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Pathogenic mechanisms underlying synaptic dysfunction
in congenital myasthenic syndromes David Beeson Weatherall Institute of Molecular Medicine Oxford
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Congenital myasthenic syndromes
Genetic Fatiguable muscle weakness Heterogeneous Endplate region
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CMS-associated genes Agrin Clustered AChR MuSK Rapsyn CHAT AChE COLQ
LRP4 CHRNA CHRNB CHRND CHRNE CHRNG MuSK Rapsyn RAPSN MUSK DOK-7 SCN4A
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Congenital myasthenic syndromes Syndrome
(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 – 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 Common mutation 1124_1127dupTGCC 325G>T 415G>C
1339_1342dupCTGG 437delC 967C>T 596delT 1504_1505insTA IVS1+14del15 1185C>G 539G>C 1378insC IVS2-1G>T 1143insC C-terminal PH PTB 473G>A 601C>T 481G>A 1263insC 1508insC 14 have common deletion 3 single mutations 496G>A 230C>T 1357_1370del14 548_551delTCCT 101_141del 1487G>T Common mutation 1124_1127dupTGCC
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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 Murine myotubes C2C12 Dok-7 mutant (Fewer and smaller clusters)
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AChR clusters in C2C12 myotubes induced by truncated Dok-7
Number of clusters /10 fields Mock Wild type 1143insC 548delTCCT 1124dupTGCC
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Type of AChR clusters formed following expression of
Dok-7 in C2C12 cells Branched C-shaped Perforated Endplate
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Number of clusters * * * * * * * * * all significant all significant
1 way ANOVA Bonferroni’s multiple comparison all significant * * * * * 1 way ANOVA Tukey's Multiple Comparison Test
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Agrin-induced clusters on myotubes derived from a Dok-7 patient
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
Legs raised Arms raised Time on treatment Time Time on treatment Disability score QMG
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- + - + Effect of ephedrine on AChR clusters
in human myotubes from a Dok-7 patient homozygous for 1124_1127dupTGCC Clusters per field Cluster length 40% 3.4-fold increase increase - + - + Ephedrine Ephedrine
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Clustered AChR NERVE signalling MuSK Agrin Rapsyn
Retrograde signalling LRP4 MuSK Agrin Clustered AChR PTB motif NPXY Rapsyn 1124_1127dupTGCC C-terminal domain PH PTB reduced MuSK-P* Impaired kinase signalling Truncated Dok-7
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Maintaining synaptic structure
Ephedrine LRP4 b2AR
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Collaborators: Dr Palace + Clinicians Yuji Yamanashi Angela Vincent + team
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Conductance of ion channel largely governed by
three rings of charged amino acids Fetal Adult Extracellular ring K M2 Q M2 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 NH2 COOH 20 40 60 80 100 120 20 40 60 80 100 120 eP282R surface binding surface binding BuTX BuTX - - a a WT P282R DF266 control eDF266
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Electrophysiological methodology
Cell-attached patch recordings Transfected HEK 293 cells Constant low concentration of acetylcholine Wildtype AChR recordings Closed Open Log10 duration (ms) N (sqrt) Burst length 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
Wildtype AChR eDF266 AChR Ohm’s Law: V = I ×R R = V ÷ I Conductance (g) = 1/R
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Kinetic abnormalities of the AChR
Excess Na+,Ca 2+ Insufficient Na+ PROLONGED ACTIVATION SHORTENED REDUCED CONDUCTANCE
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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 33
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Dok-7 induced AChR clusters
Myotubes – no Dok-7 Dok-7 WT Murine myotubes C2C12 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 Rare Ophthalmoplegia Spontaneous improvement
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CMS-associated genes Agrin Clustered AChR MuSK Rapsyn CHAT AChE COLQ
CHRNA CHRNB CHRND CHRNE CHRNG RAPSN MUSK DOK-7 CHAT SCN4A
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Mild arthrogryposis RAPSN mutation
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Muscle AChR NH2 COOH Adult Fetal e g
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Transgenic slow channel mouse with AChR-EGFP
NFP /synaptophysin eL221F-EGFP merge
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Dok-7 induced AChR clusters in C2C12 cells
Myotubes – no Dok-7 Dok-7 mutant Dok-7 WT (Fewer and smaller clusters) Murine myotubes C2C12
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