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Identification of an agrin mutation that causes congenital myasthenia and affects synapse function C Huzé, S Bauché, P Richard, F Chevessier, E Goillot,

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Presentation on theme: "Identification of an agrin mutation that causes congenital myasthenia and affects synapse function C Huzé, S Bauché, P Richard, F Chevessier, E Goillot,"— Presentation transcript:

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:

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) IP: HA IB: pTyr IB: HA Wild-type Mutated Fold activation Agrin (nM) 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:

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


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