The age at onset of symptoms ranged from the first day of life to 5 years (mean, 1.6 years; median, 1 year). The clinical features are summarized in , presented in detail in Supplemental Table 1
, and illustrated in . The clinical course varied from mild static weakness limited to limb-girdle muscles to severe generalized progressive disease. All experienced short-term fatigability on exertion. As noted by others,4,5
10 patients experienced intermittent worsenings lasting from days to weeks. They differ from previous reports4,5
in that three patients (Patients 7, 8, and 11) had been hypomotile in utero (see Discussion), and one (Patient 13) was unusual in having severe ptosis, facial weakness, and bulbar symptoms, but only slight shoulder muscle weakness (see ). Seven patients had significant respiratory embarrassment. The overall course was progressive in 12 patients. Phenotypic features of Patient 9 were published previously.17
Different therapeutic agents were tried in the 16 patients at different dosages and in different combinations (see Supplemental Table 1
). Among 10 patients (Patients 1–5, 7, 8, 10, 11, and 14) initially treated with pyridostigmine, 6 patients (Patients 1, 3, 4, 8, 10, and 11) did not respond favorably, and the conditions of 3 patients (Patient 2, 5, 7) worsened; the condition of 1 patient (Patient 14) improved, but then worsened after the subsequent addition of 3,4-diaminopyridine. Three patients (Patients 9, 12, and 14) were treated with pyridostigmine plus 3,4-DAP; the condition of one patient (Patient 9) improved, but that of two patients (Patients 12 and 14) worsened. Four patients (Patients 8, 13, 15, and 16) were treated with 3,4-DAP alone; the conditions of two patients (Patients 8 and 15) improved, one patient (Patient 13) did not respond favorably, and the condition of 1 patient (Patient 16) worsened. Ephedrine alone improved the conditions of three patients (Patients 1, 2, and 6), and ephedrine plus with 3,4-DAP benefited two patients (Patients 4 and 7). Patient 3, who did not respond to pyridostigmine at age 6 years, showed an improvement with Albuterol in his twenties. Worsenings after exposure to cholinergic agents were noted within a few days to several weeks after start of exposure or dose escalation. Although the different dosage regimens and combination of agents are confounding variables, the above observations imply that cholinergic agent are of uncertain benefit or can worsen the symptoms of Dok-7 myasthenia.
Clinical Features in 16 Patients
Fig 1 Phenotypic variability of Dok-7 myasthenia. Patient 10 has mild weakness and atrophy of limb girdle muscles, and mild eyelid ptosis (A). Patient 6 has severe diffuse weakness and atrophy of limb and axial muscles (B). Patient 1 shows mild asymmetric ptosis (more ...)
External intercostal muscle specimens in 14 patients showed type 1 fiber preponderance in 14, type 2 fiber atrophy in 8, isolated necrotic or regenerating fibers suggesting a myopathy in 4, pleomorphic decreases of oxidative enzyme activity in 12, and target formations suggesting denervation in 4 (see Supplementary Fig 1
AChE-reactive synaptic contact areas on the muscle fibers were frequently punctate and small relative to fiber size (); others consisted of multiple small regions (see ) linked by terminal nerve sprouts (see ); still others were near normal or normal in size but lacked a normal pretzel configuration (see ).
Fig 2 Synaptic contact areas visualized with the cholinesterase reaction. Single small (B), multiple small (D–F), and perforated (A, C) contact areas were observed. Nerve sprouts are recognizable (D, asterisk) as faint brown lines connecting contact (more ...)
Because the degeneration of the postsynaptic junctional folds resembled that in the slow-channel syndrome,18
and because these alterations are accompanied by Ca2+
accumulation in the junctional sarcoplasm,19
we stained Dok-7 EPs with Alizarin red but detected no accumulation of Ca2+
in the junctional sarcoplasm.
Dok-7 was localized in EP-containing cryostat sections available from Patients 1, 4, 10, 12, and 14, non-weak control subjects, and patients with primary AChR deficiency caused by low-expressor mutations in AChR subunits. The sections from the Dok-7 patients and control subjects were also reacted for AChR, and the sections from patients with primary AChR deficiency were also immunostained for AChE.7
EP expression of Dok-7 was readily detected in the control subjects, and in favorable sections, expressions of AChR and Dok-7 were topographically distinct (). Dok-7 expression was also detected in Patient 4, who is homozygous for the common 1124_1127dupTGCC mutation (see ). Dok-7 expression was robust at EPs of mildly affected Patient 10 (see ) and was markedly attenuated at EPs of severely affected Patient 12 (see ). However, EP expression of Dok-7 was also strong in Patient 14, who was moderately severely affected (see ), and was decreased in Patient 1, who was only mildly affected (see ). Finally, decreased EP expression of Dok-7 was present in patients with primary EP AChR deficiency. In summary, we found no consistent correlation between the clinical state and EP expression of Dok-7, decreased EP expression of Dok-7 is not specific for Dok-7 myasthenia, and robust EP expression of Dok-7 does not preclude Dok-7 myasthenia.
Fig 3 End-plate (EP) localization of Dok-7 (green signal, left column), acetylcholine receptor (AChR; red signal, center column except T), acetylcholinesterase (AChE; T), and merge (right column) in control subject (C), in Patients 1, 4, 10, 12, and 14, and (more ...)
Electron micrographs of 613 EP regions of 409 EPs of Patients 1 to 14 were inspected for changes of EP conformation (). Some EP regions appeared normal (), but many displayed one or more of the following abnormalities: degeneration of junctional folds, frequently severe (, ); partial occupancy by nerve terminal () or absence of nerve terminal (see ); highly simplified junctional folds (see ); and degeneration of subsynaptic organelles (see ). Some nerve terminals were partly (see ) or completely encased by Schwann cell, and some were degenerating (see ). Nerve sprouts appeared near degenerating or simplified EPs (see ).
Frequencies (%) of Conformational Changes at End Plates in Patients and Control Subjects
Fig 4 Electron micrographs of normal (A) and degenerating (B) neuromuscular junction in same patient. (B) Most junctional folds are replaced by globular debris (asterisk), causing widening of the synaptic space. This predicts a decreased synaptic response to (more ...)
Fig 5 End plates (EPs) with presynaptic and postsynaptic abnormalities. (A) A highly abnormal EP region devoid of nerve terminal. Some junctional folds are degenerating (asterisk). The subsynaptic sarcoplasm harbors large myeloid structures. Nerve sprouts (s) (more ...)
Fig 6 Acetylcholine receptor (AChR) localization with peroxidase-labeled α-bungarotoxin (α-bgt). (A) Junctional folds are preserved and show a normal density and distribution of AChR. (B) Folds at bottom left react strongly for AChR but are (more ...)
Consistent with the earlier observations, morphometric analysis of 340 EP regions showed a significantly reduced postsynaptic area. The length of the postsynaptic membrane, normalized for either the length of the primary synaptic cleft or the size of the postsynaptic area, was significantly reduced (). The AChR index (length of the AChR-reactive postsynaptic membrane normalized for the length of primary synaptic cleft) was also significantly decreased. However, the density and distribution of AChR on nondegenerate junctional folds was normal (see ).
Morphometric Analysis of End-Plate Regions
We detected no consistent correlation between conformational changes at the EPs and the clinical state, except that among five severely affected patients (Patients 5, 6, 7, 9, and 12), EPs of Patients 6, 7, and 9 displayed the greatest frequency of conformational changes.
In Vitro Electrophysiology Studies and Counts of Acetylcholine Receptors per End Plate
The overall means for MEPPA
, and m
were reduced, but the decrease in m
did not reach statistical significance. However, EPPA
in absence of curare, predicted from m
, was significantly reduced. The number of AChRs per EP, estimated from the number of [125
I] binding sites per EP, was decreased to approximately 50% of normal (; see also Supplementary Figs 2 and 3
). This decrease is attributed to the frequently small EPs and to focal loss of AChR from degenerating folds. However, for each parameter of neuromuscular transmission, the distributions of patient and control values overlapped (see Supplemental Figure 2
Microelectrode Studies of Neuromuscular Transmission and [125I]α-Bungarotoxin Binding Sites per End Plate
The extensive degeneration of the junctional folds was similar to that observed in the slow-channel syndrome in which the AChR channel opens in markedly prolonged bursts, but patch-clamp recordings from EPs of seven patients indicated that the duration of channel opening bursts was close to normal (see ), and that the AChR channels opened to a normal conductance of approximately 60 picosiemens.
We detected no correlation between the parameters of neuromuscular transmission and the clinical state except that three of five severely affected patients (Patients 6, 7, and 9) had the lowest MEPPA and predicted EPPA values.
Mutation Analysis of DOK7
We identified 11 mutations in genomic DNA and 6 in cDNA isolated from EP-enriched muscle specimens (; and ; and Supplementary Fig 4
); 10 of these rearrangements are novel. Exon 3-4S appears in the National Cancer for Biotechnology Information (NCBI) database, and four of the observed rearrangements were reported previously,3,5
but only the common 1124_1127dupTGCC mutation had been functionally characterized.3
Rearrangements in DOK7 Observed in 16 Patients
Genomic structure of DOK7 and identified rearrangements in 16 patients. (inset) Intron 1 retention (thick horizontal line).
Fig 8 Scaled linear models of wild-type DOK7 and predicted peptides of mutant transcripts. White and shaded regions represent wild-type and missense residues. Solid circle shows location of missense mutation. The pleckstrin homology (PH) and phosphotyrosine-binding (more ...)
Mutations in Genomic DNA
Among the mutations identified in genomic DNA, 7, including the common 1124_1127dupTGCC mutation, reside in exon 7, 2 reside in exon 5, a splice-site mutation appears in intron 1, and another in intron 3 (see and ). Two mutations in Patient 13 are unusual: one is an insertion-deletion mutation in exon 7 (1139_41delinsA), and the other is a readthrough mutation (1513T>C) that extends the open reading frame by 182 missense residues.
Rearrangements in Complementary DNA
In Patients 2, 5, 9, and 10, a protracted search showed no second mutation in exons or at splice junctions. We therefore isolated messenger RNA from intercostal muscle specimens and searched for mutations in cDNA and cloned cDNA. In Patients 2 and 5, the second allele includes intron 1, predicting 23 missense residues followed by a stop codon (see and ). Inclusion of intron 1 was not detected in 100 control cDNA samples. Interestingly, Patients 2 and 5 also carry a 15-nt deletion in intron 1 (54+14_28delGGGGGG-GGGGGGCGC), with one parent of each patient also carrying the 15-nt deletion. Deletion of 15 nt in intron 1 was not detected in genomic DNA of 100 healthy control subjects.
In Patient 9, analysis of 44 cDNA clones demonstrated that 91% show in-frame skipping (S) of exons 3 to 6 (Ex3-6S), and 9% harbor the 1001_1011dup mutation observed in genomic DNA (see and ). Ex3-6S was not present in 100 control cDNA samples by allele-specific PCR.
In Patient 10, cDNA clones derived from messenger RNA by two separate reverse transcription yielded concordant results. Both sets of cDNA harbored six different transcripts. The combined frequencies in a total of 79 clones were Ex3-4S (17%); Ex2S (10%); Ex2-4S (11%); the common 1124_1127dupTGCC frameshift mutation (32%); both Ex2S and the common frame-shift mutation (9%); and no mutation (21%), consistent with cDNA mosaicism (see and ). Ex2S was not detected in 100 healthy control subjects. We next searched by allele-specific PCR for Ex3-4S in cDNA isolated from 20 control muscles and found that 9 of 20 samples also showed Ex3-4S consistent with polymorphism. We next determined the frequency of the Ex3-4S in the three control samples that showed the highest level of expression in allele-specific PCR and found that only 1 of 12, 1 of 16, and 3 of 23 clones were Ex3-4S. In a further search for the cause of the multiple abnormal splice variants in Patient 10, we sequenced entire introns 2 and 3, and found no mutation that is likely to cause aberrant transcripts.
To further investigate the consequences of the splice-site mutations in Patients 8 and 12, we also cloned their cDNA. In Patient 8, 55-2A>C (IVS1-2A>C) results in recognition of a new splice site after the first 11 nt of exon 2, yielding 69 missense residues and a stop codon. Patient 12 harbors 331+1G>T (IVS3+1G>T) (see and ). Examination of 42 cDNA clones shows Ex3-4S in 55%, Ex3S in 24%, Ex2-4S in 2%, the common frameshift mutation in 12%, and no mutation in 7%. Ex3S was not observed in 100 control cDNA samples by allele-specific PCR.
Except for 1263delC, expression studies were performed to assess the pathogenicity of all mutations found in exons 5 and 7 of genomic DNA, and the 3 in-frame exon-skipping transcripts (Ex3S stemming from the 331+1G>T splice-site mutation, Ex3-4S, and Ex3-6S) detected in cDNA (see ).
Expression Studies in Human Embryonic Kidney Cells
HEK cells were transfected with wild-type DOK7 cDNA, the three in-frame exon skipped (S) constructs (Ex3S, Ex 3-4S, Ex3-6S), and with the 1139_41de-linsA, 1263insC, 1513T>C, 1001_1011dup, 596delT, 601C>T, and 1378insC constructs. Densitometric analysis of immunoblots demonstrated significantly reduced expression of the Ex3S, Ex3-4S, 1513T>C, 596delT, and 601C>T mutants (). To further investigate the consequences of these mutations, we examined their ability to phosphorylate MuSK. Only the in-frame Ex3S, Ex 3-4S, Ex3-6S transcripts that remove the PH, or both the PH and PTB, domains failed to phosphorylate MuSK (). Phosphorylation of MuSK by the remaining DOK7 mutants was not significantly different from that by wild type.
Fig 9 Immunoblot demonstrating expression of wild-type (WT) and mutant DOK7 transcripts in human embryonic kidney (HEK) cells. Expression levels are normalized for cotransfected β-galactosidase. Bars indicate means and standard errors of four transfections. (more ...)
Fig 10 Immunoblot demonstrating MuSK and phosphorylated MuSK in affinity-purified extracts of human embryonic kidney (HEK) cells transfected with MUSK and wild-type or exon-skipped constructs of DOK7. Products of transcripts lacking exons 3, 3 to 4, or 3 to (more ...)
Expression Studies in C2C12 Myotubes
To assess the effects of identified mutations on the size, numerical density, and complexity of AChR clusters expressed by differentiated C2C12 myotubes, we transfected C2C12 myoblasts with FLAG-labeled constructs of 1001_1011dup, 1139_41delinsA, 1263insC, 1378insC, 1513T>C, and the previously characterized common 1124_1127dupTGCC mutation. The mutations expressed at the lowest level in HEK cells (596delT and 601C>T) and the exon-skipping mutations that failed to phosphorylate MuSK were not investigated in this system.
After 6 to 7 days in the differentiation medium, the axial length and density of the AChR clusters was significantly smaller in myotubes transfected with mutant than wild-type transcripts except for 1263insC (; see Supplementary Table 2
). Cluster complexity was evaluated by systematically traversing wells containing transfected myotubes and classifying the 200 first-encountered AChR clusters. For each construct, simple plaques were predominant; the more differentiated (complex) plaques comprising C-shaped, perforated, or branching moieties16
accounted from 17 to 47% of the observed plaques. The frequency of the complex forms was significantly lower in myotubes transfected with mutant than wild-type transcripts (see Supplementary Fig 5
Fig 11 Localization of acetylcholine receptor (AChR; red; A, B), Flag-Dok-7 (green; C, D), merge (E, F) in C2C12 myotubes transfected with Flag-tagged wild-type (A, C, E) and flag-tagged 1139_1141delinsA-DOK7 complementary DNA (B, D, F). Apotome optics, 0.43μm (more ...)
Phosphorylation of the Acetylcholine Receptor β Subunit at Patient End Plates
An important function of activated MuSK is to phosphorylate the AChR β subunit, which, in turn, promotes anchoring and clustering of AChR.20,21
We therefore immunostained intercostal muscle EPs of Patients 1, 4 to 7, and 9 to 12, and of 4 control subjects for the phosphorylated epitope of the AChR β subunit. We found that the reaction for this epitope at patient EPs was as intense as at control EPs (see Supplementary Fig 6
). This was of particular interest in Patient 4 who carries the common 1124_1127dup mutation at homozygosity, because this mutation was reported to reduce AChR β subunit phosphorylation in myotubes.3
In the remaining patients, presence on one allele of the similar common mutation must be sufficient to maintain AChR β-subunit phosphorylation when the second allele lacks the PH and PTB domains.