**Congenital Myasthenic Syndromes – Molecular Bases of Congenital Defects of Proteins at the Neuromuscular Junction**

Kinji Ohno1, Mikako Ito1 and Andrew G. Engel2

*1Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, 2Department of Neurology, Mayo Clinic, Rochester, Minnesota, 1Japan* 

#### **1. Introduction**

174 Neuromuscular Disorders

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Congenital myasthenic syndromes (CMS) are heterogeneous disorders caused by mutations in molecules expressed at the neuromuscular junction (NMJ) (Fig. 1). Each mutation affects the expression level or the functional properties or both of the mutant molecule. No fewer than 11 defective molecules at the NMJ have been identified to date. The mutant molecules include (i) acetylcholine receptor (AChR) subunits that forms nicotinic AChR and generate endplate potentials (Ohno *et al.*, 1995; Sine *et al.*, 1995), (ii) rapsyn that anchors and clusters AChRs at the endplate (Ohno *et al.*, 2002; Milone *et al.*, 2009), (iii) agrin that is released from nerve terminal and induces AChR clustering by stimulating the downstream LRP4/MuSK/Dok-7/rapsyn/AChR pathway (Huze *et al.*, 2009), (iv) muscle-specific receptor tyrosine kinase (MuSK) that transmits the AChR-clustering signal from agrin/LRP4 to Dok-7/rapsyn/AChR (Chevessier *et al.*, 2004; Chevessier *et al.*, 2008), (v) Dok-7 that interacts with MuSK and exerts the AChR-clustering activity (Beeson *et al.*, 2006; Hamuro *et al.*, 2008), (vi) plectin that is an intermediate filament-associate protein concentrated at sites of mechanical stress (Banwell *et al.*, 1999; Selcen *et al.*, 2011), (vii) glutamine-fructose-6 phosphate aminotransferase 1 encoded by *GFPT1*, the function of which at the NMJ has not been elucidated (Senderek *et al.*, 2011), (viii) skeletal muscle sodium channel type 1.4 (NaV1.4) that spreads depolarization potential from endplate throughout muscle fibers (Tsujino *et al.*, 2003), (ix) collagen Q that anchors acetylcholinesterase (AChE) to the synaptic basal lamina (Ohno *et al.*, 1998; Ohno *et al.*, 1999; Kimbell *et al.*, 2004), (x) 2-laminin that forms a cruciform heterotrimeric lamins-221, -421, and -521 and links extracellular matrix molecules to the -dystroglycan at the NMJ (Maselli *et al.*, 2009), (xi) choline acetyltransferase (ChAT) that resynthesizes acetylcholine from recycled choline at the nerve terminal (Ohno *et al.*, 2001). AChR (Lang & Vincent, 2009), MuSK (Hoch *et al.*, 2001; Cole *et al.*, 2008), and LRP4 (Higuchi *et al.*, 2011) are also targets of myasthenia gravis, in which autoantibody against each molecule impairs the neuromuscular transmission.

CMS are classified into three groups of postsynaptic, synaptic, and presynaptic depending on the localization of the defective molecules. Among the eleven molecules introduced

Congenital Myasthenic Syndromes – Molecular Bases

**2.2 Physiology of the nicotinic muscle AChR** 

than for Na+.

**3. Postsynaptic CMS** 

**3.1 Endplate AChR deficiency** 

plectin, and GFPT1.

*CHRNE*, but not *CHRNG*.

of Congenital Defects of Proteins at the Neuromuscular Junction 177

Nicotinic AChRs are pentameric ligand-gated ion channels. The family of pentameric ligand-gated ion channels includes cationic AChRs, cationic serotonergic receptors (5HT3), anionic glycine receptors, and anionic GABAA and GABAC receptors (Keramidas *et al.*, 2004). Heteromeric neuronal nicotinic AChRs are comprised of various combinations of (2-7) and subunits (2-4), whereas homomeric AChRs are formed only by a single subunit (e.g., 7-9) (Mihailescu & Drucker-Colin, 2000). On the other hand, nicotinic muscle AChRs have only two forms: fetal AChR that carries the , , , and subunits encoded by *CHRNA1*, *CHRNB1, CHRND, CHRNG*, respectively, in the stoichiometry 2; and adult-type AChR that carries the subunit instead of the subunit in the stoichiometry 2 (Mishina *et al.*, 1986). The subunit is encoded by *CHRNE*. Nicotinic muscle AChR harbors two binding sites for ACh at the interfaces between the and / subunits (Lee *et al.*, 2009; Mukhtasimova *et al.*, 2009). Binding of a single ACh molecule opens the channel pore but for a short time. Binding of two ACh molecules stabilizes the open state of AChR, and AChR stays open for a longer time. Only cations pass through the channel pore of nicotinic AChRs. Unlike sodium, potassium, or calcium channels, AChRs, in general, have no selectivity for cations, but 7 AChRs have 10-20 times higher permeability for Ca2+

Postsynaptic CMS is classified into four phenotypes: (i) endplate AChR deficiency due to defects in AChR, rapsyn, agrin, MuSK, Dok-7, plectin, glutamine-fructose-6-phosphate aminotransferase 1, (ii) slow-channel congenital myasthenic syndrome, (iii) fast-channel

Endplate AChR deficiency is caused by defects in AChR, rapsyn, agrin, MuSK, Dok-7,

Endplate AChRs deficiency can arise from mutations in *CHRNA1*, *CHRNB1*, *CHRND*, and

Two different groups of mutations of the AChR subunit genes cause endplate AChR deficiency. The first group includes null mutations in *CHRNE* encoding the subunit. The null mutations are caused by frameshifting DNA rearrangements, *de novo* creation of a stop codon, and frameshifting splice-site mutations, or mutations involving residues essential for subunit assembly. Large-scale in-frame DNA rearrangements also abolish expression of the AChR subunit (Abicht *et al.*, 2002). Mutations in the promoter region (Ohno *et al.*, 1999) and most missense mutations (Ohno *et al.*, 1997) do not completely abolish expression of the subunit but the molecular consequences are indistinguishable from those of null mutations. Lack of the subunit can be compensated for by the presence of the fetal subunit that is normally expressed in embryos (Engel *et al.*, 1996). The patients can survive with -AChR even in the absence of -AChR. If a null mutation resides in the other AChR

congenital myasthenic syndrome, and (iv) sodium channel myasthenia.

**3.1.1 Endplate AChR deficiency due to defects in AChR subunits** 

above, AChR, rapsyn, MuSK, Dok-7, plectin, and NaV1.4 are associated with the postsynaptic membrane. Agrin, ColQ, and 2-laminin reside in the synaptic basal lamina. The only presynaptic disease protein identified to date is choline acetyltransferase (ChAT). A target molecule and its synaptic localization of glutamine-fructose-6-phosphate aminotransferase 1 (GFPT1) are still unresolved but the phenotypic consequence is the postsynaptic AChR deficiency. This chapter focuses on molecular bases of these three groups of CMS.

Fig. 1. Schematic of molecules expressed at the NMJ
