Electrophysiological and morphological characterization of a case of autosomal recessive congenital myasthenic syndrome with acetylcholine receptor deficiency due to a N88K rapsyn homozygous mutation

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Abstract

Congenital myasthenic syndromes are rare heterogeneous hereditary disorders, which lead to defective neuromuscular transmission resulting in fatigable muscle weakness. Post-synaptic congenital myasthenic syndromes are caused by acetylcholine receptor kinetic abnormalities or by acetylcholine receptor deficiency. Most of the congenital myasthenic syndromes with acetylcholine receptor deficiency are due to mutations in acetylcholine receptor subunit genes. Some have recently been attributed to mutations in the rapsyn gene. Here, we report the case of a 28-year-old French congenital myasthenic syndrome patient who had mild diplopia and fatigability from the age of 5 years. His muscle biopsy revealed a marked reduction in rapsyn and acetylcholine receptor at neuromuscular junctions together with a simplification of the subneural apparatus structure. In this patient, we excluded mutations in the acetylcholine receptor subunit genes and identified the homozygous N88K rapsyn mutation, which has already been shown by cell expression to impair rapsyn and acetylcholine receptor aggregation at the neuromuscular junction. The detection of the N88K mutation at the heterozygous state in five of 300 unrelated control subjects shows that this mutation is not infrequent in the healthy population. Electrophysiological measurements on biopsied intercostal muscle from this patient showed that his rapsyn mutation-induced fatigable weakness is expressed not only in a diminution in acetylcholine receptor membrane density but also in a decline of endplate potentials evoked at low frequency.

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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