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