symptoms were aggravated at the end of the day or when he
Reduction of AChR and rapsyn at the NMJ
Histochemical examinations of muscle cryostat sections showed predominance of type I fibres and atrophy of type II fibres by ATPase staining (not shown). On fluorescence confocal microscopy, examination of NMJs stained for acetylcholinesterase by FAS-2 and for AChR by α-BTX showed that the α-BTX fluorescence was considerably diminished as compared with controls. Furthermore, the area of the NMJ stained for AChR was also diminished as compared with that stained for acetylcholinesterase (Fig. 1).
Discussion
We report a case of autosomal recessive CMS with AChR deficiency due to a N88K rapsyn homozygous mutation. This N88K mutation was for the first time reported in four patients by the Engel group [2] and subsequently in several patients by our group and others [3], [4], [5], [6]. On the whole, 26 patients including the present case shared this mutation, which was either homozygous like in the present case report or compound heterozygous (50% of patients in each group) [2], [3], [4], [5], [6].
Acknowledgments
We would like to thank Dr Stanley C. Froehner and Dr Eric Krejci for their generous gifts of anti-rapsyn antibody and fluorochrome-tagged fasciculin, respectively. Dr Eriko Yasaki was a recipient of a post-doctoral fellowship from the Association Française contre les Myopathies (AFM) and Dr Cassandra Prioleau from the INSERM (Poste Vert). Julien Barbier was supported by a fellowship from the Direction des Systèmes de Forces et de la Prospective. This work was supported by INSERM, Assistance
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Clinical variability of early-onset congenital myasthenic syndrome due to biallelic RAPSN mutations in Brazil
2018, Neuromuscular DisordersCitation Excerpt :From 73 reported RAPSN cases with outcome data, all except one (an early-onset RAPSN CMS case reported by Burke et al. [15]) presented some level of disability after long-term follow-up. In those cases, symptomatic treatment is usually required for life, especially if respiratory distress is present [8,12–22]. It is worth noting that there is no consensus regarding the prevention of episodic apneas with acetylcholinesterase inhibitors and whether there is a safe time point at which to cease it [12–14].
Congenital myasthenic syndromes
2008, Handbook of Clinical NeurologyCitation Excerpt :Ultrastuctural studies show shallow postsynaptic folds, few secondary clefts, and smaller than normal nerve terminals and postsynaptic regions but the structural integrity of the pre- and postsynaptic regions is preserved (Fig. 10.26A and B) (Ohno et al., 2002, 2003b). Mutations have now been detected in each translated domain of RAPSN as well as in the untranslated RAPSN E-box (Ohno et al., 2002, 2003b; Burke et al., 2003; Dunne and Maselli, 2003; Müller et al., 2003, 2004b, 2006b; Yasaki et al., 2004; Cossins et al., 2006) (Fig. 10.24A and B). Among the first four identified patients with rapsyn deficiency, one carried L14P in TPR1 and N88K in TPR3; two were homozygous for N88K; and one carried N88K and 553ins5, which frameshifts in TPR5.
Developmental increase in the amount of rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability
2007, Developmental BiologyCitation Excerpt :It will be important to experimentally test these ideas by perturbing the level of rapsyn expression during the first month of postnatal life. Postnatal synaptic development also involves growth of secondary postjunctional membrane folds, and these folds were reduced in patients with point mutations to rapsyn (Ohno et al., 2002; Yasaki et al., 2003). Thus, it is conceivable that changes in AChR aggregation (detected by FRET) and in AChR turnover may involve altered postjunctional folding.
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