**4.2 CMS due to a defect in 2 laminin**

186 Neuromuscular Disorders

or in any other disease. There are three classes of ColQ mutations. First, mutations in the proline-rich attachment domain (PRAD) hinder binding of ColQ to AChE. Sedimentation analysis of AChE species of the patient muscle and transfected cells shows complete lack of ColQ-tailed AChE. Second, mutations in the collagen domain, most of which are truncation mutations, hinder formation of triple helix of ColQ. Sedimentation analysis of muscle and transfected cells demonstrate a truncated single-stranded ColQ associated with a homotetramer of AChE. Third, the mutations in the C-terminal domain have no deleterious effect on formation of the asymmetric ColQ-tailed AChE, but they compromise anchoring of ColQ-tailed AChE to the synaptic basal lamina as elegantly shown in vitro overlay binding of mutant and wild-type human recombinant ColQ-tailed AChE to the frog endplate

Fig. 4. ColQ anchors to the synaptic basal lamina by binding to perlecan and MuSK.

CMAP also occurs in slow channel syndrome.

amplitude of the endplate potential (Engel *et al.*, 1977).

EMG studies show a decremental response as in other CMS. In addition, most patients have a repetitive CMAP response on a single nerve stimulus. The repetitive CMAP decrements faster than the primary CMAP. It can be overlooked unless a well rested muscle is tested by single nerve stimuli. The prolonged dwell time of unhydrolyzed ACh in the synaptic space prolongs the endplate potential; when this exceeds the absolute refractory period of the muscle fiber action potential, it elicits a repetitive CMAP. As mentioned above, a repetitive

Some aspects of the pathophysiology of endplate AChE deficiency resemble those of the SCCMS. As in the SCCMS, neuromuscular transmission is compromised by three distinct mechanisms. First, staircase summation of endplate potentials causes a depolarization block, which inactivates a proportion the voltage-gated skeletal sodium channel, NaV1.4. (Maselli & Soliven, 1991). Second, prolonged exposure of AChR to ACh during physiologic activity desensitizes a fraction of the available AChRs (Milone *et al.*, 1997). Third, repeated openings of AChR cause calcium overloading to the endplate, which culminates in an endplate myopathy (Groshong *et al.*, 2007). Unlike in the SCCMS, the nerve terminals are abnormally small and often encased by Schwann cells. This decreases the quantal content and hence the

(Kimbell *et al.*, 2004).

Laminins are cruciform heterotrimeric glycoproteins composed of , , and chains and are assembled from products of five , four , and three genes. The laminin molecules are named according to their chain composition. For example, laminin-321 contains 3, 2, and 1 chains (Aumailley *et al.*, 2005). Three laminins are present at the synaptic basal lamina, laminin-221, laminin-421, and laminin-521. Each contains the 2 subunit. Laminin-421 is restricted to the primary synaptic cleft and promotes the precise alignment of pre- and postsynaptic specializations. Laminin-521 lines the primary and secondary clefts, promotes presynaptic differentiation, and prevents Schwann cells from entering the synaptic cleft. The synaptic laminins provide a stop signal for axons at developing endplates and organize presynaptic differentiation (Sanes, 1997). Mice deficient for *Lamb2* that encodes 2 laminin show reduced terminal branching of presynaptic motor axons, with a decreased number of active zones, no clustering of the synaptic vesicles above the active zones, and extension of Schwann cell processes into the primary synaptic cleft, and decreased spontaneous and evoked quantal release (Noakes *et al.*, 1995; Patton *et al.*, 1998). In addition to its presence at the endplate, 2 laminin is also highly expressed in renal glomeruli and the eye. *LAMB2* mutations in humans cause Pierson syndrome characterized by ocular malformation including small non-reactive pupils, loss of accommodation, and abnormalities of the lens, cornea and retina and by fatal nephrotic syndrome that requires renal transplantation (Zenker *et al.*, 2004).

Maselli and coworkers reported a 20-year-old woman with Pierson syndrome caused by two heteroallelic frameshifting mutations (1478delG and 4804delC) in *LAMB2* who also had a severe CMS (Maselli *et al.*, 2009). The nephrotic syndrome was corrected by a renal transplant at age 15 months. The patient had respiratory distress in infancy, delayed motor milestones, a decremental EMG response, limited ocular ductions, bilateral ptosis, severe proximal limb weakness, scoliosis, and required assisted ventilation at night and sometimes during the day. AChE activity was spared at the NMJ. Electron microscopy of the NMJ showed small axon terminal size and encasement of nerve endings by the Schwann cell, widening of the primary synaptic clefts with invasion of the synaptic space by processes of Schwann cells, moderate simplification of postsynaptic membranes, and decreased number of synaptic vesicles. Both morphological and microelectrode studies were similar to those observed in *Lamb2*-mice (Noakes *et al.*, 1995). Notably, symptoms were worsened by pyridostigmine but were improved by ephedrine.

Congenital Myasthenic Syndromes – Molecular Bases

the enzyme structurally unstable (Cai *et al.*, 2004).

next generation sequencers may speed this effort.

**6. Conclusions** 

**7. Acknowledgments** 

Association to A.G.E.

**8. References** 

ChAT deficiency is proven or suspected (Byring *et al.*, 2002).

of Congenital Defects of Proteins at the Neuromuscular Junction 189

vesicular packaging of ACh at the nerve terminal. Kinetic studies of mutant ChAT enzymes disclosed variable decreases in affinity for choline and/or acetyl-CoA, as well as variable reduction the catalytic rate (Ohno *et al.*, 2001) (Fig. 5). Moreover, some recombinant mutants expressed at a reduced level in COS cells. Two patients carried a functionally null mutation on one allele, but ChAT encoded on the other allele was partially functional. Heterozygous parents that carried the null allele were asymptomatic indicating that humans can tolerate up to but not exceeding 50% reduction of presynaptic ChAT activity. None of our patients has autonomic symptoms or signs of central nervous system involvement other than that attributed to anoxic episodes. This suggests that the ChAT activity and/or substrate availability are rate limiting for ACh synthesis at the motor nerve but not at other cholinergic synapses. Indeed, stimulated quantal release at the endplate is higher than at other cholinergic synapses, which points to selective vulnerability of the NMJ to reduced ACh resynthesis. Crystal structure of ChAT resolved at 2.2 Å revealed that some of the reported *CHAT* mutations in CMS patients are not at the substrate-binding or the catalytic site of ChAT. Hence these mutation exert their effect by an allosteric mechanism or render

In most patients, anticholinesterase medications are of benefit in ameliorating the myasthenic symptoms and preventing the apneic crises but few patients fail to respond to cholinergic therapy remaining permanently paralyzed and remain respirator dependent. Prophylactic anticholinesterase therapy is advocated even for patients asymptomatic between crises. Parents of affected children must be indoctrinated to anticipate sudden worsening of the weakness and possible apnea with febrile illnesses, excitement, or overexertion. Long-term nocturnal apnea monitoring is indicated in any patient in whom

We reviewed the clinical and molecular consequences of defects in 11 genes associated with CMS. Molecular studies of CMS began with identification of a missense mutation in the AChR subunit in a SCCM patient (Ohno *et al.*, 1995). Since then, mutations in seven postsynaptic, three synaptic, and one presynaptic proteins have been discovered. In some CMS the disease gene has been elusive and await discovery. Resequencing analysis with the

Works in our laboratories were supported by Grants-in-Aid from the MEXT and the MHLW of Japan to K.O., and by NIH Grant NS6277 and a research Grant from Muscular Dystrophy

Abicht, A., Stucka, R., Schmidt, C., Briguet, A., Höpfner, S., Song, I.-H., Pongratz, D., Müller-

myasthenic syndrome. *Brain*, Vol. 125, No., pp. 1005-1013, ISSN 0006-8950

Felber, W., Ruegg, M. A. & Lochmüller, H. (2002). A newly identified chromosomal microdeletion and an N-box mutation of the AChR gene cause a congenital

## **5. Presynaptic CMS**

Choline acetyltransferase (ChAT) is the only presynaptic molecule that is known to be defective in CMS.

#### **5.1 CMS with episodic apnea due to defects in choline acetyltransferase (ChAT)**

ACh released from the nerve terminal is hydrolyzed into choline and acetate by AChE at the synaptic basal lamina. Choline is taken up by the nerve terminal by a high-affinity choline transporter on the presynaptic membrane (Apparsundaram *et al.*, 2000; Okuda *et al.*, 2000). ChAT resynthesizes ACh from choline and acetyl-CoA (Oda *et al.*, 1992). After the synaptic vesicles are acidified by the vesicular proton pump (Reimer *et al.*, 1998), the resynthesized cationic ACh is packed into a synaptic vesicle by the vesicular ACh transporter (vAChT) in exchange for protons (Erickson *et al.*, 1994).

Fig. 5. Choline acetyltransferase (ChAT). (A) Genomic structure of *CHAT* and identified mutations. A gene for vesicular acetylcholine transporter (vAChT) is in the first intron of *CHAT*. (B) Kinetics of wild-type and mutant ChAT enzymes. ChAT synthesizes acetylcholine using choline and acetyl-CoA. L210P abrogates an affinity of ChAT for acetyl-CoA (AcCoA), and R560H abolishes an affinity of ChAT for choline.

We determined the complete genomic structure of *CHAT* encoding ChAT, and identified ten mutations in five CMS patients with the characteristic clinical features of sudden episodes of apnea associated with variable myasthenic symptoms (Ohno *et al.*, 2001). Additional *CHAT* mutations were later reported by other groups (Maselli *et al.*, 2003; Schmidt *et al.*, 2003; Barisic *et al.*, 2005; Mallory *et al.*, 2009; Yeung *et al.*, 2009; Schara *et al.*, 2010). All of our patients showed a marked decrease of the endplate potential after subtetanic stimulation that recovered slowly over 5 to 10 min, which pointed to a defect in the resynthesis or vesicular packaging of ACh at the nerve terminal. Kinetic studies of mutant ChAT enzymes disclosed variable decreases in affinity for choline and/or acetyl-CoA, as well as variable reduction the catalytic rate (Ohno *et al.*, 2001) (Fig. 5). Moreover, some recombinant mutants expressed at a reduced level in COS cells. Two patients carried a functionally null mutation on one allele, but ChAT encoded on the other allele was partially functional. Heterozygous parents that carried the null allele were asymptomatic indicating that humans can tolerate up to but not exceeding 50% reduction of presynaptic ChAT activity. None of our patients has autonomic symptoms or signs of central nervous system involvement other than that attributed to anoxic episodes. This suggests that the ChAT activity and/or substrate availability are rate limiting for ACh synthesis at the motor nerve but not at other cholinergic synapses. Indeed, stimulated quantal release at the endplate is higher than at other cholinergic synapses, which points to selective vulnerability of the NMJ to reduced ACh resynthesis. Crystal structure of ChAT resolved at 2.2 Å revealed that some of the reported *CHAT* mutations in CMS patients are not at the substrate-binding or the catalytic site of ChAT. Hence these mutation exert their effect by an allosteric mechanism or render the enzyme structurally unstable (Cai *et al.*, 2004).

In most patients, anticholinesterase medications are of benefit in ameliorating the myasthenic symptoms and preventing the apneic crises but few patients fail to respond to cholinergic therapy remaining permanently paralyzed and remain respirator dependent. Prophylactic anticholinesterase therapy is advocated even for patients asymptomatic between crises. Parents of affected children must be indoctrinated to anticipate sudden worsening of the weakness and possible apnea with febrile illnesses, excitement, or overexertion. Long-term nocturnal apnea monitoring is indicated in any patient in whom ChAT deficiency is proven or suspected (Byring *et al.*, 2002).
