David Beeson

1
Pathogenic mechanisms underlying synaptic
dysfunction in congenital myasthenic syndromes
David Beeson Weatherall Institute of Molecular
Medicine Oxford
2
Congenital myasthenic syndromes

• Genetic
• Fatiguable muscle weakness
• Heterogeneous

Endplate region
3
CMS-associated genes
CHAT
AChE
COLQ
Agrin
Clustered AChR
LRP4
MuSK
CHRNA CHRNB CHRND CHRNE CHRNG
Rapsyn
RAPSN MUSK DOK-7
SCN4A
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Congenital myasthenic syndromes
(Studied in Oxford)
Syndrome Kinships
AChR deficiency (CHRNE) 112
AChR deficiency (RAPSN) 51
CMS with proximal weakness (DOK7) 55
Slow channel (CHRNA/B/D/E) 22
Fast channel (CHRNA/D/E) 12
AChE deficiency (COLQ) 15
Presynaptic (CHAT) 8
Additional referrals no mutations found
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Neuromuscular synapse (a complex structure)
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Postsynaptic specialisation
NERVE
Agrin
Clustered AChR
LRP4
MuSK
Rapsyn
Dok-7
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Clinical features of Dok-7 CMS
Inheritance - recessive Onset - 1.5 4 years,
sometimes respiratory problems at
birth Symptoms - limb girdle pattern of
weakness, ptosis, but eye muscles
unaffected
Unresponsive - pyridostigmine
Responsive - ephedrine
salbutamol
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Dok-7 mutations
325GgtT
415GgtC
1339_1342dupCTGG
437delC
967CgtT
1504_1505insTA
596delT
IVS114del15
1185CgtG
1378insC
539GgtC
1143insC
IVS2-1GgtT
473GgtA
601CgtT
481GgtA
1508insC
1263insC
496GgtA
230CgtT
1357_1370del14
548_551delTCCT
101_141del
1487GgtT
1124_1127dupTGCC
Common mutation
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In vitro clustering assay
Myoblasts Transfect with mutant cDNA
C2C12 RAPSN -/- MUSK -/-
Differentiate
Myotubes
AChR
Agrin
AChR clusters
Myotubes
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Dok-7 induced AChR clusters in C2C12 cells
Myotubes no Dok-7
Dok-7 WT
Dok-7 mutant
(Fewer and smaller clusters)
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AChR clusters in C2C12 myotubes induced by
truncated Dok-7
/10 fields
Number of clusters
Mock
Wild type
1143insC
548delTCCT
1124dupTGCC
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Type of AChR clusters formed following expression
of Dok-7 in C2C12 cells
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Number of clusters
all significant




1 way ANOVA Bonferronis multiple comparison
all significant





1 way ANOVA Tukey's Multiple Comparison Test
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AChR clusters in cultured human myotubes
Agrin-induced clusters on myotubes derived from a
Dok-7 patient
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Can patients with DOK7 mutations be treated?

• Unresponsive to cholinesterase medication
• Some show some benefit fro 3,4-DAP
• Remarkable response to ephedrine
• 
  salbutamol

Wheelchair/scoliosis to running and
jumping Wheelchair to running 200 metres
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Dok-7 CMS patients respond to treatment with
ephedrine
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Effect of ephedrine on AChR clusters in human
myotubes from a Dok-7 patient homozygous for
1124_1127dupTGCC
40
3.4-fold
increase
increase
-
-


Ephedrine
Ephedrine
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NERVE
Retrograde signalling
LRP4
MuSK
Agrin
Clustered AChR
PTB motif NPXY
Rapsyn
1124_1127dupTGCC
reduced MuSK-P
Impaired kinase signalling
Truncated Dok-7
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Maintaining synaptic structure
Ephedrine
LRP4
b2AR
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Dr Palace Clinicians Yuji Yamanashi Angela
Vincent team
Collaborators
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(No Transcript)
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Conductance of ion channel largely governed by
three rings of charged amino acids
Fetal
Adult
Extracellular ring
K

• Larger conductance
• Shorter openings

Intermediate ring
Cytoplasmic ring
K
Imoto et al.
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Case Study

• Clinical features
• 47 yr woman
• Onset at birth generalised weakness and ptosis
• Progressive course
• Fatigable limb weakness Severely
  restricted eye movements
• Responsive to pyridostigmine
  (high dose)
• Respiratory arrest aged 45, hypoxic brain injury

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DNA screening revealed two mutations

• Epsilon subunit
• eP282R missense
• eDF266 in frame deletion

Surface expression
eP282R
surface binding
surface binding
BuTX
BuTX
-
-
a
a
WT
control
P282R
DF266
eDF266
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Electrophysiological methodology

• Cell-attached patch recordings
• Transfected HEK 293 cells
• Constant low concentration of acetylcholine

Wildtype AChR recordings
Hundreds/thousands of bursts are measured
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Size of eDF266 opening are reduced
Pipette potential
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Slope conductance reduced
Ohms Law V I R R V I Conductance (g)
1/R
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Kinetic abnormalities of the AChR
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A
B
Figure 3 Time (seconds) for individual patients
in A Arms raised 90 degrees and B Legs raised
45 degrees (data points are mean of right side
and left side scores).
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Novel AChR abnormality

• 47 year old female with symptoms weakness
  since birth
• Progrssive bulbar, repiratory and limb muscle
  involvement
• Positive response to pyridostigmine, but no
  improvement with 3,4-DAP
• At 45 suffered respiratory arrest with
  resultant hypoxic brain damage
• No family history

Heteroallelic for mutations in CHRNE (e subunit)
eP282R eDF266
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Agrin-induced clusters on myotubes derived from a
Dok-7 patient
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AChR deficiency
Normal
Mutant
20 mm
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Inverted screen test
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Dok-7 induced AChR clusters
Myotubes no Dok-7
Dok-7 WT
Dok-7 common mutation
(Fewer and smaller clusters)
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Comparison of AChR deficiency phenotypes with
early onset presentation
Clinical feature Early Onset rapsyn mutations AChR deficiency ?-subunit mutations
Arthrogryposis Common Absent
Episodic crises Common Rare
Ophthalmoplegia Absent Common
Spontaneous improvement Common Rare
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(No Transcript)
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Mild arthrogryposis
RAPSN mutation
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Muscle AChR
Fetal
Adult
e
?
?
?
?
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Transgenic slow channel mouse with AChR-EGFP
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Dok-7 induced AChR clusters in C2C12 cells