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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Identification of an agrin mutation that causes congenital myasthenia and affects synapse functionC 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
Agrin
2 laminin
††
Nav1.4
ChAT
MuSK
Dok-7AChR
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:
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
Agrin ACh
Dok-7
LRP4
MuSKMuSKrapsyn
AChR
Synapse-specific Synapse-specific transcription transcription
Extra-synaptictranscription
Neural agrin classical functions
AChR
AChR RNA extrasynaptic nucleus
synaptic nucleus
In the embryo: AChR aggregation
In the adult: synaptic nucleus transcription
The patientClinical 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
c.5125g>c
Wild-type
Proband III-5
The patientMutation 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
1st degree cousins
5Gly1709ArgGly1709Arg
4Gly1709ArgGly1709Arg
3
no mutation
1
Gly1709Arg
2
Gly1709Arg
5
no mutation
3
[Gly1709Arg]
4
Gly1709Arg
6
ND
1
ND
2
ND
III
Control Patient N=28 N=21
The patientMuscle biopsy
Remodeling Neoformed
Normal Denervated
Abnormal neuromuscular junctions
Dishevelled aspect of neurofilament staining
Fragmentation of NMJs %
of
tota
l num
ber
of
NM
Js
n=21 NMJs
Num
ber
of
fragm
ents
Functional evaluationProduction of mutated and wild-type recombinant agrin
SS
LG1 LG2 LG3EG
NtA
S/T S/TSEALELE
A/y B/z
α-DystroglycanAChR aggregation
Laminin
Heparin Integrin
EGEG
EG
TM
SS NtA LG1 LG2 LG3EG
A/y B/z
EGEG
EG
Gly1709Arg
FSMini-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 FS FS FS FS FSFSFS FS
Recombinant agrin
Rat soleus muscle
Nerve Functional evaluationEffects of mutated agrin in rat muscle
denervated
neoformedremodeling
normal
Pe
rce
nta
ge
of
tota
l nu
mb
er
of
NM
Js
Saline Wild-type Mutated N=42 N=45 N=44
Num
ber
of
fragm
ents Recapitulation in the rat of the features
observed in the patient muscle biopsy: Abnormal neuromuscular junctions Dishevelled aspect of neurofilament
staining Fragmentation of NMJs
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 1IP: HA
IB: pTyr
IB: HA
Wild-type Mutated
0
1
2
3
4
5
Fold
acti
vatio
nAgrin (nM) 0.1 0.11 1
Wild-type Mutated
Functional evaluationClassical functions of neural agrin are not affected
Functional evaluationUnderlying 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
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
ENS, LyonCaroline Huzé Annie ChaboudHeba-Aude LecuyerIsabelle Grosjean Evelyne GoillotLaurent Schaeffer
CHU Vaudois, LausanneThierry Kuntzer
Biozentrum, Basel Markus Rüegg
MPI, HeidelbergFrédéric Chevessier
Università Cattolica del Sacre Cuore, Rome Andrea Brancaccio
Hôpital de La Salpêtrière, Paris
UF Cardio- et Myogénétique Karen GaudonPascale Richard
Service d’ElectrophysiologieEmmanuel Fournier
Institut de Myologie Inserm U975 & Neuromuscular ClinicsStéphanie BauchéAsma Ben AmmarAndrée RoucheNektaria AlexandriMichel FardeauJeanine KoenigDaniel HantaïBruno Eymard
□
Acknowledgments
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