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

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Acknowledgments