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Inherited Disorders of Neuromuscular Transmission –
from gene discovery to tailored treatments
IRDiRC conference, Dublin
Forward Look Session
April 17, 2013
Hanns LochmüllerInstitute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
Clinical Criteria:� myasthenic syndrome: exercise induced muscle weakness� early onset of disease: birth / first years of life (< 2 years)� no response to immunosuppressive treatment� treatment with anticholinesterase drugs may lead to
improvement
CMS: Diagnostic Criteria
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
Neonatal:� poor suck and cry, choking spells, ventilatory failure, ptosis;
symptoms worsened by crying or activity
Infancy, childhood:� delayed motor milestones; seldom learn to run � cannot climb steps well; abnormal fatigability on exertion � cannot keep up with peers in sports� Ptosis; fixed or fluctuating ocular palsies� Spinal deformities, reduced muscle bulk
(Uehara and Desaki, in Salpeter, 1987; Ed.)
The neuromuscular junction - structure
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
Ca++ channel
ChAT deficiency
AChR
ColQ
Sodium channel, voltage-gated
laminin beta 2
agrin
axon
synaptic vesicle
acetylcholinesterase
potassium channel
AcetylCoA
Cholin
ACh
muscle
Lrp4
presynaptic
endplate-AChEdeficiency
ChAT CHAT
synaptic
10q11
COLQ 3p25
LAMB2 3p21 laminin
Kinetic changes of AChR/ endplate-AChRdeficiency
CHRNA1 2q24
rapsyn
CHRNB1CHRNDCHRNE
17p112q3317p13
agrin AGRN 1pter
postsynaptic
rapsyn (impaired clustern of AChR)
MuSK muscle-specific kinase
RAPSN 11p11
MUSK 9q31
Dok-7 downstream of kinase 7 DOK7 4p16
SCN4A 17q23Nav
GFPT1 GFPT1 2p13glutamine:fructose-6-phosphate amidotransferase
CHRNEno mutation
RAPSN
MUSK DOK7
CHRNA1
COLQ
CHRND
CHRNB1
CHAT
GFPT1
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
Identified genetic defects in a large CMS cohort(Munich, 1997-2011)
Most frequent !
CHRNE
CHRNE mutations leading to AChR deficiency: clinical clues
�Onset of disease: early infancy, typically within the first year of life
�Clinical symptoms: ptosis, fixed ophthalmoparesis, weakness of bulbar muscles, in general mild phenotype and benign course of disease
�Very rare: respiratory insufficiency, severe generalized weakness
�Anticholinesterase medication: clearly positive response to short-term and long-term treatment
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
CHRNEno mutation
RAPSN
MUSK DOK7
CHRNA1
COLQ
CHRND
CHRNB1
CHAT
GFPT1
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
LG-CMS represent a significant proportion of total CMS cases
� Onset usually in the first years of life, but late-onset is possible
� first symptom: limb girdle weakness(waddling; sometimes inward rotation of knee and feet)
� other frequent symptoms: ptosis, respiratory crisis (often with need for asisted ventilation)
� Mestinon ineffective for the long-term(sometimes worsening)
CMS with DOK7 mutations: clinical clues
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
LG-CMS with tubular aggregates – a distinct entity
�Limb girdle weakness�No ophthalmoplegia, no ptosis�Benefit from esterase inhibitors�No mutations in DOK7�Mutations in GFAT or DPAGT
Tubular aggregates
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
Reduced number and size of AChR clusters after downregulation of Dok-7
Motor neurons mergeAChR
Non
-inje
cted
zDok
7MO
inje
cted
Neuromuscular junctions at 48 hpf
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
No
n-i
nje
cted
zDo
k7M
O in
ject
ed
AChR Slow muscle merge
Slow-twitch muscle fibre morphology is altered after downregulation of Dok-7
Slow-twitch muscle fibres and acetylcholine receptors at 48 hpf
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
DOK-7 knockdown in Zebrafish - summary
� Dok-7 deficient zebrafish have less and smaller
neuromuscular junctions and motility defects
� Dok-7 deficiency leads to misalignment of slow muscle
fibres. This is a previously unrecognised function of Dok-7
outside the NMJ
� This function is MuSK independent, as MuSK deficiency
causes a more severe loss of acetylcholine receptors, but
leaves slow muscle fibres intact
� Zebrafish are a good animal model for studying the
neuromuscular junction
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
Defect Gene Therapy
Cholin-Acetytransferase Deficiency CHAT AChEI, prophylactic usefor apnea prevention!
Endplate AChE Deficiency COLQ ephedrine? AVOID AChEI!
Receptor Deficiency CHRNA-E AChEI, 3,4 DiaminopyridineSlow Channel Syndrome CHRNA-E Fluoxetine, AVOID AChEI!Fast Channel Syndrome CHRNA-E AChEI, 3,4 DiaminopyridineRapsyn Defect RAPSN AChEI, prophylactic use?Sodium channel, voltage-gated SCN4A AChEI?? (1 family)MuSK (muscle-specific kinase) MUSK AChEI ?? (1 family)
Dok-7 (downstream of kinase 7) DOK7 ephedrine, albuterol/salbutamol?GFPT1/GFAT1 GFPT1 AChEI
CMS – Differential Therapy
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
Summary
�CMS are clinically very variable, genotype-phenotype correlations are difficult to establish. However, there are some clues pointing towards the underlying genetic defect
Hanns Lochmüller - Congenital Myasthenic Syndromes (CMS)
�Detection of the genetic defect is important for the selection of the appropriate drug therapy
�Clinical vigilance for neuromuscular transmission defects is key to accurately diagnose and treat patients with CMS
�Standard clinical trials may be difficult, but standardized follow-up of patients and international collaboration may help to improve standards of care and therapy
16|TREAT-NMDBioBank
Patient Registries
Care & Trial Site Registry
Outcome Measures
Standards ofDiagnosis &
CareTACT
Website & Communications
Joint Research for DMD
Standard Operating
Procedures
Three year
work plan
www.treat-nmd.eu
Action Plan
3 year plan
Milestone-driven approach
Maintains network
momentum & establish
new goals
Website &
Communication
Extensive website
250,000 annual page hits
70,000 visitors annually
Monthly newsletter sent
to 3,500 recipients
Proven communication
platform
Secretariat - Kate Bushby
Volker Straub
Funding – EC (operating grant)
TACT
TREAT-NMD Advisory
Committee for
Therapeutics,
Expert multidisciplinary
body
Independent and objective
guidance on advancing
new therapies for
neuromuscular diseases
Chair – Dominic Wells
Funding - US (Dept of Defense)
Joint research
Regular meetings to
consolidate efforts and
jointly tackle common
problems
Topics based on necessity,
hosting to be rotated
between partners
Leads - Eric Hoffman
Annemieke Aartsma-Rus
Filippo Buccella
Funding - COST
SOPs
Unified experimental
protocols improve the
comparability of studies
Drawn up by a group of
independent researchers
(listed in each protocol)
Approx 40 sops updated
regularly
EuroBioBank
Unique network of 18
members
Stores & distributes
440,000 quality DNA, cell
and tissue samples
Leads - Marina Mora
Lucia Monaco
Marco Crimi
Funding - Fondazione Telethon
Patient Registries
Standardized genetic &
clinical core data for trial
recruitment
Interface can vary
between countries whilst
still able to share core data
Ethical & governance best
practice
>10,000 DMD patients
across 30 countries
Leads - Jan Verschuuren
Hugh Dawkins
Funding - AFM & EC
(operating grant)
Care & Trial Site
Registry
Information about each
registered trial site kept in
one location for ease of
comparison
Addresses organisational
difficulties of identifying
appropriate sites when
setting up a trial
Coordinated by University
Medical Center Freiburg
Outcome measures
Tests to decide whether
treatment being tested in
a trial is having any effect
Vital to use the correct
outcome measure to
prove if a treatment works
Working to harmonise the
use of most appropriate
outcome measures for
different diseases
Lead - Eugenio Mercuri
Funding - Telethon & Parent
Organizations
Standards of
Diagnosis & Care
International consensus
publication recommended
standards of care
DMD-SMA-CMD-LGMD
Family guides
in 25 different languages
translations verified
Printed booklets or
download from
website
Leads - Thomas Sejersen
Kathy North
2007-2011EU funded Network
2012 onwardsAlliance funded
through multiple
streams with global
partners & membership
GovernanceChair – Hanns Lochmüller
Vice – Annemieke Aartsma-Rus
Executive Committee
Supported by academic advisory
board (“task force”) of NMD
leaders