Selcen D, Milone M, Shen X-M, et al. Dok-7 myasthenia: phenotypic and molecular genetic studies in 16 patients

Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN 55905, USA.
Annals of Neurology (Impact Factor: 9.98). 07/2008; 64(1):71-87. DOI: 10.1002/ana.21408
Source: PubMed


Detailed analysis of phenotypic and molecular genetic aspects of Dok-7 myasthenia in 16 patients.
We assessed our patients by clinical and electromyographic studies, by intercostal muscle biopsies for in vitro microelectrode analysis of neuromuscular transmission and quantitative electron microscopy EM of 409 end plates (EPs), and by mutation analysis, and expression studies of the mutants.
The clinical spectrum varied from mild static limb-girdle weakness to severe generalized progressive disease. The synaptic contacts were single or multiple, and some, but not all, were small. In vitro microelectrode studies indicated variable decreases of the number of released quanta and of the synaptic response to acetylcholine; acetylcholine receptor (AChR) channel kinetics were normal. EM analysis demonstrated widespread and previously unrecognized destruction and remodeling of the EPs. Each patient carries 2 or more heteroallelic mutations: 11 in genomic DNA, 7 of which are novel; and 6 identifiable only in complementary DNA or cloned complementary DNA, 3 of which are novel. The pathogenicity of the mutations was confirmed by expression studies. Although the functions of Dok-7 include AChR beta-subunit phosphorylation and maintaining AChR site density, patient EPs showed normal AChR beta-subunit phosphorylation, and the AChR density on the remaining junctional folds appeared normal.
First, the clinical features of Dok-7 myasthenia are highly variable. Second, some mutations are complex and identifiable only in cloned complementary DNA. Third, Dok-7 is essential for maintaining not only the size but also the structural integrity of the EP. Fourth, the profound structural alterations at the EPs likely contribute importantly to the reduced safety margin of neuromuscular transmission.

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    • "In the absence of MuSK, neuromuscular synapses fail to form (DeChiara et al. 1996). Mutations that impair MuSK kinase activity or signaling steps downstream from MuSK cause congenital myasthenia, characterized by structurally and functionally defective synapses, leading to muscle weakness and fatigue (Beeson et al. 2006; Muller et al. 2007; Selcen et al. 2008). "
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    ABSTRACT: Muscle-specific kinase (MuSK) is essential for each step in neuromuscular synapse formation. Before innervation, MuSK initiates postsynaptic differentiation, priming the muscle for synapse formation. Approaching motor axons recognize the primed, or prepatterned, region of muscle, causing motor axons to stop growing and differentiate into specialized nerve terminals. MuSK controls presynaptic differentiation by causing the clustering of Lrp4, which functions as a direct retrograde signal for presynaptic differentiation. Developing synapses are stabilized by neuronal Agrin, which is released by motor nerve terminals and binds to Lrp4, a member of the low-density lipoprotein receptor family, stimulating further association between Lrp4 and MuSK and increasing MuSK kinase activity. In addition, MuSK phosphorylation is stimulated by an inside-out ligand, docking protein-7 (Dok-7), which is recruited to tyrosine-phosphorylated MuSK and increases MuSK kinase activity. Mutations in MuSK and in genes that function in the MuSK signaling pathway, including Dok-7, cause congenital myasthenia, and autoantibodies to MuSK, Lrp4, and acetylcholine receptors are responsible for myasthenia gravis.
    Cold Spring Harbor perspectives in biology 05/2013; 5(5). DOI:10.1101/cshperspect.a009167 · 8.68 Impact Factor
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    • "Dok-7 mutants generally have abnormally small and simplified NMJs but show normal AChR and AChE functions, correct MuSK activation, and AChR clusterization, although Dok-7 acts in concert with MuSK in activating rapsyn to concentrate AChRs at the junctional folds. It has therefore been suggested that the altered size and integrity of NMJs observed were probably the consequence of a high and widespread degeneration and remodeling of the EPs [86, 87]. "
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    ABSTRACT: Skeletal muscle innervation is a multi-step process leading to the neuromuscular junction (NMJ) apparatus formation. The transmission of the signal from nerve to muscle occurs at the NMJ level. The molecular mechanism that orchestrates the organization and functioning of synapses is highly complex, and it has not been completely elucidated so far. Neuromuscular junctions are assembled on the muscle fibers at very precise locations called end plates (EP). Acetylcholine receptor (AChR) clusterization at the end plates is required for an accurate synaptic transmission. This review will focus on some mechanisms responsible for accomplishing the correct distribution of AChRs at the synapses. Recent evidences support the concept that a dual transcriptional control of AChR genes in subsynaptic and extrasynaptic nuclei is crucial for AChR clusterization. Moreover, new players have been discovered in the agrin-MuSK pathway, the master organizer of postsynaptical differentiation. Mutations in this pathway cause neuromuscular congenital disorders. Alterations of the postynaptic apparatus are also present in physiological conditions characterized by skeletal muscle wasting. Indeed, recent evidences demonstrate how NMJ misfunctioning has a crucial role at the onset of age-associated sarcopenia.
    03/2012; 3(1):13-23. DOI:10.1007/s13539-011-0041-7
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    • "In contrast, hypomorphic alleles of Dok-7, which truncate Dok-7 and lead to a loss of Y396 and Y406, retain sufficient activity to mediate the formation of synapses. These synapses are nonetheless structurally and functionally defective; in particular, the synapses are small and motor axons fail to reliably terminate, leading to muscle weakness and fatigue in adults, the hallmark features of CMS (Beeson et al. 2006; Muller et al. 2007; Selcen et al. 2008). Thus, these truncated forms of Dok-7, which cannot recruit Crk/Crk-L, mediate the formation of simplified and poorly functional synapses. "
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    ABSTRACT: Agrin, released by motor neurons, promotes neuromuscular synapse formation by stimulating MuSK, a receptor tyrosine kinase expressed in skeletal muscle. Phosphorylated MuSK recruits docking protein-7 (Dok-7), an adaptor protein that is expressed selectively in muscle. In the absence of Dok-7, neuromuscular synapses fail to form, and mutations that impair Dok-7 are a major cause of congenital myasthenia in humans. How Dok-7 stimulates synaptic differentiation is poorly understood. Once recruited to MuSK, Dok-7 directly stimulates MuSK kinase activity. This unusual activity of an adapter protein is mediated by the N-terminal region of Dok-7, whereas most mutations that cause congenital myasthenia truncate the C-terminal domain. Here, we demonstrate that Dok-7 also functions downstream from MuSK, and we identify the proteins that are recruited to the C-terminal domain of Dok-7. We show that Agrin stimulates phosphorylation of two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins: Crk and Crk-L. Furthermore, we show that selective inactivation of Crk and Crk-L in skeletal muscle leads to severe defects in neuromuscular synapses in vivo, revealing a critical role for Crk and Crk-L downstream from Dok-7 in presynaptic and postsynaptic differentiation.
    Genes & development 11/2010; 24(21):2451-61. DOI:10.1101/gad.1977710 · 10.80 Impact Factor
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