Post on 12-Feb-2022
Hereditary myasthenic syndromes:
new genes and better treatment
David Beeson
Weatherall Institute of Molecular Medicine
• Acute Crises
• Introduction and Diagnosis
• Eye movements
• Later onset progressive forms
• Treatment
Congenital myasthenic syndromes
• Genetic disorders of the NMJ
• Rare 1:100,000
• Mendelian inheritance
• Heterogeneous
• Treatable
• Additional cases genetically
undiagnosed
The neuromuscular junction
Key proteins at the NMJ
NEUROMUSCULAR TRANSMISSION
MUSCLE
VGCC
AChR
NERVE
VGNaC
-40mV
-50mV
-70mV
Threshold
Membrane potential
Clustered AChR
Agrin
MuSK Rapsyn
CMS-associated proteins
DOK-7
ChAT
Na 1.4
LRP4
Choline ACh
PT
B
subunits
ColQ
At least 26 different
genes can be involved
GFPT1
DPAGT1
ALG2/14
GMPPB
Laminin 2
PREPL
ChTT
VAChT
Col13A11
Features of CMS • Genetically determined myasthenias
• Rare
• Muscle weakness:
- ptosis, EOM, face, limbs, trunk, bulbar, respiratory
- fatiguable
• Onset birth/infancy
• Stable throughout life
• Consanguinity / Family history
• EMG: decrement, jitter, blocking
• No antibodies
• Response to anti-cholinesterases & 3,4-DAP
CMS or what?
• hypermobility syndromes
• dyspraxias (Developmental Coordination Disorder)
• sero-negative MG
• chronic fatigue syndrome
• congenital myopathy
• SMA
• muscular dystrophy
Useful distinguishing features From non-myasthenic conditions
• EMG – decrement > jitter/block, non-stim vs stim SFEMG
• fatiguable ptosis
• response to pyridostigmine
• fluctuation of weakness (better in morning)
From autoimmune MG
• early onset (< 3yrs) & Family History
• ankle dorsi-flexion weakness
• ophthalmoplegia static/lack diplopia
• non-acute onset
• ‘symmetrical’ ptosis
• no antibodies (common in childhood MG)
PRE
CHAT
SYNAPTIC
COLQ
POST
AChR
Deficiency
AChR ε subunit
RAPSN
Glycosylation defects
Kinetic Abn AChR
Slow channel
Fast channel
Dispersion
DOK7ε
(MUSK)
(AGRIN)
Ophthalmoparesis
AChR deficiency
(-subunit mutation)
24/24 severely restricted EOM
• Sister born 4 years later
• Slow feeder
• At 1 month full EOM
• 3 months later some restriction
• 6 months eye movements worse
• 10 months complete ophthalmoplegia
Restriction of eye movement develops over time
Variability in eye movements in CMS
AChR mutations:
Deficiency severe ophthalmoplegia
Fast channel 90% ophthalmoplegia (70% severe)
SCS 2/3 partial ophthalmoplegia
Reduced clustering of AChR
RAPSN ‘all’ full range EOM
maj. divergent squint
DOK7 93% normal EOM
COLQ partial, normal, complete CHAT normal Glycosylation normal
AChR Deficiency
AChR ε subunit
RAPSN
Glycosylation defects
Dispersion
DOK7ε
(MUSK)
(AGRIN)
SYNAPTIC
COLQ
Kinetic Abn AChR
Slow channel
Fast channel
PRE
CHAT
POST
Early life Crises
Hospitalisation:12/14, Ventilation:10/14, Sibling deaths:3/14
Induced by minor infections
Between attacks well
Significant morbidity and mortality
RAPSN CMS
Sudden life-threatening crises infancy & early childhood
SYNAPTIC
COLQ
Dispersion
DOK7ε
(MUSK)
(AGRIN)
(LRP4)
AChR Deficiency
AChR ε subunit
RAPSN
Glycosylation defects
Kinetic Abn AChR
Slow channel
Fast channel
PRE
CHAT
POST
DELAYED PRESENTATION
PROGRESSIVE MYOPATHIC FEATURES
worse with pyridostigmine
Cohort of patients with limb girdle pattern of weakness
• Sparing of face and ocular muscles
• Onset often childhood
• EMG decrement
• Pyridostigmine responsive
• Tubular aggregates
Data analysis from whole exome/genome sequencing
Sequenced coding regions of the genome
Identified all variants.
Filtering out known SNPs
All genes with two or more mutations in the same gene
Genes shared between both patients
GFPT1/DPAGT1/ALG2/ALG14/GMPPB
N-linked glycosylation pathway
GMPPB
• Clinical features
– Limb-girdle muscle weakness
– Usually no ptosis
– No ophthalmoplegia
– No facial involvement
– No bulbar involvement
EASY MISS MYASTHENIC
FEATURES
Glycosylation pathway mutations
GMPPB - myasthenia
• Fatiguable muscle weakness
• Myasthenic features are treatable
• Selective neurophysiological abnormalities
Dr Mathew Pitt. GOSH, London
Broadening of the phenotype
• Sparing of face and ocular muscles
• Onset can be in adulthood
• Can have raised CK
• Variable severity – overlapping myopathy
• Variable neurophysiology/muscle groups
• Pyridostigmine responsive, addition
salbutamol/ephedrine beneficial
Aberrant glycosylation
AChR
levels
CMS LGMD CDG
AChR
subunits
Other NMJ
components dystroglycan Other components in
CNS and other tissues
Stability and
structure of
synapse
Myofiber
structure
CNS and other
tissues
involvement signal
transmission
Dystrophic
changes in
muscle
CNS
organisation
CNS
involvement
25% 1267delG
>90% N88K
60% 1124_1127dupTGCC
60 % G153S
25 Genes: >400 Mutations
Estimate <10 % no known mutation
Oxford CMS Cohort – genetic diagnosis 543 patients
Better treatment?
(The DOK7 story)
Dok-7
Clustered AChR
NERVE
Agrin
MuSK Rapsyn
Aneural postsynaptic specialisation
X
(Yuji Yamanashi)
C-terminal PH PTB
1263insC
1339_1342dupCTGG
1143insC
548_551delTCCT
1124_1127dupTGCC
601C>T 1508insC
1378insC
1357_1370del14
DOK7 mutations
Common mutation
IVS1+14del15
IVS2-1G>T
1504_1505insTA 437delC
481G>A
539G>C
496G>A
596delT
230C>T
473G>A
1185C>G
415G>C 967C>T
101_141del 1487G>T
325G>T
Differentiate
+ Dok-7
Mutant Dok-7
Myoblasts
Transfect with cDNA
Myotubes AChR
AChR clusters
In vitro clustering assay
C2C12
Myotubes
METHODS C2C12 mouse
myoblasts
Retroviral
infection
Differentiation
into myotubes
AChR cluster
induction DOK7
Myotubes – no DOK7
DOK7 common mutation
DOK7 WT
(Fewer and smaller clusters)
DOK7-induced AChR clusters
Type of AChR clusters formed following expression of
Dok7 in C2C12 cells
Branched C-shaped Perforated Endplate
Dok-7 mutations lead to fewer and less complex clusters
pB
AB
ED
ok-
7 W
T
T77
MG
109C
R15
8QG
161R
G18
0A11
27in
sTG
CC
0
100
200
300
No
A
Ch
R clu
sters p
er m
m2
branched
pBABE
WT
T77M
G10
9C
R15
8Q
G16
1R
G18
0A
1127
insT
GCC
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
perc
en
t o
f clu
ste
rs
Number Complexity
(J.Cossins)
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
Respond – ephedrine/salbutamol
(Palace et al., 2007))
Clinical features DOK7 CMS
0
40
80
120
160
200
240
Baseline First Dose 2 Month 6-8 Month
0
20
40
60
80
100
Baseline 1st dose 2 months 6-8months
Legs raised
Arms raised
Time on treatment T
ime
Tim
e
Baseline 1st dose 2 months 6-8 months0
3
6
9
12
15
18
21
24
*
QM
G (
max 3
9)
Time on treatment
Disability score
QM
G
DOK7 CMS patients respond to
treatment with ephedrine
Lashley et al., 2010
EPHEDRINE 4 months 15mg EPHEDRINE 1 yr 45mg
Improvement over months
Originally non-ambulant
DOK7 CMS treated from an early age
Age 3, severe weakness, unable to walk, in and out of ITU
Good response if treated from an early age
Age 9, almost normal strength
Nerve terminal
ACh AGRN
MUSK
DOK7 Stabilise
Rapsyn
Nerve terminal
ACh Agrin
MuSK
Dok-7
Stabilise
2AR
Salbutamol
Ephedrine
Compensation 2nd messenger
How is the medication working?
Rapsyn
Impaired signaling
A group of patients with AChR deficiency who are
severely disabled despite optimum therapy
Excellent initial response to anticholinesterase therapy
but effectiveness tails off over time
Fetal
g
Adult
Muscle
Nerve
AChR deficiency syndrome
Normal Mutation
1206ins19
M1 M2 M3 M4
Extracellular
Intracellular
NH2
Y15X
S143L
E157del
541delG
553del7
627ins2
1267delG
1045ins10
1033insT
1030insC
911delT
1258del23
P121L
P245L
M292del
S278del
R311Q 1293insG
P331L
509insA
1208ins19
C128S
1369delG
Y458XIVS8-1G/A
IVS11+74del85
R217L
A411P
T159P Y15H
COOH
IVS9+1G/T
70insG94ins37127ins5
I216del
826ins10
Y104X -11-10LLdel
R64X
Promoter-156C->T
1206ins19
M1 M2 M3 M4
Extracellular
Intracellular
NH2
Y15X
S143L
E157del
541delG
553del7
627ins2
1267delG
1045ins10
1033insT
1030insC
911delT
1258del23
P121L
P245L
M292del
S278del
R311Q 1293insG
P331L
509insA
1208ins19
C128S
1369delG
Y458XIVS8-1G/A
IVS11+74del85
R217L
A411P
T159P Y15H
COOH
IVS9+1G/T
70insG94ins37127ins5
I216del
826ins10
Y104X -11-10LLdel
R64X
Promoter-156C->T
Partial compensation
-subunit mutations (recessive)
Bungarotoxin-labeled
Engel AG, Lambert EH, Santa T: Study of long-term anticholinesterase
therapy. Effects on neuromuscular transmission and on motor end-plate
fine structure. [Internet]. Neurology 1973, 23:1273–81.
Neurotransmission leads to disassembly of endplate AChR clusters
Focus on the factors that counteract disassembly
Control Mutation
Nerve terminal
ACh
Excess
Destabilised
postsynaptic structure
Agrin
MuSK
Dok-7
Stabilise
2AR
Salbutamol
Ephedrine
Compensation 2nd messenger
2 agonists for patients on AChE inhibitors?
Rapsyn
Pyridostigmine
0
10
20
30
Baseline 6-8 months
Mea
n c
ha
ng
e f
rom
bas
eli
ne
(% l
ow
er
lim
it o
f n
orm
al)
Armraise,right
Armraise,le
Legraise,right
Legraise,le
Headli
FVC
Grip,rigth
Grip,le
A
B
C
p2
p5
p3
p
4 p2
p1 p6
p6
p3
p1
p5
p4
QMG scores
Adding salbutamol or ephedrine to severe AChR deficiency patients
(Rodriguez-Cruz et al., 2015)
Dok-7
Clustered AChR
NERVE
Agrin
MuSK Rapsyn
LRP4
Disorders affecting
synaptic structure and stability Disorders affecting
synaptic function
Muscle
Nerve
981215 (sldq1-10.asc)981215 (sldq1-11.asc)
981215 (sldq1-12.asc)
L221F
Wild type
L221F (981202-plq3)L221F (981202-plq5)
L221F (981202-plq10)
Normal openings
Prolonged openings
981215 (sldq1-10.asc)981215 (sldq1-11.asc)
981215 (sldq1-12.asc)
L221F
Wild type
L221F (981202-plq3)L221F (981202-plq5)
L221F (981202-plq10)
Normal openings
Prolonged openings
Slow channel syndrome
AChR deficiency
Treatment summary
Destabilising effects of enhanced
neurotransmission through
anticholinesterase medication
Mestinon
Stabilisation through
AGRN pathway
2 ADR agonists
Tailored personalised therapy
Summary
• CMS
• Rare and heterogeneous
• Genetic disorders
• Neuromuscular transmission
• > 20 genes
• Fatigable muscle weakness
• Autoimmune MG
85%
(Genetic dx) Schematic representation of the NMJ
15% (NGS)
Clinical features
• Onset at birth
• Breathing and feeding difficulties
• Early episodes of clinical deterioration
• Bilateral ptosis + normal eye movements
• Dysmorphic features, abnormal chest
• No response to pyridostigmine
CMS due to COL13A1 mutation
CMS due to MuSK mutations
• 18 years old patient
• Onset at birth
• Severe muscle weakness
• Respiratory failure + tracheo
• Wheelchair bound
• Off-treatment
• Life-threatening deterioration
with pyridostigmine
Response to treatment
Pre-treatment Post-treatment
(salbutamol 4mg bd)
Good response to salbutamol
• distinct genotype / phenotype associations
• may not respond or get worse with pyridostigmine
• can present adulthood
• can look myopathic
• seronegative myasthenia:
check clustered AChR abs
weak ankle dorsiflexion consider CMS
• respond well to specific & different treatments
• important to obtain specific genetic diagnosis/mechanism
• perform RNS /SFEMG in weak muscles of limb girdle
weakness
CMS ‘take home messages’
TREATMENT
Acknowledgements
Col13a1-001 = 3093 bp
Col13a1-011 = 2276 bp
The lab team
Clinical team
Yugi Yamanashi
Angela Vincent
Hanns Lochmuller
Close up view
8 pA
5 ms
Richard Webster, University of
Oxford
Working out how the AChR is affected
Excess Na+,Ca 2+ Insufficient Na+
PROLONGED
ACTIVATION
SHORTENED
ACTIVATION
REDUCED
CONDUCTANCE
Insufficient Na+
Abnormalities of the AChR function
Slow channel Fast channel Conductance syndrome