Congenital myasthenic syndrome caused by two non‐N88K rapsyn mutations
RA Maselli, H. Dris, J. Schnier
Jul 1, 2007
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17
Citations
Journal
Clinical Genetics
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Key Takeaway Key Takeaway: V45M mutations in the rapsyn gene, which are not associated with N88K mutations, cause congenital myasthenic syndrome in this patient.
Abstract
To the Editor: Mutations in the human rapsyn gene (RAPSN) are associated with a congenital myasthenic syndrome (CMS) characterized by deficient clustering of acetylcholine receptors (AChRs) (1–5). With rare exceptions, reported patients with CMS caused by RAPSN mutations are homozygotes for the N88K mutation or carry N88K combined with another mutation (6, 7). We describe here a patient with a CMS caused by two heteroallelic non-N88K RAPSN mutations. The patient is a 16-month-old girl from a nonconsanguineous couple. At birth, she was hypotonic and required mechanical ventilation and gastric tube feeding. The examination revealed normal cognition, bilateral ptosis, normal ocular movements and mild facial and proximal limb weakness. Electromyogram with repetitive nerve stimulation showed 25% decrement. In vitro electrophysiologic studies performed in an anconeus muscle biopsy demonstrated marked reduction of amplitudes of miniature endplate potentials (0.43 0.09 mV, n 1⁄4 14 vs 1.26 0.36 nA, n 1⁄4 14 in a control, p , 0.001) and a moderate decrease of the endplate potential quantal content (3.55 1.76, n 1⁄4 6 vs 7.74 3.0, n 1⁄4 6 in a control, p , 0.05). Endplate acetylcholinesterase reaction was normal. Electron microscopy of neuromuscular junctions demonstrated a marked simplification of the post-synaptic membrane with no abnormality of subsynaptic sarcoplasm or the nerve terminal. Direct sequencing of the AChR a-, b-, d-, and esubunit genes using genomic DNA revealed no mutations. However, analysis of RAPSN revealed a heterozygous G/A change in exon 1 at nucleotide 133 and in exon 2 at nucleotide 284 predicting V45M and E162K (Fig. 1a and b). V45M is located in the first tetratricopeptide region (TPR), while E162K is located in the linker between the fourth and fifth TPRs. V45 and E162 are conserved (Fig. 1c). Restriction analysis revealed that the patient and her father were heterozygotes for V45M, while the patient and her mother were heterozygotes for E162K (Fig. 1d). To evaluate the effect of V45M and E162K on the protein function, we introduced these mutations in a wild-type RAPSN–GFP construct and transfected HEK cells with either wild type or mutants as described elsewhere (1). Data extracted from three to four experiments revealed that cells transfected with the wild-type RAPSN–GFP showed green fluorescence arranged in clusters in 49.3 2.8% of the cells (n 1⁄4 269), while the rest displayed green fluorescence homogeneously. Cells transfected with RAPSN V45M-GFP or RAPSN E162K-GFP displayed green fluorescence arranged in clusters in 50.6 5.2% (n1⁄4 290) and 51.5 2.5% (n1⁄4 210), respectively. Cotransfection of HEK cells with cDNA of the four AChR-subunits with wild-type RAPSN– GFP revealed colocalization of green fluorescence granules with red fluorescence granules representing rhodamine-alpha-bungarotoxin bound to AChRs in 68.4 3.1% of the cells (n 1⁄4 68) (Fig. 2a–c and j). In contrast, cotransfection of the AChR-subunit cDNAs with RAPSN V45MGFP or RAPSN E162K-GFP revealed coclustering of greenand red-fluorescence granules in only 26.6 3.0% (n 1⁄4 76) and 24.3 2.8% (n 1⁄4 67) of the cells, respectively (Fig. 2d–j) (p , 0.001). Hence, although neither V45M nor E162K mutants hinder rapsyn self-association, both mutants markedly decrease the ability of rapsyn to cluster with AChRs. Interestingly, it was recently reported that two rapsyn mutations not found associated with N88K also diminish coclustering of AChRs with rapsyn without impairing rapsyn self-association (7). V45M and E162K are located in the TPR domain of rapsyn, which controls rapsyn selfassociation; however, because neither truncates the TPR domain, it is not surprising that they do not hinder rapsyn self-association. However,