Our patient, who is currently a 20-year-old woman, was born to non-consanguineous parents. During the neonatal period she experienced several episodes of respiratory distress, and she was found to have persistently constricted pupils and massive proteinuria. A renal biopsy at that time demonstrated findings consistent with a microcystic nephrosis. All of her motor developmental milestones were delayed, and although her nephrotic syndrome was corrected at the age of 15 months after a successful kidney transplant from her father, her motor deficit persisted. At the age of 7 years a muscle biopsy revealed non-specific changes, but an electromyogram with repetitive stimulation at 3 Hz of the left median nerve resulted in a 24% decrement of the compound muscle action potential amplitude, which became more pronounced with higher frequencies of stimulation. Serum antibodies against the acetylcholine receptor (AChR) were negative, and a brain magnetic resonance image (MRI) was normal. A trial with a cholinesterase inhibitor resulted in profound weakness requiring hospitalisation and ventilatory support; however, she responded well to treatment with ephedrine. When she was 9 years old she required corrective surgery for severe ptosis. At that time an ophthalmologic examination revealed pronounced miosis, myopia and impaired visual acuity in both eyes. Fundoscopic examination revealed hypoplastic macular areas and poor foveal reflex, but her electroretinogram was normal. A neurologic examination revealed normal cognition, impaired visual acuity and reactive pinpoint pupils. Her external ocular movements were limited to 15–20° in the horizontal axis, and she had bilateral ptosis. She showed no bulbar deficit, but the motor examination revealed severe proximal limb weakness. She had spine surgery for scoliosis at the age of 12, and since then she has used bi-level positive airway pressure during night sleep and intermittently during the day.
To elucidate the nature of the CMS in this patient, we performed an anconeus muscle biopsy at age 10, which included in vitro microelectrode recordings as previously described.16
The most remarkable finding of the microelectrode recordings was the profound reduction of the quantal content of end plate potentials (EPPs) evoked by nerve stimulation at 1 Hz relative to the controls (, t
−5.88, p<0.001). Nerve stimulation at higher frequencies resulted in an even more severe reduction of the EPP quantal content as shown by the estimate of the ratio of EPP quantal content using 20 Hz stimulation to 1 Hz stimulation, which was decreased relative to controls. Frequencies and amplitudes of miniature endplate potentials (MEPPs) were also diminished (t
3.55, p<0.05). However, the half-decay phases of MEPPs were not different from recordings performed in age matched controls (). At two endplates MEPPs were recorded under voltage clamp conditions. The decay time constants of the resultant miniature endplate currents (MEPCs) were not different from controls (mean (SEM) 3.71 (0.15) ms, n
2 in the patient; and 3.58 (0.16) ms, n
11 in controls). Finally, to verify that the endplate acetylcholinesterase was active we measured the amplitudes and half-decay phases of the MEPPs recorded before and after exposure of the preparation to 0.1 μM neostigmine. The average amplitude and half-decay phase of MEPPs recorded before neostigmine exposure, 1.16 (0.67) mV and 2.94 (0.67) ms, respectively, increased to 1.42 (0.62) mV and 6.08 (0.88) ms after exposure of the muscle to neostigmine, thus indicating that the endplate acetylcholinesterase was active.
Except for occasional small angular fibres and type I fibre predominance there were no other histological abnormalities. The acetylcholinesterase reaction performed in teased muscle fibre bundles revealed, as in control muscles, only one endplate per muscle fibre; however, the mean endplate area of 169.9 (16.6) μm2 (17) in the patient was reduced in comparison with the mean endplate area of 227.03 (11.9) μm2 (40) in controls.
The most consistent structural abnormality of the NMJ shown by electron microscopy were: (1) small axon terminal size and encasement of nerve endings by the Schwann cell; (2) severe widening of the primary synaptic clefts with invasion of the synaptic space by processes of Schwann cells; and (3) moderate simplification of the postsynaptic membranes (). The small nerve terminals, which often appeared divided into multiple very small segments and retracted from the postsynaptic membrane, were in all cases partially or completely encased by processes of the Schwann cells. As a result of the encasement of the nerve terminal and invasion of the synaptic space by the Schwann cell, the areas of apposition between the nerve terminal and the postsynaptic membrane were extremely small, and even in these areas where presynaptic membranes were directly apposed to postsynaptic membranes the primary synaptic cleft was notably widened. Occasional active zones were seen in the nerve terminal, but they were not consistently apposed to the secondary clefts, as seen in the controls. Some nerve terminals showed a relatively normal number of synaptic vesicles, while others showed few synaptic vesicles. Overall, there was a mild reduction in the density of synaptic vesicles compared to the controls. In the postsynaptic region, there was simplification of the postsynaptic membranes, which was variable and minor compared to the extreme hypoplasia of the axon terminals. There was no other postsynaptic abnormality, with the exception of two sub-sarcolemmal nuclei containing degenerating membranous debris.
Figure 1 Ultrastructural findings at the neuromuscular junction (NMJ). Control (A) and the patient (B–F). (A) Normal NMJ from a control demonstrating normal nerve terminal size and highly complex postsynaptic membrane folding with well formed secondary (more ...)
The morphometric analysis of the NMJ was performed as previously described16
and revealed that relative to the controls, our patient showed diminished average axon terminal area and number of synaptic vesicles per area (). The width of the primary synaptic cleft was notably increased and the area of direct apposition of the nerve terminal to the postsynaptic membranes was extremely small (). There was also a reduction of the endplate index (ratio of postsynaptic membrane length/presynaptic membrane length), although the average number of secondary folds per μm of primary cleft was not decreased.
After obtaining a signed consent approved by the institutional review board of the University of California Davis, we amplified and sequenced genomic DNA from all 32 exons of the human LAMB2 gene and encountered two novel LAMB2
mutations (, supplemental ). Both mutations are single base pair deletions, resulting in frameshifts. 1478delG
occurs in exon 11, while 4804delC
occurs in exon 29. 1478delG
results in an early stop codon at amino acid 496, and 4804delC
creates a termination codon at amino acid 1653. Mutational analysis in the patient’s family revealed that the 1478delG
mutation derives from the patient’s unaffected mother. In contrast, the 4804delC
mutation is derived from the patient’s unaffected father and is also carried by the patient’s unaffected brother. Mutational analysis in CHRNA1, CHRNB, CHRND
, CHRNE, RAPSN, MUSK, DOK7
was performed as previously described16
and showed no abnormalities.
Figure 2 Mutational analysis findings. (A) Schematic view of the 32 coding regions of human LAMB2 showing the positions of the identified mutations in exons 11 and 29. White regions correspond to untranslated portions of the gene. The 100 bp marker corresponds (more ...)
Because both genetic defects encountered in LAMB2
are deletions leading to frame shifts, we predicted that the protein would be truncated and fail to assemble with the rest of the subunits. Indeed, both Western blot, performed as previously described,17
in kidney and muscle tissues, and immunohistochemistry in kidney tissue showed no expression of laminin β2 (, supplemental ).
Figure 3 Western blot analysis. Results of a Western blot using a rabbit polyclonal antibody directed against an epitope corresponding to amino acids 1549–1798 of human laminin β2 (Santa Cruz Biotechnology, Santa Cruz, California, USA) and frozen (more ...)