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

ABSTRACT 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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    • "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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    • "Several studies provide evidence of significant inter-familial phenotypic variability in DOK7 myasthenia [8]. Our study highlights the additional intra-familial phenotypic variability in DOK7 congenital myasthenia. "
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    ABSTRACT: DOK7 mutations cause a congenital myasthenic syndrome (OMIM 254300) characterized by a "limb-girdle" phenotype. We identified 7 French-Canadian patients with a previously undiagnosed proximal myopathy. A genome wide scan was performed. Homozygosity mapping identified a locus on chromosome 4p16.2 containing DOK7. Sequencing of DOK7 revealed homozygous 1124_1127dupTGCC mutations in all individuals. SNP genotyping of 42kb surrounding DOK7 in our cohort and in 9 patients of various European origins demonstrated a shared haplotype suggesting a common ancestral European mutation. In our cohort, fatigability was not prominent; rather patients reported prolonged periods of increased weakness. Abnormalities on repetitive nerve stimulation and single fiber EMG were not invariably present. There was considerable intra-familial phenotypic variability, and we report an asymptomatic individual. DOK7 mutations should be considered in patients with early-onset myopathy, even in the absence of symptoms suggesting a possible myasthenia.
    Neuromuscular Disorders 07/2010; 20(7):453-7. DOI:10.1016/j.nmd.2010.05.007 · 2.64 Impact Factor
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