A Mutation Causes MuSK Reduced Sensitivity to Agrin and Congenital Myasthenia Asma Ben Ammar 1,2 , Payam Soltanzadeh 1 , Ste ´ phanie Bauche ´ 1,3 , Pascale Richard 4 , Evelyne Goillot 5 , Ruth Herbst 6 , Karen Gaudon 4 , Caroline Huze ´ 5 , Laurent Schaeffer 5 , Yuji Yamanashi 7 , Osamu Higuchi 7 , Antoine Taly 8 , Jeanine Koenig 1 , Jean-Paul Leroy 1 , Fayc ¸al Hentati 2 , Hossein Najmabadi 9 , Kimia Kahrizi 9 , Manouchehr Ilkhani 10 , Michel Fardeau 11 , Bruno Eymard 1,12 , Daniel Hantaı¨ 1,12 * 1 Inserm, UMRS 975, UPMC, Institut du Cerveau et de la Moelle e ´pinie `re, Groupe Hospitalier Pitie ´ -Salpe ˆtrie `re, Paris, France, 2 Laboratoire de Neurobiologie Mole ´culaire et Neuropathologie, Institut National de Neurologie, Universite ´ Tunis El Manar, La Rabta, Tunis, Tunisia, 3 Ecole Pratique des Hautes Etudes, Paris, France, 4 APHP, UF Cardioge ´ne ´tique et Myoge ´ne ´tique, Service de Biochimie Me ´tabolique, Groupe Hospitalier Pitie ´-Salpe ˆtrie `re, Paris, France, 5 Equipe Diffe ´ renciation Neuromusculaire, IFR128, UMR5161, ENS Lyon, CNRS, INRA, Universite ´ de Lyon, Lyon, France, 6 Medical University of Vienna, Center for Brain Research, Vienna, Austria, 7 Division of Genetics, Department of Cancer Biology, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan, 8 Laboratoire de Conception et Application de Mole ´ cules Bioactives, UMR 7199 CNRS-Universite ´ de Strasbourg, Illkirch, France, 9 University of Social Welfare and Rehabilitation Sciences, Genetics Research Center, Tehran, Islamic Republic of Iran, 10 Shahid Beheshti University of Medical Sciences, Department of Neurology, Tehran, Islamic Republic of Iran, 11 Unite ´ de Morphologie Neuromusculaire, Institut de Myologie, Groupe Hospitalier Pitie ´-Salpe ˆtrie `re, Paris, France, 12 APHP, Centre de Re ´fe ´rence en Pathologie Neuromusculaire Paris-Est, Institut de Myologie, Groupe Hospitalier Pitie ´ -Salpe ˆtrie `re, Paris, France Abstract Congenital myasthenic syndromes (CMSs) are a heterogeneous group of genetic disorders affecting neuromuscular transmission. The agrin/muscle-specific kinase (MuSK) pathway is critical for proper development and maintenance of the neuromuscular junction (NMJ). We report here an Iranian patient in whom CMS was diagnosed since he presented with congenital and fluctuating bilateral symmetric ptosis, upward gaze palsy and slowly progressive muscle weakness leading to loss of ambulation. Genetic analysis of the patient revealed a homozygous missense mutation c.2503A.G in the coding sequence of MUSK leading to the p.Met835Val substitution. The mutation was inherited from the two parents who were heterozygous according to the notion of consanguinity. Immunocytochemical and electron microscopy studies of biopsied deltoid muscle showed dramatic changes in pre- and post-synaptic elements of the NMJs. These changes induced a process of denervation/reinnervation in native NMJs and the formation, by an adaptive mechanism, of newly formed and ectopic NMJs. Aberrant axonal outgrowth, decreased nerve terminal ramification and nodal axonal sprouting were also noted. In vivo electroporation of the mutated MuSK in a mouse model showed disorganized NMJs and aberrant axonal growth reproducing a phenotype similar to that observed in the patient’s biopsy specimen. In vitro experiments showed that the mutation alters agrin-dependent acetylcholine receptor aggregation, causes a constitutive activation of MuSK and a decrease in its agrin- and Dok-7-dependent phosphorylation. Citation: Ben Ammar A, Soltanzadeh P, Bauche ´ S, Richard P, Goillot E, et al. (2013) A Mutation Causes MuSK Reduced Sensitivity to Agrin and Congenital Myasthenia. PLoS ONE 8(1): e53826. doi:10.1371/journal.pone.0053826 Editor: Israel Silman, Weizmann Institute of Science, Israel Received May 14, 2012; Accepted December 5, 2012; Published January 9, 2013 Copyright: ß 2013 Ben Ammar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Assistance Publique-Ho ˆ pitaux de Paris (PHRC AOM 1036), Re ´seaux Inserm, ANR-Maladies Rares (#ANR-07-MRAR-001), Association Franc ¸aise contre les Myopathies (AFM), and Comite ´ Mixte Franco-Tunisien pour la Coope ´ ration Universitaire (CMCU Project #05G0809). A.BA. was the recipient of a doctoral fellowship from the AFM, P.S. of a postdoctoral fellowship from the AFM, and D.H. of a Contrat hospitalier de recherche translationnelle AP- HP Inserm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: daniel.hantai@upmc.fr Introduction The NMJ is a highly specialized structure formed by a motoneuron, a terminal Schwann cell and a muscle fiber. The contact between the nerve terminal and the muscle membrane constitutes a privileged zone in which neurotransmission occurs. At this synapse, the nerve terminal organizes postsynaptic differentiation by releasing a proteoglycan called agrin which binds to its receptor, a low-density lipoprotein receptor (LDLR)- related protein (Lrp4) located on the postsynaptic membrane [1– 3]. Lrp4 forms a complex with the muscle-specific tyrosine kinase (MuSK) which plays a central role in the organization of the postsynaptic scaffold. MuSK activation is required to recruit downstream signaling components that trigger the local aggrega- tion and synthesis of postsynaptic nicotinic acetylcholine receptors (nAChRs) and several other proteins, such as the cytoskeletal protein rapsyn [4–6]. MuSK is a tyrosine kinase receptor with an ectodomain containing three immunoglobulin (Ig)-like domains and a Friz- zled-like cysteine-rich domain (initially described as a C box plus a fourth Ig-like domain), a transmembrane-spanning region, and the intracellular region including a juxtamembrane domain, a kinase domain, and a short C-terminal tail [7–9]. In general, ligand binding to the extracellular portion of tyrosine kinase receptors results in autophosphorylation of specific tyrosine residues, which in turn increases the catalytic activity of the receptor and creates binding sites for other signaling proteins. The PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e53826