Congenital myasthenic syndrome due to a TOR1AIP1 mutation: a new disease pathway for impaired synaptic transmission

Abstract

Congenital myasthenic syndromes are inherited disorders characterized by fatiguable muscle weakness resulting from impaired signal transmission at the neuromuscular junction. Causative mutations have been identified in genes that can affect the synaptic function or structure. We identified a homozygous frameshift deletion c.127delC, p. Pro43fs in TOR1AIP1 in two siblings with limb-girdle weakness and impaired transmission at the neuromuscular synapse. TOR1AIP1 encodes the inner nuclear membrane protein lamin-associated protein 1. On muscle biopsy from the index case, lamin-associated protein 1 was absent from myonuclei. A mouse model with lamin-associated protein 1 conditionally knocked out in striated muscle was used to analyse the role of lamin-associated protein 1 in synaptic dysfunction. Model mice develop fatiguable muscle weakness as demonstrated by using an inverted screen hang test. Electromyography on the mice revealed a decrement on repetitive nerve stimulation. Ex vivo analysis of hemi-diaphragm preparations showed both miniature and evoked end-plate potential half-widths were prolonged which was associated with upregulation of the foetal acetylcholine receptor γ subunit. Neuromuscular junctions on extensor digitorum longus muscles were enlarged and fragmented, and the number of subsynaptic nuclei was significantly increased. Following these findings, electromyography was performed on cases of other nuclear envelopathies caused by mutations in LaminA/C or emerin, but decrement on repetitive nerve stimulation or other indications of defective neuromuscular transmission were not seen. Thus, this report highlights the first nuclear membrane protein in which defective function can lead to impaired synaptic transmission.

Keywords: congenital myasthenic syndromes; envelopathy; myasthenia; neuromuscular junction; nuclear envelope protein.

Graphical Abstract

Figure 1

Clinical, histological and genetic analysis of cases 1 and 2. (A) Mild muscle wasting (arrow) in medial gastrocnemius is shown in individual 1. (B) H&E staining of quadriceps muscle biopsy from individual 1 (when he was 9 years old) shows mild variation in fibre size (i) and an occasional central nucleus indicated by arrow (ii). ATPase pH 4.3 indicates a predominance of type I muscle fibres (iii). Scale bar in (i) and (iii) = 100 µm and in (ii) is 50 µm. (C) Example RNS trace from affected individual 1 from the trapezius showing the characteristic envelope or U shape that is typical of decrement recording from a NMJ with a postsynaptic myasthenic disorder. Muscle was stimulated with a train of 10 stimuli at 3 Hz and CMAPs recorded. The traces from each stimulus are overlaid. Decrement is recorded at the fourth stimulus. (D) Exome sequencing of genomic DNA from individual 1 showing homozygous deletion in exon 1 at position c.127delC (p.P43fs). (E) Sanger sequencing of exon 1 showing segregation of c.127delC within the family. (F) Schematic diagram showing location of mutation (*) within the protein. Mutation is predicted to truncate LAP1B whilst leaving LAP1C unaffected. TM = transmembrane domain.

Figure 2

Analysis of LAP1 expression in muscle biopsy from trapezius and muscle cells from individual 1. (A) LAP1 is expressed in myonuclei in a control muscle biopsy but is undetectable in a myonuclei in a muscle biopsy from trapezius muscle from individual 1. Scale bar = 50 µm. (B) LAP1 can be detected in nuclei within endothelial cells in the patient biopsy. Inset shows the selected region of interest at higher magnification. Scale bar = 50 µm. (C) Western blot showing expression of LAP1B and LAP1C in muscle cells cultured in differentiation medium. Both isoforms are expressed in control cells but LAP1B is absent in muscle cells from individual 1.

Figure 3

Transmission electron micrographs showing abnormal myonuclei in patient biopsy. The red regions of interest in A and D are shown enlarged in B and E, respectively. Convoluted nuclear envelope is visible in all nuclei shown. Other abnormalities include detached chromatin, where there are gaps between inner nuclear envelope and electron dense chromatin (B), nuclear envelope blebs (*) in B, and cytoplasmic channels (white arrows) in C and D with some infiltration of mitochondria (E). M = mitochondria; N = nucleus. Scale bars: 2 µm in (A), (C) and (D); 0.5 µm in (B); 1 µm in (E).

Figure 4

Analysis of strength and electrophysiology of M-LAP1^−/− mice. (A) Strength test of M-LAP1^−/− mice and littermate controls using an inverted screen hang test. The time the mice hung onto the mesh was measured, and the endpoint was 10 min. Six mice for each group were measured, error bars show standard deviation. (B) EMG data from gastrocnemius of M-LAP1^−/− and littermate control mice. Summary of EMG data showing how decrement (represented as the value of the 8th stimulus as a percentage of the first) varies with stimulation frequency for mice aged 6, 9, and 12 weeks (n = 3 mice control 6 and 12 weeks and M-LAP1^−/− 6 weeks; 4 mice control 9 weeks; 7 mice M-LAP1^−/− 9 and 12 weeks). (C) Single electrode analysis of muscle membrane potential in diaphragm/phrenic nerve preparations from 12-week-old mice. Summary of the data showing mEPP and EPP amplitudes, mEPP and EPP half widths and quantal content. (n = four mice per group; *P < 0.05, **P < 0.01, ***P < 0.001). (D) Measurements of endplate current using two-electrode voltage clamp potential in diaphragm/phrenic nerve preparations from 12-week-old mice. Summary of decay (tau) and mEPC amplitude. Also included is data from an AChR deficiency mouse model in which only foetal AChRs are expressed, for comparison. (n = three mice per group; ***P < 0.001).

Figure 5

Analysis of synaptic and extrasynaptic AChR expression, and expression of key NMJ genes in M-LAP1^−/− mice and littermate controls. (A) Synaptic and extrasynaptic AChR expression levels in hemidiaphragm preparations were measured using ¹²⁵I-α-BuTx. Synaptic levels of AChR are unchanged in the M-LAP1^−/− mice, but AChR expression is increased in extrasynaptic regions. Three mice age 12 weeks per group; *P < 0.05. (B) RNA-seq analysis of differential expression of key NMJ genes. RNA extracted from EDL muscles from 12 week old M-LAP1^−/− mice and littermate controls were compared, three mice per group. (C) qPCR analysis of expression of genes encoding the AChR subunits, MuSK, Dok7 and rapsyn. Expression of each gene was compared with expression of Hprt1, which was used as a housekeeping gene control. The fold change between the control and M-LAP1^−/− mice was then calculated.

Figure 6

Morphology of NMJs in EDL muscles from M-LAP1^−/− mice and littermate controls at 6 and 12 weeks of age. (A–C) Morphological analysis of NMJs at 6 and 12 weeks of age in EDL muscles from M-LAP1^−/− mice and littermate controls. (A) The NMJs were visualised by staining AChRs with 594-α-BuTx (red), scale bar=20µm. (B, C) Length of NMJs and the number of fragments which made up each NMJ were measured. Each point represents a NMJ. Two mice per group, number of NMJs per mouse: 6 week control 20,20; 6 week M-LAP1^−/− 21,16; 12 week control 20,11; 12 week M-LAP1^−/− 14,17. ANOVA analysis with Tukey’s multiple comparison, ****P < 0.0001. (D, E) Synaptic nuclei number in M-LAP1^−/− and control mice EDL muscle. D Examples of NMJs from 12 week old control and M-LAP1^−/− mice showing the nuclei (left panels, stained with DAPI) and AChRs (middle panels, stained with 594-α-BuTx). Right panels show merged images. In this example, the control NMJ has eight subsynaptic nuclei and the M-LAP1^−/− NMJ has 23. Scale bar=20 µm. (E) Quantification of number of subsynaptic nuclei in 6 week old and 12 week old control and M-LAP1^−/− mice. Each point represents a NMJ. Three mice per group, number of NMJs per mouse: 6 week control 20,18,17; 6 week M-LAP1^−/− 19,19,16; 12 week control 23,18,19; 12 week M-LAP1^−/− 20,17,20. ANOVA analysis with Tukey’s multiple comparison, P values as shown. (F, G) Transmission electron microscopy analysis of NMJs in EDL muscle from 12 week-old M-LAP1^−/− mice and littermate controls. (F) Example electron micrographs showing abnormal endplates in which there is a reduction in the sole plate, meaning that there is very little post-synaptic sarcoplasm and the nerve terminal appears to almost touch the sarcomeres (indicated by white asterisks). Scale bars = 2 µm. (G) Contingency graph showing a reduction of sole plate area in some M-LAP1^−/− mice but not in littermate controls. Number of NMJs analysed per group = 26, three mice per group. Chi-square (and Fisher’s exact test); P = 0.0042.