1. Identification of an agrin mutation that causes congenital myasthenia and affects synapse function C Huzé, S Bauché, P Richard, F Chevessier, E Goillot, K Gaudon, A Ben Ammar, A Chaboud, I Grosjean, HA Lecuyer, V Bernard, A Rouche, N Alexandri, T Kuntzer, M Fardeau, E Fournier, A Brancaccio, MA Rüegg, J Koenig, B Eymard, L Schaeffer, D Hantaï

2. Agrin  2 laminin †  Na v 1.4 ChAT MuSK Dok-7 AChR  Rapsyn ColQ Congenital myasthenic syndromes  group of rare genetic diseases affecting neuromuscular transmission  heterogeneous for inheritance and pathophysiology  Several causing genes coding for proteins of the neuromuscular junction:

3. Agrin  Heparan sulphate proteoglycan  Encoded by AGRN (chromosome 1p36.33)  Stably associated with synaptic basal lamina  Binds laminin (N-ter), interacts with  -dystroglycan and LRP4 (C-ter)  Several splice forms: with A/y and B/z inserts when synthetized by motor neurons without in non-neural cells including muscle and Schwann cells Bezakova and Rüegg, Nature Rev Mol Cell Biol (2003) 4:295-309

4. Agrin ACh Dok-7 LRP4 MuSK rapsyn AChR Synapse-specific transcription  Extra-synaptic transcription  Neural agrin classical functions AChR AChR RNA extrasynaptic nucleus synaptic nucleus  In the embryo: AChR aggregation  In the adult: synaptic nucleus transcription

5. The patient Clinical data  42-year-old woman  unable to run since early childhood  at last examination:  mild bilateral lid ptosis  mild facial deficiency  mild weakness of the proximal lower limbs  no muscle atrophy, no scoliosis, no contractures  EMG repetitive stimulation at 3 Hz: clear decrement  Her brother presented similar symptoms  Treatment:  Cholinesterase inhibitors and 3,4-diaminopyridine: ineffective  Ephedrine: sustained increase in muscle performance and endurance

6. c.5125g>c Wild-type Proband III- 5 The patient Mutation analysis  36 exons of AGRN were sequenced  Homozygous missense transversion c.5125G>C in exon 29  Gly to Arg substitution in the LG2 domain of agrin  Conserved residue among species and isoforms  Absence in >200 control chromosomes 7 Gly1709Arg I II 1 st degree cousins 5 Gly1709Arg 4 Gly1709Arg 3 no mutation 1 Gly1709Arg 2 Gly1709Arg 5 no mutation 3 [Gly1709Arg] 4 Gly1709Arg 6 ND 1 ND 2 ND III

7. Control Patient N=28 N=21 The patient Muscle biopsy Remodeling Neoformed Normal Denervated  Abnormal neuromuscular junctions  Dishevelled aspect of neurofilament staining  Fragmentation of NMJs % of total number of NMJs n=21 NMJs Number of fragments

8. Functional evaluation Production of mutated and wild-type recombinant agrin SS LG1 LG2 LG3 EG NtA S/T SEA LE A/y B/z α-Dystroglycan AChR aggregation Laminin Heparin Integrin EG TM SS NtA LG1 LG2 LG3 EG A/y B/z EG Gly1709Arg FS Mini-Agrin Agrin  Chick mini-agrin construct (Moll et al., Nature 2001)  Site-directed mutagenesis  Production of wild-type and mutant mini-agrin in 293-EBNA cells  Conditioned medium purified by affinity chromatography on nickel agarose column FS

9. Recombinant agrin Rat soleus muscle Nerve Functional evaluation Effects of mutated agrin in rat muscle denervated neoformed remodeling normal Percentage of total number of NMJs Saline Wild-type Mutated N=42 N=45 N=44 Number of fragments Recapitulation in the rat of the features observed in the patient muscle biopsy:  Abnormal neuromuscular junctions  Dishevelled aspect of neurofilament staining  Fragmentation of NMJs

10.  AChR clustering in C2C12 myotubes in culture  MuSK phosphorylation in 293T cells in culture expressing both LRP4 and MuSK mutant agrin wild-type agrin Agrin (nM) - - 0.1 0.1 1 1 0.1 0.1 1 1 IP: HA IB: pTyr IB: HA Wild-type Mutated 0 1 2 3 4 5 Fold activation Agrin (nM)0.1 1 1 Wild-typeMutated Functional evaluation Classical functions of neural agrin are not affected

11. Functional evaluation Underlying mechanisms?  Binding of agrin to  -dystroglycan is not modified by the mutation  Changes in muscle cytoskeleton?  -dystrobrevin and  -syntrophin KO mice have frayed axon terminals  Anomalies in axon terminals may impair neurotransmission: agrin binding to  3Na+/K+-ATPase? ELISA

12.  First identification of a mutation in agrin causing CMS  This mutation does not affect the classical function of agrin in the formation of the post-synaptic compartment  Novel function of agrin in the maintenance of the neuromuscular junction? Conclusion Huzé et al. Am J Hum Genet 2009; 85: 155-167

13.  ENS, Lyon Caroline Huzé Annie Chaboud Heba-Aude Lecuyer Isabelle Grosjean Evelyne Goillot Laurent Schaeffer  CHU Vaudois, Lausanne Thierry Kuntzer  Biozentrum, Basel Markus Rüegg  MPI, Heidelberg Frédéric Chevessier  Università Cattolica del Sacre Cuore, Rome Andrea Brancaccio Hôpital de La Salpêtrière, Paris  UF Cardio- et Myogénétique Karen Gaudon Pascale Richard  Service d’Electrophysiologie Emmanuel Fournier  Institut de Myologie Inserm U975 & Neuromuscular Clinics Stéphanie Bauché Asma Ben Ammar Andrée Rouche Nektaria Alexandri Michel Fardeau Jeanine Koenig Daniel Hantaï Bruno Eymard □ Acknowledgments