Neuromuscular Junction Disorders and Channelopathies S62
Mitochondrial Disease S67
Other Diseases and Diagnostics S84
Author index S101
Invited Speakers
Thursday 4th April 2019
Clinical services for Neuromuscular Disease patients
Prof Sir Doug Turnbull
Wellcome Centre for Mitochondrial Research and MRC International Centre for
Genomic Medicine in Neuromuscular Diseases, Newcastle University
Neuromuscular diseases collectively are common but many
individual disorders are rare. Whilst there are generic measures that support many
patients, individualised care is crucial. Developing a model enabling patients with rare
forms of neuromuscular disease to be seen by a team that is highly skilled in managing
their condition is important. Within the NHS, the development of the NHS Highly
Specialised Services has been crucial to the implementation of this model of care. It
allows patients with specific neuromuscular diseases to be seen (and investigated) in
centres of excellence, allowing improvements in care and support. There are many other
advantages of this model of care including the developing expertise of the clinical team
caring for the patients, the production of clinical guidelines and the collection of
clinical cohorts so important for clinical trials. This innovative model of care, combined
with a universal health system, means that the UK has arguably the best standards of care
for all patients with neuromuscular disease.
From Gene Discovery to Therapy
Francesco Muntoni
Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health,
30 Guilford Street, London WC1N 1EH, UK.
The current generation of clinician scientists has
benefitted enormously from the technological advances in molecular genetics that have
expedited the identification of novel disease genes in ways that until a few years ago
were unimaginable. This has also led not only to the exponential identification of novel
disease genes but also novel molecular targets for therapeutic intervention.
Regarding the first phase of the genomic studies, the UK
has benefitted from the network of clinical centres involved in rare neuromuscular
diseases and funded via the Highly Specialised Services, and from the links with other
European centres and with other large networks in US and Australia. The large collection
of well characterized patients and the introduction for the first time of deep phenotyping
using not only detailed muscle pathology but also muscle MRI, allowed the UK Centres to
identify ~ 20 years ago some of the most common genes involved in congenital and in limb
girdle muscular dystrophies, such as dysferlin and FKRP.
In the last few years the genomic revolution has provided
a completely different dimension to the gene identification, allowing the characterization
of ultrarare conditions which identification would have been impossible until just a few
years ago.
The improved understanding of mechanism of disease has
also led to the development of novel therapeutic interventions. For example, for a common
condition such as Duchenne muscular dystrophy, the understanding of the rules that
regulate individual exon splicing, and the ability to modulate splicing using antisense
oligonucleotides, has led to the first genetic therapeutic option for the affected
individuals. More recently, genomic studies using large population of DMD patients is
providing for the first time both the molecular explanation for difference in clinical
course between different patients. These studies are providing insight on potential novel
therapeutic targets; and will also help to understand the extent of any therapeutic
intervention for patients
The FOR DMD trial: Individualized vs standardized treatment
R. Griggs1
and Michela Guglieri2
1University of Rochester School of Medicine and Dentistry, Rochester,
NY, USA; 2The John Walton Muscular Dystrophy Research Centre, Newcastle
University, UK
Over 16 years ago Professor Katie Bushby noted the
“chaos” (at least 29 regimens) in corticosteroid (CS) treatment of Duchenne Muscular
Dystrophy (DMD). She also set out to establish consensus guidelines for DMD care. Working
with multinational clinicians and patient advocacy groups, she enlisted the Muscle Study
Group (MSG) to collaborate with TREAT-NMD to address the wide variation in the use of CS
by studying the long-term risks/benefits of the 3 most commonly prescribed regimens, daily
prednisone, intermittent prednisone , and daily deflazacort, in a randomized,
double-blind, comparative effectiveness trial. The resulting FOR DMD clinical trial
enrolled 196 CS-naïve boys (age 4-7) from sites in 5 countries and involved nearly 200
investigators/staff. In 2020, after participants have completed 3-5 years of follow-up,
the final results of the study will be analyzed. The deliverables expected of the study
include: (1) identifying the superior CS regimen; (2) establishing the outcomes of
systematically-applied consensus guidelines on side effects (metabolic, ocular, bone) and
on DMD complications; and (3) exploring the relationship of genotype to complications of
DMD and to benefits/side effects of CS treatment.
Knowing the superior regimen for long-term CS treatment
in DMD remains of great importance, since novel treatments will likely require concomitant
CS. Importantly, in advance of the final results we already know that 3-5 years of all 3
CS regimens have been well tolerated. This study is a tribute to Katie Bushby’s vision and
persistence and her skill in collaborating with the MSG and TREAT-NMD.
Acknowledgements: The FOR DMD investigators and steering
committee; TREAT-NMD; the MSG. Funding by NINDS grant: U01 NS061799; the Parent Project
for Muscular Dystrophy; the Muscular Dystrophy Association; Italian Telethon; Association
Française contre les Myopathies-AFM: and by Marathon, PTC, Sarepta, Santhera
Pharmaceuticals.
Vision DMD: developing an alternative to steroids
Michela
Guglieri1, Paula R. Clemens2,
Benjamin D. Schwartz3, John van den Anker 4,5, Laurel J. Mengel
Gaw3, Laurie S. Conklin4,5, Jana Haberlova6, Dimitrios
Athanasiou7, Elizabeth Vroom7, Rebecca Crow1, Andrea L.
Smith8, Lauren P. Morgenroth8, Adrienne Arrieta8,
Ritchie Head9, Christina Olsen9, John M. McCall4,
Kanneboyina Nagaraju4,10, Jesse M. Damsker4, Bushby K1,
Eric P. Hoffman4,10
1John Walton Muscular Dystrophy Research Centre, Newcastle University,
Newcastle upon Tyne, UK
2University of Pittsburgh and Department of Veterans Affairs Medical
Center, Pittsburgh, PA, USA
3Camden Group, LLC, St. Louis, MO, USA
4ReveraGen Biopharma, Rockville, MD, USA
5Children’s National Health System, Washington DC, USA
6Fakultni Nemocnice v Motole; Czech Republic
7United Parent Project Muscular Dystrophy
8TRiNDS LLC, Kensington, MD, USA
9Ceratium UK
10Binghamton University- SUNY, Binghamton, NY, USA
Corticosteroids are routinely prescribed as part of the
care recommendations in Duchenne muscular dystrophy (DMD). They are effective but the
burden of side effects impacts clinical outcomes and detracts from patient quality of
life. Developing safe and better tolerated treatments is an important goal for the
management of DMD and other chronic inflammatory diseases. Vamorolone (VBP15), is a
first-in-class ligand of the glucocorticoid receptor that shows the anti-inflammatory
efficacy of corticosteroids (inhibition of innate immunity via activator protein 1 and
nuclear factor kB) while reducing or eliminating many adverse effects of corticosteroids
(transcriptional transactivation of genes due to binding the glucocorticoid response
element).
Vamorolone demonstrated both efficacy and a reduction in
adverse effects in DMD patients compared to traditional corticosteroids in a
first-in-patient 24-week, open-label study (VBP15-003) following an acute treatment trial
(VBP15-002), demonstrating that oral administration of vamorolone at dose levels of 0.25,
0.75, 2.0 and 6.0 mg/kg/day was safe and well-tolerated over a 24-week treatment
period.
A randomised, double blind, parallel group, placebo and
active controlled study in 120 ambulant DMD boys ages 4 to <7 years is currently
recruiting and aims to confirm the safety, efficacy, pharmacodynamics and population
pharmacokinetics of vamorolone at daily doses of 2.0 mg/kg and 6.0 mg/kg versus prednisone
0.75 mg/kg/day and placebo over a 24-week treatment period and to evaluate persistence of
effect over a total treatment period of 48 weeks. The study is being conducted in 34 sites
across 11 countries.
Vamorolone has the potential to replace chronic
corticosteroid treatment for many disorders where adverse effects detract from the quality
of life of patients. Vamorolone studies in younger and older DMD patients are planned as
well as studies in other neuromuscular conditions.
Vamorolone has been developed by ReveraGen Biopharma
under a venture philanthropy model, with funding from international non-profit
foundations, and the US and EU governments.
EURO-NMD, a reference network for neuromuscular diseases
T. Evangelista
NeuromuscularMorphology Unit, Myology Institute, GHU Pitié-Salpêtrière ; AP-HP;
Sorbonne Université Paris, France
John Walton Muscular Dystrophy Research Centre and MRC Centre for Neuromuscular
Diseases, Institute of Genetic Medicine, Newcastle University, Newcastle upon
Tyne
Rare diseases (RD) are defined in the European Union as
life-threatening or chronically debilitating conditions that affect less than 5 per 10 000
people. It is well established that the small number of patients and the geographic
dispersion is an obstacle to the diagnosis, access to care, research and improvement of
medical expertise. The European Union has tried, over the years, to combat the lack of
specific health policies for rare diseases in the different Member States, through the
establishment of an overall strategy for Member States throughout the European Union.
The process behind the establishment of the European
Reference Networks (ERNs) was a long one that started around 2004 with the creation of the
Rare Diseases Task Force later replaced by the EUCERD Joint Action (EJA), led by Kate
Bushby. These specialised working groups were responsible for the policy work and
recommendations that were at the origin of the publication, in March 2014, of the
Delegated and Implementing Acts by the European Commission (EC). These provide a framework
for the creation of ERNs. ERNs are defined as networks of health care providers/centres
providing highly specialised healthcare, with the purpose of improving access to
diagnosis, treatment and care for patients across Europe. After the publication of the
Delegated and Implementing Acts in March 2014, the EJA conducted a study to suggest a
coherent grouping of the different RD in thematic areas. The suggested grouping intended
to be a rational approach to RD ERN planning and to ensure coverage of all RD.
The work to establish EURO-NMD started in 2013 under Kate
Bushby’s lead. At that time we organised and ENMC workshop that has set the grounds for
the neuromuscular ERN –“ 200th ENMC International Workshop “European Reference Networks:
Recommendations and Criteria in the Neuromuscular field”, 18–20 October 2013”. EURO-NMD
builds and expands on the successful CARE-NMD and utilised the TREAT-NMD Alliance
portfolio of resources.
EURO-NMD is a European Reference Network for the thematic
grouping of rare neuromuscular diseases (NMDs), a broad group of related disorders that
represent a major cause of mortality and lifelong disability in children and adults. NMDs
collectively affect an estimated 500,000 EU citizens and result in significant costs for
families and the healthcare system. EURO-NMD unites 61 of Europe’s leading NMD clinical
and research centres in 14 Member States and includes highly active patient organizations.
More than 100,000 NMD patients are seen annually by the ERN.
The network aims are the harmonization and implementation
of standards for clinical and diagnostic best practice; improving equity of care provision
across Member States; decreasing time to diagnosis; increasing cost efficiency through
better care pathways and access to specialist training and education. These aims will be
addressed through the application of eHealth services, development and application of care
guidelines, facilitation of translational and clinical research and development of
educational programs.
The DMD-Hub, collaboration to address and increase trial capacity in the UK
Emma
Heslop1, Michela Guglieri1,
Cathy Turner1, Becky Crow1, Anna Peel1, Emily
Crossley2, Alexandra Johnson2 Mariacristina Scoto3,
Francesco Muntoni3 and Volker Straub1
1 John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle University and Newcastle Hospitals NHS Foundation Trust, Newcastle
upon Tyne, UK (Emma.Heslop@ncl.ac.uk)
2 Duchenne UK, London, UK
3 The Dubowitz Neuromuscular Unit, UCL Great Ormond Street Institute of
Child Health, London, UK
With Duchenne muscular dystrophy (DMD) clinical research
at an unprecedented stage in terms of the number of possible therapeutic approaches coming
to trials, the need to increase trial capacity for DMD trials in the UK and improve trial
readiness was identified. Specifically, clinicians at established UK clinical trial
centres involved in multiple DMD studies were reaching capacity, while centres that did
have capacity lacked the expertise and needed support to run industry-sponsored clinical
trials.
Kate Bushby was instrumental in leading a collaboration
with the community to take decisive action. The ‘Newcastle plan’ workshop was organized to
bring together 75 participants from the DMD patient and research community, as well as
industry to address the issues. As a result, 8 DMD patient organizations agreed to fund 16
posts at a cost of £1.2 million to address the immediate capacity issue at experienced
sites.
As part of a longer-term strategy the DMD-Hub was set up
as a partnership between UK centres of excellence and Duchenne UK. With additional
investment of £1.1 million from Duchenne UK, the DMD-Hub is facilitating the sharing of
expertise and has successfully developed a network of trial-ready centres in the UK now
able to take on interventional trials in DMD.
To date the DMD-Hub has funded 12 additional posts at 6
trial sites in Newcastle, Liverpool, Leeds, Birmingham, Bristol and Glasgow. In 2019 there
are plans to support an additional 2 sites in London and Manchester. The Hub is committed
to working with further sites (including Oswestry, Cambridge and adult sites) to
facilitate them to take on upcoming industry- and academic-led trials. Ongoing training
for other sites is expected to open up additional opportunities in subsequent years.
The DMD-Hub website (dmdhub.org) is a key resource for
industry, clinicians and patients. It hosts an interactive map of the UK detailing
clinical trial opportunities for patients, contains a repository of information and tools
for sites and acts as a one-stop shop for industry / sponsors interested in conducting
trials in the UK.
Partnerships with industry and sites are being enabled by
the DMD-Hub to facilitate trial planning and innovative funding models are being
implemented at DMD-Hub sites to ensure sustainability.
Future areas of interest for the DMD-Hub include
addressing the issues related to gene therapy trials and the development of an Adult-Hub
network to include the non-ambulant population.
The mission of the DMD Hub is to ensure all patients with
DMD, including children and adults, have access to clinical research opportunities.
Promoting rare disease policies across Europe
V.
Hedley1, K. Bushby1
1John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle University
Leadership of the initial TREAT-NMD ‘Network of
Excellence’ resulted in the increasing involvement of the Newcastle Muscle Team
researchers in projects and advisory bodies dedicated to ‘rare diseases’ more broadly.
Professor Kate Bushby, in particular, quickly became acknowledged as a leading expert in
the field of rare disease policy. Rare diseases have, for many years, been recognised as a
priority area for European cross-border collaboration: although the number of patients
living with any single condition will -by definition- be small, the fact that there are
over 7000 individual conditions classed as ‘rare’ equates to ca. 30 million patients
across Europe. The rarity of these conditions poses particular challenges to patients and
families, but also to health and social systems.
As vice-chair of the European Union Committee of Experts
in Rare Diseases (2010-2013), and subsequent independent expert in the Commission Expert
Group on Rare Diseases (2014-2016), Kate and her team developed a unique reputation for
Newcastle University as a seat of expertise not only in specific thematic areas such as
neuromuscular diseases, but also in the formulation and implementation of policies
supporting better diagnostics, treatment and care for all conditions classed as rare. By
focusing on the commonalities, Kate and her team led the development of policies and
recommendations across a broad range of topics, from national frameworks to rare disease
registries and data capture, from cross-border genetic testing to network-building. The
fruits of those policies are highly visible today, most notably in the creation of 24
European Reference Networks for rare diseases, and in the adoption of national plans and
strategies by 25 European Member States: this impact is very evident in the UK, as well as
in mainland Europe.
Kate’s legacy in the rare disease field has continued, as
her colleagues in the John Walton Muscular Dystrophy Research Centre continue to lead and
shape international initiatives and projects with many ambitious goals: to identify future
determinants of health and wellbeing for rare diseases; to reduce the diagnostic odyssey;
to optimise the knowledge base and identify innovative solutions applied by one country
which could be expanded to others; and generally to continue to strive towards
collaborative policies with the power to alleviate the many difficulties and inequalities
rarity creates.
What makes Genetics Human?
Professor Sir John Burn
Professor of Clinical Genetic, Institute of Genetic Medicine, Newcastle
University, Newcastle upon Tyne, NE1 3BZ
Times of rapid change can expose fault lines in society.
Just as the mass of refugees from the Middle East elicited contrasting reactions which in
turn stressed the existing partnerships in Europe, so the arrival of high throughput
sequencing and point of care DNA testing will test the assumptions of Europe’s nation
states. Termination of pregnancy and population screening for genetic disorders test the
diversity of opinion from the more individualistic and secular traditions of the North and
West of Europe across to the countries of the East and South of Europe where traditional
Catholic teaching holds sway. Memories of misuse and fears about impact on insurance deter
many from whole genome sequencing and genetic databases while others, notably in the UK,
press ahead with industrial scale sequencing in healthcare.
The emergence of non-invasive testing based on fetal DNA
in maternal circulation will increase terminations of pregnancy for genetic disorders.
Similarly, the availability of molecular targeted cancer treatments such as PARP
inhibitors, PD1 blockers and BRAF specific therapy will prompt adoption of testing for
germline defects in high penetrance “cancer genes” routine.
Point of care DNA testing for pharmacogenetic
susceptibility, DNA/RNA tumour markers and the presence of pathogen sequences will
democratise genomics. By testing cheaply and quickly for diagnostically relevant
information at the clinical interface it becomes possible to use DNA technology to improve
outcomes without eliciting negative reactions. Current restrictions on use of DNA
technology in Europe will soon seem out of step with pressing clinical utility. A balance
must be found which allows high throughput and point of care DNA tests to be ordered by a
wide range of healthcare professionals and, in some cases, by members of the public while
ensuring the hard won skills of professional geneticists remain available and their
services financially viable.
Invited Speakers
Friday 5th April 2019
Genome Editing for Duchenne Muscular Dystrophy
Christopher E. Nelson1,2, Jacqueline N.
Robinson-Hamm1,2, Jennifer E. Kwon1,2, Karen Bulaklak, Veronica
Gough1,2, Joel D. Bohning1,2, Matthew Gemberling1,2,
Charles A. Gersbach1,2,3
1Department of Biomedical Engineering, Duke University
2Center for Genomic and Computational Biology, Duke University
3Department of Orthopaedic Surgery, Duke University Medical
Center
The advent of genome editing technologies, including the
RNA-guided CRISPR/Cas9 system, has enabled the precise editing of endogenous human genes.
We have applied these tools to the correction of mutations that cause genetic disease. For
example, we engineered CRISPR/Cas9-based nucleases to correct the human dystrophin gene
that is mutated in Duchenne muscular dystrophy patients. When we delivered these nucleases
to cells from patients with this disease, the correct gene reading frame and expression of
the functional dystrophin protein were restored in vitro and following cell
transplantation into mouse models in vivo (Ousterout et al., Nature Communications 2015).
When delivered directly to a mouse model of this disease, gene editing by the CRISPR/Cas9
system led to gene restoration and improvement of biochemical and mechanical muscle
function (Nelson et al., Science 2016). In more recent studies we have shown that genome
editing and dystrophin protein restoration is sustained in the mdx mouse model of DMD for
one year after a single intravenous administration of AAV-CRISPR. We also confirmed
immunogenic host response to Cas9 when administered via AAV vectors to adult mice, but
show that the humoral and cellular immune response can be avoided by treating neonatal
mice (Nelson et al., Nature Medicine 2019). Additionally, we have observed unintended
genome and transcript alterations induced by AAV-CRISPR that should be considered for the
development of AAV-CRISPR as a therapeutic approach. More recently, we have developed
novel animal models of this disease for the preclinical development of therapies that will
correct human disease-causing mutations. New constructs have been developed and validated
for significant levels of gene correction and dystrophin restoration in this model. These
studies demonstrate the potential for genome editing to be used to treat Duchenne muscular
dystrophy and other neuromuscular disorders, and also highlight aspects of host response
and alternative genome editing outcomes for further study.
Alternate gene therapy approaches to DMD
Kevin M. Flanigan
Center for Gene Therapy, The Research Institute of Nationwide Children’s Hospital,
Columbus, Ohio
Duchenne muscular dystrophy (DMD) occurs due to mutations
in the DMD gene that result in an absence of the dystrophin protein,
which plays a critical role in stabilization of the muscle sarcolemmal membrane via
linkage to the transmembrane dystrophin-associated protein complex, and ultimate to the
extracellular matrix. Gene therapy for DMD has largely concentrated on AAV-mediated
delivery of microdystrophin gene constructs that have been engineered to fit within the
approximately 5 kb size limit of AAV genomes, and typically encode critical dystrophin
domains. Here we discuss two alternate gene therapy approaches. One approach utilizes AAV
delivery of the GALGT2 gene, encoding β-1,4-GalNAc transferase.
Endogenous GALGT2 is typically expressed in muscle only at the
neuromuscular synapse and myotendonous junction, where utrophin replaces dystrophin in a
utrophin-associated protein complex. Overexpression of GALGT2 results in
expression of utrophin throughout the muscle fiber, as well as upregulation of other
proteins associated with muscle membrane stabilization, and a clinical trial of delivery
of an rAAVrh74.MCK.GALGT2 vector via isolated limb infusion is underway. A second approach
is directed toward patients with the most common single exon duplication, using AAV
delivery of non-coding U7 small nuclear RNAs (snRNAs) with sequences targeting exon 2
splice donor and acceptor sites that results in highly efficient exclusion of one or both
copies of exon 2 from the mature mRNA transcript. In this case, either transcript is
therapeutic, as exclusion of exon 2 results in translational initiation from a downstream
internal ribosome entry site leading to a highly functional N-deleted protein isoform
found in mildly affected Becker muscular dystrophy patients who have walked into their
eight decade. Both approaches are complementary to microdystrophin therapies, to which
they may also have theoretical advantages in subsets of patients.
AAV-mediated Gene Therapy in Neuromuscular Disease: Clinical Immunology
Considerations
Byrne, Barry J
University of Florida, Powell Gene Therapy Center
Early onset neuromuscular disease (NMD) is often
associated with severe or null mutations that lead to greater disease burden. The success
of gene replacement strategies in this setting may be influenced by limited endogenous
protein expression leading to anti-transgene immune response and loss of effective copy
number in skeletal muscle due to somatic growth. In one example, Pompe disease is due to a
deficiency or absence of the lysosomal enzyme acid alpha glucosidase (GAA), resulting in
lysosomal glycogen accumulation that impacts striated muscle and the CNS, including
defects of the neuromuscular junction (NMJ). Respiratory failure is the leading cause of
morbidity and mortality in Pompe patients. AAV vectors expressing GAA have been evaluated
in a phase I/II study in ventilator-dependent and independent pediatric Pompe patients.
These studies are based on the finding that accumulation of glycogen in spinal motor
neurons contributes to weakness and diaphragmatic dysfunction observed in Pompe disease.
In a number of preclinical studies, we have found that restoration of GAA activity in
muscle and neural tissue is able to reverse ventilatory insufficiency by reversing motor
neuron dysfunction and restoring the integrity of the NMJ. The principle defect in the
motor unit is related to deficiency of NMJ structure and function. New evidence also
indicates the need for early intervention related to neural dysfunction since motor
neurons show evidence of apoptosis in the murine model of Pompe. These deficits are
present early in the mouse model and restoration of GAA activity in the muscle and neurons
before 6 months of age leads to restoration of in situ force production. After 18 months
of age, the loss in motor neurons leads to permanent deficits in force production of the
tibialis anterior.
Clinical studies of AAV-mediated gene therapy have been
pursued to address the fundamental aspects of gene therapy in a neuromuscular disease
where patients are identified by severe early onset or newborn screening. Findings in
non-clinical and clinical studies related to immune management in conjunction with AAV
systemic delivery have paved the way for clinical studies in adults and younger subjects
who are candidates for therapeutic AAV administration. The loss of neuromuscular junction
formation is a major contributor to weakness and ventilatory failure and these deficits
can be prevented by early administration of AAV-GAA. Studies which utilize next generation
AAV vectors for systemic administration have led to efficient targeting of muscle and
motor neurons for the early treatment of Pompe disease. Related studies in Duchenne
muscular dystrophy also highlight the importance of identification and management of
pre-existing anti-AAV antibodies which are able to reduce the efficacy of systemic AAV
vectors. Practical considerations for the implementation of systemic AAV administration
will be discussed for early onset neuromuscular disease.
Preventing transmission of pathogenic mitochondrial DNA mutations
Louise Hyslop1,2, Yuko Takeda, Lyndsey
Craven3 , Meena Choudhary1,2, Nilendran Prathalingam1,
Doug Turnbull3, Mary
Herbert1,2
Newcastle Fertility Centre1 & Wellcome Trust Centre for
Mitochondrial Research, Institute of Genetic Medicine2 & Institute of
Neuroscience3, Biomedicine West Wing, Centre for Life, Times Square,
Newcastle upon Tyne.
Newcastle Fertility Centre1 & Wellcome Trust Centre
for Mitochondrial Research, Institute of Genetic Medicine2 & Institute of
Neuroscience3, Biomedicine West Wing, Centre for Life, Times Square, Newcastle upon
Tyne.
Mutations in mitochondrial DNA (mtDNA) are inherited
exclusively from our mothers and can cause a broad range of debilitating and fatal
diseases. Reproductive technologies designed to uncouple the inheritance of the
mitochondrial genome from the nuclear DNA genome may enable affected women to have a
genetically related child with a greatly reduced risk of mtDNA disease. Such technologies
involve transplantation of the nuclear DNA from the egg of an affected woman to an
enucleated egg from an unaffected donor. Nuclear genome transplantation can be performed
either before or after fertilisation. The latter involves transfer of pronuclei, which
separately contain the maternal and paternal genomes. In parallel with legal and
regulatory reform to permit therapeutic application of so called “mitochondrial donation”
techniques in the UK, we have conducted preclinical studies to test the safety and
efficacy of pronuclear transplantation (PNT). Our findings indicate that PNT is compatible
with development of embryos to the blastocyst stage, with no detectable effect on
chromosome segregation and gene expression. Moreover, we find that the amount of mtDNA
co-transferred with the pronuclei is minimal, accounting for 0-5% of the mtDNA contained
in PNT blastocysts. Remarkably, this tiny amount mtDNA outcompetes the cytoplast donor
mtDNA in some embryonic stem (ES) cell lines derived from PNT blastocysts. While the
relevance of this to development in vivo remains to be established, the finding highlights
the importance of ongoing research to minimise resurgence of pronuclear-associated mtDNA.
In the meantime, the Human Fertilisation and Embryology Authority (HFEA) have approved the
cautious use of PNT in the UK and our clinic has been granted the first licence for its
therapeutic application.
Mitochondrial donation: predicting who might benefit?
Gráinne S Gorman
Wellcome Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle
University, Newcastle upon Tyne, UK
Primary mitochondrial (mt) DNA diseases affect up to 1in
5000 live births and can often have devastating clinical sequelae. There are currently no
curative treatments with the focus of therapeutic strategies largely on palliation of
symptoms. The emergence of assisted reproductive techniques, such as Mitochondrial
Donation, offer the first real opportunity to prevent the transmission of some serious
forms of mtDNA disease from mother to child. The Human Fertilization and Embryo Authority
(HFEA), the statutory regulatory authority charged with regulating human embryo research
in the UK, has devised, a detailed regulatory process for issuing individual licenses to
couples considered suitable for Mitochondrial Donation. Clear licensing prerequisites
include a risk of transmitting serious mtDNA disease and a predicted mtDNA mutant load in
oocytes that would make Pre-implantation Genetic Diagnosis (PGD) unsuitable. In women of
child bearing age with a confirmed diagnosis of mitochondrial disease due to pathogenic
mtDNA mutations, reproductive counselling is considerably more complicated due to issues
of mtDNA heteroplasmy, threshold effect and variable penetrance of specific mutations.
Predicting mutant load in children is particularly complex as seriously affected children
with high heteroplasmy levels can be born to asymptomatic mothers harbouring low levels of
deleterious mtDNA mutations. Here I will discuss the complexities around such predictions
and present new work on the derivation of a statistical model to forecast the proportion
of children with mtDNA heteroplasmy levels expected below a cut-off value that aligns with
current PGD practice guidelines for the most common heteroplasmic mtDNA pathogenic variant
in adults. Our findings have not only considerable implications for women at risk of
transmitting serious mtDNA mutations but are now being considered in the HFEA
deliberations for the granting of treatment licenses for Mitochondrial Donation.
Posters and Platform Presentations
‡indicates a platform or fl ash presentation
Key:
Abstract CategoryAbstract prefix
Muscular Dystrophies D
Peripheral neuropathy PN
Motor Nerve Disorders MND
Neuromuscular Junction Disorders and Channelopathies NMJ&C
Mitochondrial Disease MD
Other Diseases and Diagnostics OD
Dystrophy
Selenoprotein related myopathy (SEPN1-RM): A comprehensive analysis of progression
over time to guide clinicians and clinical trials
A. Silwal1, M.Scoto1, D.
Ridout2, A. Schmidt3, A. Laverty4, M.
Henriques4, L. D’Argenzio5, M. Main1, R.
Mein1, A. Sarkozy1, M. Manzur1, F. Abel3,
F. Al-Ghamdi6, C. Genetti6, D. Ardicli7, G
Haliloglu7, H Topaloglu7, A.
Sarkozy1, A. Beggs6, F.
Muntoni1
1The Dubowitz Neuromuscular Centre, University College London, Great
Ormond Street, Institute of Child Health and MRC Centre for Neuromuscular Diseases,
Neurosciences Unit, Great Ormond Street Hospital, London, UK
2Population, Policy and Practice Programme, UCL Great Ormond Street
Institute of Child Health, London, UK
3Respiratory Department, Sleep and Non-Invasive Ventilation, Great
Ormond Street Hospital for Children NHS foundation Trust, London, UK
4Principal Clinical Physiologist (Lung Function and Sleep Unit),
Great Ormond Street Hospital for Children NHS foundation Trust, London, UK
5Honorary Research Fellow
UCL, Institute of Child Health, London, UK
6Beggs laboratory congenital myopathy research program at Boston
Children’s Hospital, Harvard University
7Hacettepe University Children’s Hospital, Department of Paediatric
Neurology, Ankara, Turkey
Background: The term
SEPN1-related myopathies (SEPN-RM) refers collectively to four
autosomal-recessive disorders caused by mutations in the SELENON gene,
with similar clinical features but with distinct muscle pathologies.
Aims and Methods: We present
cross-sectional phenotypic data of a multi-centric large cohort of 61 patients with
SEPN1-RM with pathogenic variant in SELENON gene. We also present
longitudinal data on clinical course of 25 patients (21 families) aged 2.5-24 years over
a mean follow-up of 6.3 years with the aim to assess changes in motor abilities,
respiratory function and scoliosis progression based on functional scores, lung function
tests and Cobbs angle.
Results: In the cross-sectional data more
than two-thirds (47/61; 77%) of patients presented at = 2 years with hypotonia, poor
head/neck control and development delay. 45/61 (74%) patients developed scoliosis at a
mean age of 10.3 years with 18 patients undergoing scoliosis surgery (mean age 13.6
years). A total of 10/61 (16%) children lost ambulation within the first two decades of
life. Nocturnal ventilator support was initiated in 3/4th (50/61; 82%)
patients (with mean FVC 38% - n=20) at mean age of 13 years.
In the longitudinal data patient cohort, two-third of the children (n=16/25; 64%) had
weight below the 2nd centile during the follow up period and 6/61 children
(0.1%) needed nasogastric feeds and/or gastrostomy. Functional motor scores declined
from mean total score of 33 to 27 with an estimated annual change in motor ability
scores of -0.55; similar trends were also noted with timed tests indicative of a slow
motor decline over time. 21/25 (84%) patients had spinal stiffness, preceding scoliosis
by mean of 1.8 years. FVC trends showed a decline from a mean of 52% to 40% over a
4-year mean follow up. The estimated change in FVC % per year was -2.04 (SE 0.46; p
value <0.001).
Conclusions: This large cohort of SEPN1-RM
patients well describes the phenotype and details the clinical course of this rare
condition, with emphasis on a paediatric population. Our data suggest that loss of
independent ambulation occurs more frequently than previously reported; and the observed
FMS and FVC trend data provide useful information on the slope of change over time. Our
results emphasize the importance of a consistent multi-disciplinary assessment and
management of these patients, to improve outcomes and inform clinical trials.
Abstract structure:
An investigation of DMD genetic variation and conservation and its
links to intellectual disability
Alexander J. M.
Blakes1, Andrew G. L. Douglas1,
2
1Wessex Clinical Genetics Service, University Hospital Southampton
NHS Foundation Trust, Southampton, UK
2Human Development and Health, Faculty of Medicine, University of
Southampton, Southampton, UK
Background:DMD mutations in both males and females are linked to cognitive
impairment of varying degrees. The degree of impairment does not correlate with the
severity of muscle disease or with the “reading frame rule” and the pathogenic mechanism
is poorly understood. This makes it impossible to reliably predict cognitive outcomes
for specific mutations. Similarly, it is difficult to confidently ascribe pathogenicity
to DMD variants identified in children undergoing genomic testing for
intellectual disability, whether or not any muscle disease is present.
Aims: We sought to ascertain whether
publicly available genomic datasets could be used to infer regions of functional and
clinical importance within the DMD gene, with a particular focus on
potential causes of intellectual disability.
Methods: Population variant data were
obtained from the gnomAD database, while conservation scores were obtained via the UCSC
table browser. Further variant information (including both copy number and
single-nucleotide variants) and clinical phenotype data were obtained from the DECIPHER
database.
Results: Preliminary analysis reveals
regions with especially high degrees of conservation within the dystroglycan-binding
domain of the dystrophin protein. These same regions also display especially low
frequencies of population-level variation, further suggesting their functional
importance. Analysis of clinical phenotype data is ongoing and these results will also
be presented.
Conclusion: This study confirms the
functional importance of the 3’ region of the DMD gene and helps define
specific regions that may be of pathogenic relevance in DMD-related
cognitive impairment. Furthermore, this data-driven approach to the definition of
functionally important gene regions is applicable to any gene of interest and may
provide a clinically useful tool in helping to resolve the pathogenicity of genomic
variants.
e-mail address: alex.blakes@soton.ac.uk
An exploration of the relative progression of change in a range of upper limb (UL)
parameters in boys with Duchenne muscular dystrophy (DMD)
1NIHR Great Ormond Street Hospital Biomedical Research Centre, Great
Ormond Street Institute of Child Health, University College London, & Great Ormond
Street Hospital Trust, London, UK
2Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health & Great Ormond Street Hospital Trust, London, UK
3Institut de Myologie, Groupe Hospitalier Pitié Salpêtrière, Paris,
France
4John Walton Muscular Dystrophy Research Centre, Newcastle
University, Newcastle, UK
5Leiden University Medical Centre, Leiden, Netherlands
6Radboud University Nijmegen Medical Center, Donders Centre of
Neuroscience, Department of Rehabilitation, Nijmegen, Netherlands
7Great Ormond Street Institute of Child Health, University College
London, UK
Background: The progression of DMD in the
UL is not well documented in the literature, particularly in boys treated with
glucocorticoids.
Aims: To explore the progression of change
in isometric strength, joint range and function of the UL and investigate possible
relationship between the above parameters.
Methods: 21 boys with mean age 10.4 (3.25)
years (13 ambulant, 8 non-ambulant) with DMD treated with glucocorticoids were followed
longitudinally for 4.5 years. UL myometry, goniometry and the Performance of the Upper
Limb test (PUL) assessment data were extracted and analysed. The analysis included
descriptive statistics, comparisons between ambulant and non-ambulant groups and
correlation between the outcome measures.
Results: The PUL scores demonstrated an
increasing trend in the boys up to 8 years old, followed by a plateau and then declined
from approximately 11 years of age in a proximal to distal order. In our sample, the
distal domain did not change significantly but the proximal and middle domain captured
decline, particularly in the non-ambulant group. Goniometry scores demonstrated
increasing presence and severity of contractures with age. In the ambulant group,
approximately 28.3 % of the boys demonstrated shoulder contractures, 6.7% elbow
contractures and 11.7% wrist contractures, while in the non-ambulant group, shoulder
contractures were present in 62.1%, elbow contractures in 58.3% and wrist contractures
in 33.3% of the boys. Myometry absolute values for shoulder flexors, elbow flexors,
elbow extensors and wrist extensors showed no particular trend with increasing age.
Myometry, goniometry and PUL were poorly correlated.
Conclusions: In this cohort, PUL scores
declined in a proximal to distal direction. Joint contractures increased in a proximal
to distal order. Myometry did not correlate with disease progression. Neither myometry
nor goniometry were predictive factors for UL function in boys with DMD. Further
research to evaluate the use of myometry in DMD may be beneficial as this is a commonly
used outcome measure.
The common COL6A1 deep intronic c.930+189C>T mutation in
Ullrich Congenital Muscular Dystrophy is efficiently corrected by exon-skipping AONs
treatment
Sara Aguti, Dominic Scaglioni, Matthew
Beck, Tenzin Sung-rab Brookes, Haiyan Zhou and Francesco Muntoni
The Dubowitz Neuromuscular Centre, Great Ormond Street Institute of Child
Health, University College London, United Kingdom
Background: Collagen VI-related congenital
muscular dystrophies (COL6-CMD) are the second most common congenital muscular dystrophy
variants and currently there is no cure available. COL6-CMD are caused by recessive or
dominant mutations in one of the three genes encoding for the α-chains
of collagen type VI (COL6A1, COL6A2 and
COL6A3). Depending on the type of mutations, COL6-CMD ranges from the
severe Ullrich Congenital Muscular Dystrophy (UCMD) to intermediate phenotypes and the
milder Bethlem Myopathy (BM). The commonest recurrent mutation is a deep intronic
COL6A1 mutation that induce the incorporation of a novel pseudo-exon
in the transcript, causing a dominant gain of toxic function.
Aims: The aim of this study was to
identify lead AON sequences designed to target a COL6A1 deep intronic
mutation and abolishing the inclusion of the pseudo-exon in the transcript, hence
generating a normal transcript.
Materials and Methods: Skin fibroblasts
cultured from 4 UCMD patients carrying COL6A1 deep intronic
c.930+189C>T mutation were used as cellular models. 15 AONs were designed and their
efficiency was evaluated at both RNA and protein levels using quantitative real-time
PCR, immunofluorescence and flow cytometry.
Results: Two lead candidates have been
identified with promising results in skipping the pseudo-exon. These AONs specifically
suppress the aberrant splicing and the incorporation in the mature transcript of the
pseudo-exon, without altering the COL6A1 wild-type expression at RNA
level. Moreover, these two AONs lead to the restoration of collagen VI protein
production in the extra cellular matrix (ECM) from all the 4 patients derived
fibroblasts. The restoration of the protein production in the ECM was evaluated with
both immunofluorescence and flow cytometry analysis. We are currently analysing the
ultra microstructure of collagen VI protein in ECM with electron microscopy, to confirm
normal collagen VI structure restoration after AONs treatment.
Conclusion: Our findings provide a proof
of concept for AON exon-skipping as a therapeutic approach for one of the most common
mutation in COL6-CMD.
Sara Aguti, PhD
Postdoctoral Research Associate
University College London
Institute of Child Health, WC1N 1EH
London
United Kingdom
sara.aguti@ucl.ac.uk
Translational consequences of neurodegeneration in dystrophic nerves of
mdx mice, as a model for Duchenne Muscular Dystrophy
Miranda D Grounds and Vidya S Krishnan
School of Human Sciences, the University of Western Australia, Australia, 6009
miranda.grounds@uwa.edu.au
Background: In the childhood disease
Duchenne Muscular Dystrophy (DMD) and the dystrophic mdx mouse model,
intrinsic repeated bouts of skeletal muscle necrosis result in denervated neuromuscular
junctions (NMJs). We hypothesise that this ongoing NMJ denervation results in premature
progressive degenerative changes in the nerves innervating the dystrophic muscles
contributing to their loss of function.
This dystrophic research builds upon our studies of
normal age-related loss of skeletal muscle mass (sarcopenia) in healthy mice, associated
with denervation of NMJs and myofibres. Our time course study of sciatic nerves from
ageing C57Bl/6J mice (immunoblotting) showed increased levels of various neuronal
proteins by 18 months (v SMI-32, ChAT) and many others by 22 months (Tau5, p62),
indicative of neurodegeneration (2017. PMID: 27030741). Immunostaining of lumbar spinal
cords from old mice shows loss of proprioceptive muscle afferents, with consequences for
sensorimotor control of muscle function (2018. PMID: 30084046). These ongoing ageing
studies provide the techniques for the studies in dystrophic rodents.
Aim: To test the hypothesis that premature
progressive (irreversible) neuronal changes occur in the mdx mouse
model of DMD.
Methods: Protein levels in sciatic nerves
were quantified using Western immunoblotting.
Results: Our analyses of sciatic nerves of
mdx compared with normal C57Bl/10Scsn mice by immunoblotting
(n=8-10/group), show significantly increased levels (P=0.05) of Tau5 and S100 by 13
months (9 months earlier than in normal mice), confirming our hypothesis; with markedly
increased protein levels (e,g. Tau5) by 18 months (n=4-7/group.
Conclusions: Progressive neurodegenerative
changes have potentially major adverse consequence for contraction and long-term
function of dystrophic muscles. For growing DMD boys where relentless myofibre and NMJ
damage occurs over many years, it is essential for clinical translation to know the
extent to which neuronal/spinal cord function has been irreversibly altered at a
particular stage of the disease, to optimise efficacy of clinical therapies that aim to
stabilise or improve function of the DMD muscles.
For pre-clinical trials using dystrophic rodents,
neuronal degeneration may prove a valuable readout to monitor longer-term benefits of
therapies designed to prevent/reduce myonecrosis.
Neuromuscular disorders in Qatar: Genetic Distribution and Proteomics
A. Abdelaleem1, M. Elsaid2, N.
Chalhoub1, R. Al Shamy2, A. Chakrcoun1, O.
Kuzu1, Kh. A Mohamed2, O. Alalamy2, N.
Almudheki2, R. Mohamed2, Kh. Ibrahim2
1Weill Cornell Medicine-Qatar (WCM-Q), 2Hamad Medical
Corporation (HMC), Doha -Qatar
Alice Abdel Aleem, MB,B.Ch; MD
Assistant Prof. of Neurology and Neuroscience
Weill Cornell Medicine Qatar- Qatar
Brain and Mind Institute-NY-USA
Prof. Human genetics, NRC
Background: NMDs comprise an extensive
clinically and genetically heterogeneous group of uncommon inherited disorders.
Aims: The objectives of our study are to
find out about the genetic distribution of NMDs in Qatar, the frequency of the subtypes
as well as the pattern of altered circulating plasma proteins in a sample of NMDs
patients.
Subjects and methods: The first phase of
the project has recruited 58 NMDs families having a variety of congenital
myopathies/dystrophies and limb girdle muscle dystrophies. Whole genomic sequencing and
large scale Somologic proteomic screening were applied.
Results: Congenital mersoine deficient
muscle dystrophy (MDC1A) was the predominating type encountered in our NMDs cohort. All
MDC1A patients were of the non-ambulatory type, the best achieved motor skill, in a
proportion of patients, was the unsupported sitting. A founder mutation was identified
in a group of Qatari patients. Variability in clinical phenotype’s severity in terms of
respiratory and feeding compromises, osteopenic changes, and brain images were clearly
obvious among MDC1A patients with LAMA2 founder mutation as well as
other mutations. An interesting novel large N-terminal deletion identified in our study
showed the retained transcription and translation of LAMA2 in affected cases. This
finding points out the activation of an alternative LAMA2 promoter.
Sarcoglyconopathy and Fukutin Receptor related muscle
dystrophies were of the recurrent dystrophies encountered in our cohort. Other rare
subtypes were identified. WGS variants analysis revealed exciting clue to the atypical
brain structural abnormalities identified in one of our patients. The proteomic
screening approach revealed a set of 100 significantly differentially altered markers in
patients versus controls. A number of naturally non-enriched muscle proteins has been
identified specifically altered in patients indicating potential roles in promoting
secondary changes of muscle damage process.
Conclusion: The findings of this study are
the first to address the genetics of NMDs in Qatar. WGS identified nation specific
founder mutations both in LAMA2 and sarcoglycan alpha and spotted a genetic background
clarifying some of the atypical MDC1A-clinical characteristics. The “Omics”, Whole
Genome and Proteomic screening provide high capacities of better understanding of the
pathophysiology of muscle damage in dystrophies.
Global Phase 3 PolarisDMD Trial for Edasalonexent, an Oral NF-kB Inhibitor that Has
Shown Consistent Positive Effects on Muscle Function in Boys with DMD
Joanne Donovan, MD, PhD
(jdonovan@catabasis.com) for the MoveDMD
Investigators
Catabasis Pharmaceuticals
Background: Edasalonexent (CAT-1004)
inhibits NF-kB, a key link between loss of dystrophin and disease pathology, and which
plays a fundamental role in the initiation and progression of skeletal and cardiac
muscle disease in DMD.
Aims: To assess efficacy and safety of
edasalonexent in the Phase 3 PolarisDMD trial.
Methods: PolarisDMD is enrolling boys with
DMD from 4 to 7 years of age (before 8th birthday) with any mutation who are able to
stand from supine in =10 sec. Key exclusion criteria include glucocorticoid use within 6
months, or recent use of investigational therapies. Edasalonexent/placebo will be given
three times/day for 1 year, and ~125 boys will be enrolled in a 2:1 ratio,
active:placebo, followed by an open-label-extension (OLE) study in which all boys
receive edasalonexent. The North Star Ambulatory Assessment is the primary endpoint with
timed function tests as secondary endpoints. Additional assessments include ambulatory
heart rate monitoring, growth assessments and bone density.
Results: Edasalonexent has previously been
studied in boys with DMD of the same age in the Phase 2 MoveDMD trial and OLE, which
showed benefits on muscle function with clinically meaningful slowing of disease
progression through more than a year of treatment compared to the off-treatment control
period. Edasalonexent had a durable and positive impact on muscle enzyme tests including
CK and other muscle biomarkers that were significantly improved. Lower leg muscle MRI
T2, which correlates with muscle function and loss of functional abilities, was
significantly improved compared to off-treatment progression, consistent with slowing of
disease progression. Edasalonexent was well tolerated without evidence of steroid side
effects. Height and weight increased age-appropriately, and the elevations in heart rate
typically observed in DMD declined significantly toward age-normative values, suggestive
of benefit.
Conclusions: Edasalonexent has
demonstrated safety and efficacy in the MoveDMD trial, and the global Phase 3 PolarisDMD
trial is currently enrolling. Edasalonexent is a potential oral foundational therapy for
all affected by DMD.
Non-invasive assessment of fibrosis in mouse skeletal and cardiac muscle by
contrast enhanced magnetic resonance imaging using EP3533
Alexander Peter Murphy1, Elizabeth
Greally1, Dara O’Hogain2, Andrew Blamire2, Peter
Caravan3,4, Volker Straub1
1The John Walton Muscular Dystrophy Research Centre, Institute of
Genetic Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom
2Institute of Cellular Medicine & Newcastle Magnetic Resonance
Centre, Newcastle University, Newcastle upon Tyne, United Kingdom
3Martinos Center for Biomedical Imaging, Massachusetts General
Hospital and Harvard Medical School, Charlestown, MA and 4Institute for
Innovation in Imaging, Department of Radiology, Massachusetts General Hospital,
Boston, USA
Background
Increased levels of fibrosis in skeletal and cardiac muscle have been associated with a
worse prognosis in Duchenne muscular dystrophy (DMD). Anti-fibrotic medications are
being evaluated for amelioration of DMD. To date, MRI has not been used successfully to
image fibrosis within skeletal muscle as an outcome measure.
Aims and Methods:
EP3533 is a novel gadolinium-based contrast agent using a complex peptide with an
affinity to collagen 1. Compared to standard Gd (DTPA), the relaxivity of EP3533 is five
times higher per Gd atom. We acquired MRI scans from age-matched 40 week old
mdx and BL10 mice, using EP3533 to detect and quantify fibrosis
within skeletal and cardiac muscle.
Results:
Significant differences in post-contrast R1 were demonstrated between
mdx and BL10 mice using EP3533 (cardiac p=0.02, GCN p=0.04, TA
p=0.04). All muscle groups had increased signal to noise ratio (SNR) at 5 minutes, which
may be due to interference from Gd enhancement in blood vessels. Levels of SNR in the
mdx mouse reached a plateau at 60-90 minutes, and did not return to
pre-contrast levels after the scanning period (120 minutes). The BL10 mouse showed an
initial peak at 5 minutes, and then plateaued at a much lower level from approximately
10 minutes onwards. Differences between mdx and BL10 mouse muscles in
signal were demonstrated most clearly in the tibialis anterior (TA) muscles. Post-MRI
the TA and gastrocnemius (GCN) muscles and the heart were removed for fibrosis
quantification via Masson’s trichome staining and the hydroxyproline assay. R1 change at
70 minutes in all muscles at follow up scan correlated significantly with Masson’s
trichrome. R1 change at follow up correlated in three out of four muscles with
hydroxyproline. The strongest correlations to ex vivo measures of
fibrosis were in the GCN muscle
Conclusion: The
results of this study suggest that EP3533 can be used to quantify fibrosis within
skeletal and cardiac muscles of mdx mice, showing a high degree of
correlation to established histological and biochemical methods of fibrosis
quantification. The results do provide support for the potential use of EP3533 as a
non-invasive way to quantify fibrosis in vivo.
DMD HUB: Expanding and facilitating clinical trials for Duchenne Muscular Dystrophy
in the UK
Emma
Heslop1, Michela Guglieri1,
Cathy Turner1, Becky Crow1, Anna Peel1, Emily
Crossley2, Alexandra Johnson2 Mariacristina Scoto3,
Francesco Muntoni3 and Volker Straub1
1John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle University and Newcastle Hospitals NHS Foundation Trust, Newcastle
upon Tyne, UK (Emma.Heslop@ncl.ac.uk)
2Duchenne UK, London, UK
3The Dubowitz Neuromuscular Unit, UCL Great Ormond Street Institute
of Child Health, London, UK
The DMD Hub was established as a unique partnership
between Duchenne UK, the John Walton Muscular Dystrophy Research Centre in Newcastle and
the Dubowitz Neuromuscular Unit at Great Ormond Street Hospital in London, to address
trial capacity issues for Duchenne muscular dystrophy (DMD) trials in the UK.
Together we are able to address the issues and share
expertise to establish a network of trial-ready centres in the UK, able to take on
interventional trials in DMD. During the first 2 years we have supported the development
of 6 trial sites and are working to facilitate additional sites to take on upcoming
industry and academic-led studies.
The DMD-Hub website (DMDHub.org) is a key resource for
industry, clinicians and patients. It hosts the clinical trial finder, which is an
independent resource, regularly updated by a variety of reliable sources to provide
timely information on existing and pending clinical trials for DMD in the UK. Links to
centres and staff performing the trials are provided to facilitate clinical trial
recruitment. The development of a Standard Operating Procedure for clinical trial
recruitment will be linked to the clinical trial finder to facilitate fair and equitable
access to clinical trials across the UK.
The clinical trial finder currently lists 26 trials in
12 UK sites. The JWMDRC-Newcastle and GOSH-London remain the sites with the highest
number of trials, however it is clear that the DMD-Hub has successfully facilitated
additional sites to take on trials.
The DMD-Hub toolbox, also available on the DMD-Hub
website, is an ever evolving repository for training material and other resources to
respond to the needs of sites and industry in the setting up and delivery of
interventional clinical trials in DMD.
The DMD-Hub has expanded its focus to better include
the DMD adult population. A meeting was convened with key stakeholders to prepare for
the unique challenges of conducting clinical trials in this group. The DMD-Hub is
committed to working on the recommendations identified during this meeting and
supporting the development of the infrastructures needed.
A novel in-situ hybridisation (ISH) assay mapping the in-frame pseudoexon 11 (pE11)
expression in cultured dermal fibroblasts (CDF) and muscle in patients with severe
collagen VI disease due to a deep intronic mutation in COL6A1
Matthew
Beck1, Sara Aguti1, Pierpaolo
Ala1, Angela Richard-Loendt2, Darren Chambers3,
Dominic Scaglioni1, Didem Ardicli1,4, Lucy Feng2,
Rachael Main5, Haiyan Zhou1,6, Caroline Sewry2, Anna
Sarkozy1, Silvia Torelli1, Francesco Muntoni1, Rahul
Phadke2
1Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health & MRC Centre for Neuromuscular Diseases London, UK
2Division of Neuropathology, UCL Queen Square Institute of Neurology,
UCLH NHS Foundation Trust
3Dubowitz Neuromuscular Centre, Great Ormond Street Hospital for
Children & Division of Neuropathology, UCL Queen Square Institute of Neurology,
UCLH NHS Foundation Trust
4Pediatric Neurology and Rheumatology Unit, Hacettepe University
School of Medicine, Ankara, Turkey;
5DNA laboratory, GSTS Pathology, Genetics Centre, Guy’s Hospital,
London, UK;
6Genomics and Genetic Medicine Programme, UCL Great Ormond Street
Institute of Child Health, London, UK.
Email: matthew.beck.16@ucl.ac.uk
Background: A recurrent,
de-novo deep intronic mutation (intron 11) of COL6A1
(c.930 + 189C>T) leading to a dominantly acting in-frame pseudoexon insertion,
originally identified by a combination of muscle RNA and whole genome sequencing, has
been recently identified in a larger international cohort to cause severe collagen
VI-related disease (COL6-RD). cDNA analysis has shown lower expression of
pE11-containing transcripts in muscle and CDF compared to wild type transcripts, despite
the clinical severity and markedly reduced/mislocalised collagen VI, indicating a strong
dominant-negative effect.
Aim: To develop an ISH assay for spatial
and quantitative mapping of pE11, wild-type (WT) and total COL6A1 transcripts and study
effects of the mutation on collagen VI assembly in muscle and CDF.
Methods: We developed customised highly
specific short-length exon junction chromogenically tagged mRNA probes targeting E9E10
(total transcripts), E11pE11 (pseudoexon-containing transcripts) and E11E12 (WT
transcripts) for brightfield detection (BaseScope, ACD bio-techne). A panel of recently
authenticated antibodies against chain-specific (collagen VI A1, A2, A3) and multimeric
collagen VI was selected for immunoanalysis in muscle and CDF. Muscle and CDF from four
patients carrying the intronic mutation and unaffected controls (CTRL) were recruited
for the study.
Results: Our current work is focused on
optimising ISH assay conditions using housekeeping probes, and optimising collagen VI
multimeric and chain-specific antibodies in control muscle and CDF. In CDF from all 4
patients, we have observed uniform, low-level cytoplasmic expression of mutant
pE11-containing transcripts and a much higher expression of WT transcripts comparable to
CTRL.
Conclusion: Work on ISH in muscle and
immunoanalysis in muscle and CDF is in progress. We will correlate results with existing
muscle pathology, quantitative collagen VI flow-cytometry and COL6A1
RT-qPCR transcription data. Quantitative ISH for mutation mapping combined with
high-resolution microscopy could be potentially useful tools for studying effects of
dominant negative mutations on collagen VI assembly in COL6-RD, and the effect of
therapies aimed at removing the retained intron from the mature transcript.
Optimisation of a high–throughput digital script for multiplexed immunofluorescent
analysis of the sarcolemmal dystrophin and associated protein complex (DPC) and myofibre
regeneration in entire transverse sections of muscle biopsies in Duchenne muscular
dystrophy (DMD)
Dominic Scaglioni1, Matthew
Ellis2, Francesco Catapano1, Silvia Torelli1, Darren
Chambers3, Lucy Feng3, Caroline Sewry3, Jennifer
Morgan1, Francesco Muntoni1,4, Rahul Phadke3,5
1Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health, London, UK
2Department of Neurodegenerative Diseases, UCL Queen Square Institute
of Neurology, London, UK
3Dubowitz Neuromuscular Centre, UCL Queen Square Institute of
Neurology & Great Ormond Street Hospital for Children NHS Foundation Trust,
London, UK
4NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL
Great Ormond Street Institute of Child Health & Great Ormond Street Hospital for
Children NHS Foundation Trust, London, UK
5 Division of Neuropathology, National Hospital for Neurology and
Neurosurgery, UCLH NHS Foundation Trust, London, UK
Email: d.scaglioni@ucl.ac.uk
Background: The primary molecular endpoint
for a number of gene-based therapeutic clinical trials in DMD is restoration or
increased production of sarcolemmal dystrophin from the baseline, assessed by measuring
the amount of dystrophin using Western Blotting (WB) and immunohistochemistry (IHC).
With the ever-increasing numbers of DMD clinical trials there is an urgent need to
develop robust, reliable and objective dystrophin quantification methodologies by IHC
that can overcome limitations of existing methods.
Aims: To develop a fully automated digital
high-throughput method for multiplexed immunofluorescent analysis of dystrophin,
associated DPC proteins (alpha-sarcoglycan and beta-dystroglycan) and regenerating
fibres (fetal/developmental myosin-positive) in entire transverse sections of muscle
biopsies with rigorous optimisation of two key variables - exposure settings and
background signal correction.
Methods: Multiplex immunostaining of fresh
frozen sections and subsequent whole section fluorescent slide scanning (AxioScan ZEISS)
enabled the assessment and quantification by image analysis (Definiens software
platform) of entire tissue sections from DMD, Becker muscular dystrophy (BMD) and
control (CTRL) muscle biopsies. Optimal exposure settings were determined using DMD, BMD
and CRTL sections. Arbitrary thresholding was minimised using a novel dynamic
thresholding strategy of correcting the background signal per myofibre.
Results: The script generated automated
data for intensity, coverage and colocalisation for dystrophin and DPC at the
single-fibre as well as whole section level, number of dystrophin-positive fibres in the
entire section and number of regenerating fibres in correlation to the mean fluorescent
intensities of dystrophin, DPC and colocalised dystrophin-DPC in the entire section from
DMD, BMD and CTRL biopsies.
Conclusion: We present a novel digital
dystrophin quantification script capable of multiparametric analysis of dystrophin in
relation to key DPC proteins and myofibre regeneration allowing unbiased analysis of the
amount of functional dystrophin in DMD/BMD samples. Rigorous optimisation strategies are
implemented to demonstrate ‘clinical trial readiness’ and regulatory compliance.
Comparative analysis of previously published digital methodology with our new script is
ongoing.
Preliminary results from the “Observational study of clinical outcomes for
testosterone treatment of pubertal delay in Duchenne Muscular Dystrophy (DMD)
(NCT02571205)”
C.Wood1,
V. Straub1, M Guglieri1, T Cheetham2
1John Walton Muscular Dystrophy Research Centre, Newcastle
University
2Newcastle upon Tyne Hospitals Trust Paediatric Endocrinology
department
Claire.wood@ncl.ac.uk
Background: Delayed puberty is almost
universal in glucocorticoid-treated patients with DMD. We have demonstrated variations
in the way this is managed and there is a pressing need to establish the impact of
exogenous testosterone therapy on well-being, pubertal status and longer-term gonadal
function as well as the musculoskeletal system in this group of adolescents.
Aims: This single centre study followed
the progress of 15 adolescents with DMD and delayed puberty who were treated with
incremental intramuscular testosterone injections using the testosterone ester
‘Sustanon’ every 4 weeks for 2 years.
Methods: Data on a range of endpoints
including pubertal status and gonadal function was collected at 6 monthly intervals for
2 years. Patients were also assessed 3 months after the final testosterone
injection.
Results: All 15 patients completed 27
months of follow-up. All were pre-pubertal at baseline with a testicular volume (TV)
less than 4mls and baseline testosterone level <2nmol/l. 27 months later (3 months
after the last testosterone injection) the mean testicular volume was 3.3 mls (range
1.5-6mls), Tanner stage G4P4 (range G4P4-G5P5) with a mean testosterone of 10.4 nmol
(1.9-21.8). Mean FSH was 9.1 IU/L (range 1.8-38.3) and mean LH 8.6 IU/L(range
<0.5-25.1). Qualitative interviews and treatment satisfaction questionnaire responses
were favourable and no serious AEs were attributed to testosterone therapy. 2 AEs were
felt to be associated with testosterone (acne and injection site reaction). 2 patients
chose to stop testosterone injections at 18 months because of concerns about potential
effects on mobility. Both continued to progress through puberty spontaneously.
Conclusion: Testosterone is not part of
the standard of care in DMD but the described regimen was an effective and
well-tolerated treatment for pubertal delay. Further follow-up is required but
preliminary results suggest that this regimen promotes endogenous testosterone
production.
The utility of mdx:cmah as a model for assessing skeletal
development in Duchenne muscular dystrophy (DMD). A comparison of the
mdx and the mdx:cmah models
C.Wood1,2, S.C.
Wong3, V. Straub2, S.F. Ahmed3,
C.Farquharson1
1Developmental Biology Group, Roslin Institute, University of
Edinburgh
2John Walton Muscular Dystrophy Research Centre, Newcastle
University
3Developmental Endocrinology Research Group, University of
Glasgow
Claire.wood@ncl.ac.uk
Background and aims: Investigation of
compounds to treat the defect in growth and skeletal development in DMD necessitates an
appropriate pre-clinical model. The mdx mouse is commonly used but its
phenotype is mild and few medications that have shown benefit in mdx
have also shown efficacy in clinical trials. The
mdx:cmah carries a human-like mutation in
Cmah gene and has a more severe muscle phenotype1, but its
growth and bone have not been investigated. We aimed to investigate its use as a model
for assessing skeletal development in DMD.
Methods: 6 male mdx,
mdx:cmah and wild-type (WT) mice were sacrificed at 3, 5 and 7 weeks
of age. Bone mass and architecture were assessed by micro-CT and breaking strength
determined by 3-point bending. Growth was assessed by anthropometric measures and
calcein labelling at the chondro-osseous junction. Bone transcriptome was assessed using
the Qiagen osteogenesis pathway 84-gene array on humeral RNA.
Results:Mdx:cmah mice were smaller than WT at 3, but heavier
at 7 weeks (p=0.02). 5-week-old mdx:cmah mice showed an increased
growth rate (p=0.007). Gene profiling identified a 3-fold upregulation in
Mmp-10 and Bmpr1b expression in both
mdx and mdx:cmah bone v WT and increased expression
of many growth factors in mdx:cmah, including Igf-1
(30-fold v WT). Igf1R (10-fold) and
Vegfa (32-fold). Micro-CT indicated that cortical
bone area and fraction were lower in 3-week-old mdx:cmah mice
(p<0.05) but cortical bone fraction was greater in both mdx and
mdx:cmah v WT at 7-weeks (p<0.05).Tissue mineral density was higher in
mdx:cmah than WT at 3 (p<0.05) and 7 (p<0.01) weeks.
Conclusion: The mdx:cmah
mice shows clear evidence of catch-up growth that is also associated with an increase in
bone development. This pattern does not mimic the typical DMD growth trajectory. Whilst
its utility for studying growth and skeletal development may be limited, further studies
of this model can shed light on the phenomenon of catch-up growth.
Ref: Chandrasekharan et al. Sci Transl Med
(2010)
Atomic force microscopy based assessment of peptide-conjugated phosphorodiamidate
morpholino oligomer treatment of Duchenne muscular dystrophy
Yulia Lomonosova1, Graham
McClorey1, Joanna Zemla2, Malgorzata Lekka2, Matthew
M. Wood1
1Department of Paediatrics, University of Oxford, Le Gros Clark
Building, South Parks Road, Oxford, OX1 3QX, United Kingdom.
2Department of Biophysical Microstructures, Institute of Nuclear
Physics, Polish Academy of Sciences, PL-31342 Krakow, Poland.
Contact: yulia.lomonosova@paediatrics.ox.ac.uk
Background: Antisense oligonucleotide
(ASO) mediated splice modification is currently the most promising therapeutic
intervention for Duchenne muscular dystrophy. ASO can be used for targeted exon
exclusion resulting in the correction of aberrant reading frames and the production of
an internally deleted, yet largely functional, dystrophin protein. Crucial modification
resulting in a significant increase in bioavailability of the drug is conjugation of ASO
to a cell-penetrating peptide. The peptide Pip9b2 was conjugated to a phosphorodiamidate
morpholino oligomer (PMO). Atomic force microscopy (AFM) is a widely applied tool for
biomechanical studies of pathologically altered samples. In AFM, mechanical properties
of cells or tissues are quantified through the relative Young’s modulus.
Aims: To estimate effectiveness of
treatment of mdx mice using two different doses of Pip9b2-PMO. To utilize AFM for
assessment of mechanical properties of sarcolemma following the treatment.
Methods: Male mdx mice received 2
systematic intravenous injections of 7.5 mg/kg or 15 mg/kg Pip9b2-PMO at 1-week
interval. Two weeks after administration, Pip9b2-PMO-treated mice and their age- and
sex-matched controls (C57BL/10, mdx) were sacrificed.
Results: Exon-skipping efficiencies in m.
tibialis anterior were determined to be 50.4% and 82.1% resulting in restoration of
dystrophin protein to 49.3% and 53.3% following 7.5 mg/kg and 15 mg/kg correspondently.
The efficiencies of 37.5% and 55.4% in m. soleus were considerably lower than that in m.
tibialis anterior in mdx given 7.5 mg/kg or 15 mg/kg correspondently. Treated mdx showed
uniform dystrophin distribution in the sarcolemma. Elastic properties of individual
muscle fibers were probed by AFM. Myofibers of C57BL/10 yielded Young’s modulus values
of 4.2 kPa on average, whereas those of mdx were 2.3 kPa indicating lower resistance of
dystrophic muscles to deformation. Following the treatment of mdx where 50% of
dystrophin restoration was reached, the Young’s modulus didn’t differ from that in
C57BL/10, suggesting recovery of mechanical properties of myofibers. The Young’s modulus
of treated groups was considerably higher than that in untreated mdx. It was not found
any difference in elasticity between groups treated by 7.5 mg/kg and 15 mg/kg,
dystrophin protein content was equal between those groups.
Conclusion: Thus, Pip9b2-PMO treatment
provides effective dystrophin restoration in hindlimb muscles of mdx reversing myofibers
stiffness.
Funding Statement: This study was supported by Medical
Research Council.
Genome editing to correct duplications in the dystrophin gene
*Veronica Pini, Marc Moore, John Counsell,
Virginie Mariot, Helen C. O’Neill, Francesco Conti, Jenny Morgan, Francesco Muntoni
Greet Ormond Street Institute of Child Health, University College London, 30
Guilford Street, London, UK, WC1N 1EH
Background: Duchenne Muscular Dystrophy is
a severe neurodegenerative disorder caused by deletions, duplications or point mutations
in the DMD gene, which encodes dystrophin. In absence of dystrophin, muscle fibers
degenerate and patients become wheelchair bound by their early teens. Cardiac and
respiratory muscles are also affected, causing premature death by the third decade of
life. Several approaches are currently being tested in clinical trials to treat DMD, but
none of them are suitable to treat patients carrying duplications in dystrophin gene,
which account for 10-15% of DMD cases.
Aims: In the present work we developed a
genome editing approach that exploits CRISPR/Cas9 to correct duplications in the DMD
gene by using a single guide RNA.
Material and Methods: We compared nuclear
electroporation and integrating lentiviruses as tools to deliver CRISPR/Cas9 in
patient-derived myoblasts carrying in-frame duplications. The efficiency of genomic
editing was assessed as the ratio between untargeted and targeted amplicons in the T7
assay. Finally, Reverse Transcriptase-PCR and Western Blot were used to assess
dystrophin restoration at transcriptional and protein levels.
Results: We have identified a nuclease
able to efficiently target DMD intron 9, which would be suitable for gene editing in
several patients harbouring DMD duplications. The estimated genomic targeting efficiency
in Cas9-expressing cells obtained from DMD patients was around 28%. RT-PCR of treated
versus untreated cells confirmed the restoration to wild-type dystrophin transcripts and
finally Western Blot confirmed both dystrophin correction and a decrease of the mutated
protein in edited cells.
Conclusion: CRISPR/Cas9 editing tool has
proven to be suitable to remove large genomic duplications. Cells derived from DMD
patients carrying duplications in dystrophin gene have been treated and corrected by
using a single CRISPR/Cas9 gene therapy construct and, at later stages, corrected
myoblasts will be assessed for their potential to functionally restore dystrophin
production following transplantation in Duchenne Muscular Dystrophy animal models.
Marc Moore’s email:
sejjm41@ucl.ac.uk
Engineering cell fate and artificial chromosomes in human iPS cells for
next-generation gene and cell therapy of Duchenne muscular dystrophy
Giulia Ferrari1, Louise A. Moyle1,
Hidetoshi Hoshiya1, Martina Ragazzi1 Tamara
Casteels1,#, Sara M. Maffioletti1, Narumi Uno2,
Yasuhiro Kazuki2, Francesco Muntoni3, Mitsuo Oshimura2,
Francesco Saverio Tedesco1,3*
1Department of Cell and Developmental Biology, University College
London, WC1E 6DE London, United Kingdom.
2Department of Biomedical Science, Tottori University, 603-8503
Yonago, Japan.
3Dubowitz neuromuscular centre, UCL Great Ormond Street Institute of
Child Health and Great Ormond Street Hospital for Children, London WC1N 1EH, United
Kingdom.
#Current address: Research Center for Molecular Medicine of the
Austrian Academy of Sciences 1090 Vienna, Austria
Background: Duchenne muscular dystrophy
(DMD) is due to mutations of the dystrophin gene and primarily affects skeletal muscles.
Our study looks at different strategies to circumvent substantial obstacles in the
development of therapies for this incurable disease. The limited availability of large
number of cells and the large size of the dystrophin gene (2.4Mb) could be tackled by
combining human artificial chromosome (HAC)-based gene correction and iPS cell-mediated
production of transplantable myogenic cells. However, another significant hurdle is
posed by cell delivery, as skeletal muscle is the most abundant human tissue and to date
there are no systemically deliverable human iPS cell derived myogenic progenitors
generated without integrating transgenes. This challenge could be addressed by
modulating molecules involved in the developmental specification of pericytes, able to
migrate through the vascular endothelium upon intra-vascular delivery, thus
circumventing the requirement for multiple intra-muscular injections.
Aims: This study aims at generating safe,
systemically-deliverable DMD iPS cell-derived myogenic cells genetically correctable
with HACs containing the entire human dystrophin genetic locus.
Methods: DMD iPS cells have been generated
with non-integrating strategies and then differentiated into expandable myogenic cells
using both the myogenesis regulator MyoD (mRNA) and a trangene-free, small
molecule-based protocol. Molecules involved in the developmental specification of
perivascular cells able to migrate through the vascular endothelium upon systemic
delivery were tested to modulate cell migration.
Results: We generated three DMD iPS cell
lines, one of them already genetically corrected with HAC. We then derived inducible
myogenic cells similar to skeletal muscle pericyte-derived mesoangioblasts. Preliminary
results show that genomic integration-free MyoD mRNA drives a good myogenic
differentiation. We additionally show that iPS cell-derived myoblasts acquire enhanced
migration ability in vitro upon activation of signalling pathways involved in the
developmental specification of pericytes. Notably, RNA sequencing data elucidate the
gene expression shift underlying this acquired function, opening the way to the
identification of druggable targets.
Conclusion: The generation of human iPS
cell-derived myogenic cells able to migrate through an endothelial wall lays the basis
for further pre-clinical studies exploring a new, safer genomic integration-free
approach for ex vivo gene therapy of DMD.
Novel high-throughput digital analysis to quantify the amount of functional
sarcolemmal dystrophin and myofibre regeneration in Duchenne Muscular Dystrophy (DMD)
clinical trial samples (exon 53 skipping with Golodirsen)
Dominic
Scaglioni1, Francesco
Catapano1, Matthew Ellis2, Silvia Torelli1, Darren
Chambers3; Lucy Feng3; Diane Frank4, Aji
Nair4, Shawn Harriman4, Ashish Dugar4, Jennifer
Morgan1; Phadke R3, Muntoni F1,5
1Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health, London, UK
2Department of Neurodegenerative Diseases, UCL Queen Square Institute
of Neurology, London, UK
3Dubowitz Neuromuscular Centre, UCL Queen Square Institute of
Neurology & Great Ormond Street Hospital for Children NHS Foundation Trust,
London, UK
4Sarepta Therapeutics, Cambridge, MA, United States
5NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL
Great Ormond Street Institute of Child Health & Great Ormond Street Hospital for
Children NHS Foundation Trust, London, UK
Email:
d.scaglioni@ucl.ac.uk
Background: Exon skipping therapies in
Duchenne muscular dystrophy (DMD) aim to modulate the pre-mRNA splicing of the DMD
transcript using morpholino (PMO) antisense oligonucleotides (AONs) to restore the
DMD open reading frame, leading to the production of a partially
functional protein. However, the functionality of such restored dystrophin has been
questioned.
Aims: To determine if restored dystrophin
following treatment with Golodirsen (exon 53 skipping PMO) results in enhanced levels of
dystrophin-associated proteins (DPC) at dystrophin positive regions of the sarcolemma in
post-treatment muscle biopsies as a surrogate marker of functional dystrophin.
Additionally, to assess if there is a reduction in the number of regenerating myofibres
associated with restored dystrophin expression.
Methods: We developed a highly automated,
high-throughput method for multiplexed immunofluorescent analysis of dystrophin and DPC
in entire transverse sections of muscle biopsies. Using an optimised image analysis
script developed in Definiens software applied to digitally scanned whole sections, we
are able to generate data that allows colocalised quantification of dystrophin and DPC
(alpha-sarcoglycan, beta-dystroglycan) at the sarcolemma and quantify the number of
regenerating fibres (fetal/developmental myosin cocktail).
Results: Exposure settings following
rigorous optimisation experiments yielded normalised dystrophin and DPC intensity values
in control sections without significant variation across different batches. Digital
analysis performed on two samples revealed significantly higher mean alpha-sarcoglycan
intensity value in dystrophin-positive sarcolemma compared with dystrophin-negative
sarcolemma. We were also able to quantify the number of the regenerating fibres in these
samples.
Conclusion: Our high-throughput digital
analysis of samples blinded to treatment state demonstrated that the levels of DPC
proteins are higher in dystrophin-positive compared to dystrophin-negative fibres,
suggesting the dystrophin produced is able to drive assembly of a functional
dystrophin-associated protein complex. The algorithm also allowed us to quantify the
number of regenerating fibres in the entire muscle biopsy sections. Analysis of
dystrophin-DPC-myofibre regeneration data on the entire study cohort is in progress and
results will be correlated with treatment status following unblinding. We suggest that
quantitative dystrophin-DPC colocalisation data and potentially the percentage of
regenerative fibres could be used as a surrogate marker of the functionality of restored
dystrophin in DMD.
Long-term effects of cardio-active medications on left ventricular function in
patients with Duchenne muscular dystrophy-related cardiomyopathy
Alexander P. Murphy1, Volker Straub1,
Stephen Lord2, Alison Heads-Baister2
Anna Johnson 2 and John P. Bourke2 -
[alexander.murphy@ncl.ac.uk]
1The John Walton Muscular Dystrophy Research Centre, Institute of
Genetic Medicine, The International Centre for Life, Newcastle University, Central
Parkway, Newcastle Upon Tyne, NE1 4EP, United Kingdom.
2Department of Cardiology, Freeman Hospital, Newcastle upon Tyne NHS
Hospitals Foundation Trust, NE7 7DN, United Kingdom.
Background: Almost all patients with DMD
develop progressive left ventricular dysfunction. Despite this, there has been little
research into the response of this specific form of cardiomyopathy to standard ‘heart
failure’ medications, deployed uniformly.
Aims: To describe the course of
ventricular function before and after the introduction of a combination of ACE-inhibitor
and beta-blocker in a large cohort of patients with DMD-related cardiomyopathy.
Methods: This was a retrospective analysis
of serial left ventricular fractional shortening measures obtained in the four years
before and four years after initiation of combination therapy with an ACE-inhibitor and
beta-blocker at one hospital in the period January ‘95 to January 2015. Therapy was
initiated consistently at the first outpatient attendance at which left ventricular FS
< 28% (‘possible cardiomyopathy’) or FS < 25% or regional wall motion abnormality
(‘definite cardiomyopathy’) was detected. Doses were up-titrated over subsequent visits,
as tolerated. Eplerenone was not added during this time period.
Results: Results of 821 echocardiograms
(> 2 scans / patient), performed on 126 DMD patients, aged 16.2 ± 4.7 at time of
initial and 21 ± 7.3 years at their last analysed echocardiogram were reviewed. Mean age
of first cardiomyopathy detection was 17.3 ± 4 years - but significantly older in those
on steroid therapy (18.1 ± 3.4 versus 15.5 ± 4.1 years; p = 0.003). Group mean FS fell
by 0.83% per year before therapy, but this reduced to 0.38% per year during four years
on treatment. ‘Poor-attenders’ had lower FS% before dysfunction was detected.
Ventricular function - already lost before treatment, was not regained thereafter but
fell at the same rate as in those treated earlier. Neither age of detection of
cardiomyopathy nor FS% at therapy initiation affected the beneficial response.
Conclusion: Combination drug therapy
stabilises ventricular function but cannot prevent its slower decline over time. Cardiac
therapies should be initiated no later than on first detecting ventricular
impairment.
Global FKRP Registry
Lindsay Murphy*1, Jean-Pierre
Laurent2, Katherine Mathews3, Herb Stevenson4, Simone
Thiele5, John Vissing6, Maggie Walter5, Lacey
Woods7, Volker Straub1
1The John Walton Muscular Dystrophy Research Centre, Newcastle
University, UK;
2LGMD2I Research Fund, USA;
3University of Iowa, Carver College of Medicine, USA;
4University of Massachusetts Medical School; USA
5Ludwig-Maximilians University of Munich, Germany;
6University of Copenhagen, Denmark;
7Patient representative
*Presenting author:
lindsay.murphy@ncl.ac.uk
Background: The Global FKRP Registry is an
international registry for individuals with conditions caused by mutation of the
Fukutin-Related Protein (FKRP) gene: limb girdle muscular dystrophy
2I (LGMD2I, newly renamed LGMDR9) and the congenital muscular dystrophies MDC1C,
Muscle-Eye-Brain Disease and Walker-Warburg Syndrome. The registry seeks to further
understanding of the natural history and prevalence of FKRP-related muscular dystrophies
(MD).
Aims: The purpose of the registry is to
aid the rapid identification of eligible patients for clinical studies. It disseminates
FKRP-relevant information to participants; provides a source of information to
academics, industry and healthcare professionals; and supports the FKRP community.
Methods: Registration is patient-initiated
through a secure online portal. Participants give their informed consent and are invited
to complete a questionnaire about their condition. Data is reported by both patients and
their healthcare professionals and includes: age of onset, presenting symptoms, family
history, motor function and muscle strength, respiratory and cardiac function,
medication, in addition to information on patient quality of life and pain.
Results: Currently, 643 patients (54%
female, 46% male) are registered with the Global FKRP Registry, with an age range of 1
to 79 years. Registrations are from 38 countries, with greatest numbers from USA (28%),
Germany (21%) and UK (12%). Diagnoses are reported as LGMD2I (90%), MDC1C (2%), other
FKRP-related MD (3%), unspecified (5%). Seventy-one percent of patients are reported as
being ambulant, 24% as non-ambulant and 5% as unspecified. The mutations reported within
the registry are: 65% homozygous for the common mutation (c.826C>A), 29% heterozygous
for the common mutation, 5% heterozygous with two unique mutations and 1% homozygous
with a unique mutation which is not the common mutation.
Conclusion: The Global FKRP Registry is a
valuable tool for the collection of patient data which informs academics, healthcare
professionals and industry. For example, registry data may improve our understanding of
phenotype-genotype correlation and inform improvements in standards of care. The
registry represents a trial-ready cohort of individuals and supports the FKRP patient
community.
A complete cross-sectional and longitudinal study on plasma derived miRNAs revealed
novel dysregulated signatures in DMD patients
Francesco Catapano1, Kate
Maresh1, Victoria Selby1 Joana Domingos1,
Pierpaolo Ala1, Valeria Ricotti1, Lauren Phillips2,
Laurent Servais3, Imelda de Groot4, E.H. Niks5,
J.J.G.M. Verschuuren5, Volker Straub2,6, Thomas Voit7,
Jennifer Morgan1 and Francesco Muntoni1
1The Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute
of Child Health, London, United Kingdom
2John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
3Service of Clinical Research and Databases, Institute of Myology,
Paris, France
4UMC St. Radboud, Nijmegen, Netherlands
5Department of Neurology, Leiden University Medical Center, Leiden,
The Netherlands
6Northern Genetics Service, Newcastle upon Tyne Hospitals NHS
Foundation Trust, Institute of Human Genetics, International Centre for Life,
Newcastle upon Tyne, United Kingdom
7NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL
Great Ormond Street Institute of Child Health, London, United Kingdom
E-mail: k.maresh@ucl.ac.uk
Background: Duchenne muscular dystrophy
(DMD) is an X-linked recessive neuromuscular disorder affecting 1 in 5000 newborn males,
mainly caused by out-of-frame deletions or, more rarely, duplications, nonsense or other
small mutations affecting DMD gene and therefore dystrophin protein
production.
MicroRNAs are short (~20-23 nucleotides) non-coding
RNAs that regulate gene expression; their dysregulation in serum and urine has been
associated with many paediatric neuromuscular conditions including DMD.
MiRNAs are present in biofluids as free circulating
molecules or included in exosomes small microvescicles (40-100 nm) secreted by different
cell types.
Aims: To investigate the potential of
plasma derived exosomal miRNAs as a novel non-invasive biomarker in DMD and find novel
free circulating miRNAs cross-sectionally and longitudinally dysregulated, we performed
miRNA profiling and validation analysis in DMD patients and healthy controls.
Moreover, we assessed if there was any association
between miRNA levels in plasma and corticosteroid treatment.
Materials & Methods: The patients
included in this study are part of a cohort of DMD boys in a multicentre natural history
study registered in clinicaltrials.gov (NCT02780492). Samples from patients recruited in
London, Paris, Newcastle and Leiden were analysed.
qPCR microRNA profiling was performed using
Serum/Plasma Focus microRNA PCR SYBR green-based panels (Exiqon), while validations were
carried out by a qPCR TaqMan small RNA Assay (Life Technology).
Results: We detected novel miRNAs (both
free-circulating and exosomal) dysregulated in plasma from DMD patients and validated
those with the strongest abnormal expression.
We also detected a set of longitudinally dysregulated
miRNAs between different timepoints (?T1-T2=24 months) in DMD patients.
Conclusion: Our findings indicate that a
set of free-circulating miRNA is longitudinally dysregulated in plasma from DMD
patients. Moreover we detected novel dysregulated miRNAs in plasma from DMD
patients.
Activity of Stereopure Antisense Oligonucleotides in Cellular Free-Uptake Models
Predicts Exon Skipping and Dystrophin Protein Restoration in mdx23
Mice
Jason Zhang,1 Ann
Durbin,1 Irina Antonijevic,1 Gopal Bommineni,1 Annie
Chivatakarn,1 Michael Byrne,1 Lankai Guo,1 Naoki
Iwamoto,1 Pachamuthu Kandasamy,1 Nayantara Kothari,1
Jayakanthan Kumarasamy,1 Fangjun Liu,1 Kenneth Longo,1
Graham McClorey,2 Prashant Monian,1 Erin
Purcell-Estabrook,1 Carlo Rinaldi,2 Juili Shelke,1
Mamoru Shimizu,1 Chikdu Shivalila,1 Stephany Standley,1
Yuan Yin,1 Hailin Yang,1 Xiansi Zhao,1 Zhong
Zhong,1 Matthew Wood,2 Chandra Vargeese1
1Wave Life Sciences Ltd, Cambridge, MA, USA; 2Department
of Paediatrics, University of Oxford, Oxford, UK
Presenting Author: Jason Zhang,
jzhang@wavelifesci.com
Background: Wave Life Sciences has
established a targeted approach to designing stereopure nucleic acid compounds based on
sequence composition, chemical design, and backbone stereochemistry. Wave is using this
approach to develop stereopure exon skipping antisense oligonucleotides (ASOs) as
potential disease-modifying therapies for Duchenne muscular dystrophy (DMD).
Aims: To determine the effects of
investigational stereopure ASOs targeting exon 23 dystrophin
(DMD) pre-mRNA on exon skipping efficiency and dystrophin protein
restoration in the mdx23 mouse model of DMD and to determine the
relationship between in vitro and in vivo effects.
Methods:mdx23 mice received single or 4 weekly intravenous (IV) doses of ASO.
Exon skipping efficiency was determined by TaqMan® assay, dystrophin protein
restoration by western blot, and dystrophin localization by immunofluorescence
microscopy. Serum enzyme markers of muscle and liver function were assessed. Cultured
DMD patient-derived myoblasts or murine mdx23-derived myoblasts were
incubated with ASOs under free-uptake conditions.
Results: Stereopure oligonucleotides
targeting exon 23 induced dose-dependent exon 23 skipping in vitro in
mdx23-derived myoblasts. In vivo, after a single IV
dose, stereopure ASOs induced up to 16% exon skipping in target muscle tissues
(gastrocnemius, diaphragm, quadriceps, heart) and restored dystrophin protein in
gastrocnemius in mdx23 mice for =28 days postdose. Four weekly IV doses
of stereopure ASOs induced dose-dependent exon skipping of up to 20% in target muscle
tissues of mdx23 mice, yielding dystrophin protein restoration in
target tissues, with 93% restoration observed in the gastrocnemius. Serum creatine
kinase, aspartate aminotransferase, and alanine aminotransferase levels were reduced by
87%, 55%, and 79%, respectively, after 4 weekly doses. The dose relationship of exon
skipping induced by stereopure ASOs was similar in vitro and in
vivo. Transcript levels observed with free uptake of oligonucleotides in
cultured myoblasts were predictive of transcript and dystrophin protein levels in the
mdx23 mouse.
Conclusion: Stereopure exon 23 skipping
ASOs produced efficient exon skipping and dystrophin protein restoration in
mdx23 mice. Consistent results observed between in
vitro and in vivo models support our approach of selecting
clinical candidates targeting exon 51 and exon 53 using a free-uptake myoblast cell
model.
Supported by Wave Life Sciences Ltd, Cambridge, MA,
USA.
Vision-DMD: an update on the drug development program of the orphan drug Vamorolone
(VBP15) in Duchenne Muscular Dystrophy (DMD)
Michela Guglieri1, Rebecca
Crow1 Kanneboyina Nagaraju2, Paula Clemens3, Jesse
Damsker4, John McCall4, Laurel Mengel-Gaw4, Laurie
Conklin5, Jana Haberlova6 Ed Smith7, Diana
Castro8, Jean Mah9, Craig McDonald10, Nancy
Kuntz11, Richard Finkel12, Mar Tulinius14, Yoram
Nevo15, Monique Ryan16, Richard Webster17, Andrea
Smith18, Lauren Morgenroth18, and Eric Hoffman4.
1Newcastle University, Newcastle, UK
2Binghamton University, Binghamton, NY, USA
3University of Pittsburgh, Pittsburgh, PA, USA
4Reveragen Biopharma, Rockville, MD, USA
5Camden Medical Associates, St. Louis, MO, USA
6University Hospital Moto, Prague, Czech Republic
7Duke University, Durham, NC, USA
8University of Texas Southwestern, Dallas, TX, USA
9Alberta Children’s Hospital, Alberta, Canada
10University of California Davis, Davis, CA, USA
11Ann & Robert H. Lurie Children’s Hospital, Chicago, IL,
USA
12Nemours Children’s Hospital, Orlando, FL, USA
14Queen Silvia Children’s Hospital, Gothenburg, Sweden
15Schneider Children’s Medical Center, Tel Aviv, Israel
16Royal Children’s Hospital, Melbourne, Australia
17The Children’s Hospital at West, Sydney, Australia
18TRiNDS LLC, Kensington, MD, USA
VISION-DMD is the drug development program of orphan
drug Vamorolone (VBP15) in Duchenne Muscular Dystrophy (DMD)
Vamorolone is a first-in-class dissociative steroidal
drug developed by ReveraGen Biopharma under a venture philanthropy model, funded from
international non-profit foundations, US and EU governments.
Vamorolone is administered as liquid solution taken
daily.
The Phase IIa open-label, multiple ascending dose
(0.25, 0.75, 2.0, and 6.0 mg/kg/day) studies (VBP15-002 and VBP15-003) recruited 48
steroid naïve DMD boys, age 4 to<7 years. Following a 2-week treatment period and
2-week washout period (VBP15-002), participants continued to receive vamorolone for 24
weeks (VBP15-003). A 2-year long-term extension study of vamorolone was offered to
participants completing the phase IIa study.
Results of the phase IIa studies show vamorolone was
well tolerated during 24-weeks treatment at tested doses. Pharmacodynamic biomarkers
confirmed Vamorolone retains anti-inflammatory activities, but shows decrease in
frequencies and severity of corticosteroid side effects. Bone loss was not seen with any
dose of vamorolone, evidenced by serum osteocalcin. Adrenal suppression and insulin
resistance were less impacted in vamorolone-treated DMD patients, relative to published
glucocorticoid studies.
Additionally, dose-responsive improvement of muscle
function was observed for 2.0 and 6.0mg/kg/day dose groups compared to natural history
controls.
A phase IIb randomised, double blind, parallel group,
placebo and active controlled study with double blind extension in 120 DMD boys has been
initiated in 30 sites in 9 countries. 9 patients have been enrolled and 10 sites of 30
are recruiting. The study aims to evaluate safety, efficacy, pharmadynamics and
population pharmacokinetic of vamorolone at daily doses of 2.0mg/kg and 6.0mg/kg versus
prednisone 0.75mg/kg/day and placebo over 24-week treatment period and evaluates
persistence of effect over a Treatment Period of 48 weeks in ambulant DMD boys aged 4
to<7 years.
Studies conducted demonstrated the efficacy and
reduction in adverse effects in DMD patients using vamorolone compared to traditional
glucocorticoids, supporting further testing of vamorolone as a safer alternative to long
term glucocorticoids for DMD.
Studies in older and younger DMD patients are in
development.
Vamorolone potentially could replace chronic
glucocorticoids in disorders where side effects detract from patient quality of
life.
Respiratory Function Decline in Eteplirsen-treated Patients Diverges From Natural
History Comparators Over Time
Navid
Khan1; Lixin
Han1; Bernard T. Kinane2; Heather Gordish-Dressman3;
Linda Lowes4; Craig McDonald5; CINRG DNHS Investigators
1Sarepta Therapeutics, Inc., Cambridge, MA; 2Massachusetts
General Hospital for Children, Harvard Medical School, Boston, MA; 3George
Washington University School of Medicine and Health Sciences, Washington, DC;
4Nationwide Children’s Hospital, Columbus, OH; 5University of
California, Davis, Davis, CA
Background: Patients with Duchenne
muscular dystrophy (DMD) experience progressive degeneration of skeletal muscles,
including those involved in respiration, and respiratory decline is linked to mortality.
The onset of respiratory decline precedes increasing levels of clinical management as
the percent predicated forced vital capacity (FVC%p) falls below established thresholds.
The goal of treatment is to delay the time to reach these thresholds.
AIM: Motor function in eteplirsen-treated
patients in Studies 201 and 202 diverges from natural history after 1 year of treatment.
This analysis was conducted to determine if respiratory function follows a similar
pattern.
Methods: We evaluated eteplirsen treatment
effect in Studies 201 and 202 on FVC%p over time compared with well-matched natural
history controls from the Cooperative International Neuromuscular Research Group
Duchenne Natural History Study (CINRG DNHS) to determine if respiratory decline exhibits
a pattern similar to what has been described for motor function. In Studies 201 and 202,
pulmonary function tests were performed in all patients (N=12) every 24 weeks over the
course of 216 weeks of eteplirsen therapy. FVC%p was plotted over time for both studies.
This was compared with FVC%p of baseline age and FVC%p-matched CINRG DNHS patients over
the course of 4 years to determine if eteplirsen-treated patients diverged from the
CINRG DNHS patients. Two cohorts of CINRG DNHS patients were used as comparators: the
All CINRG cohort (n=75), and the Genotyped CINRG cohort (n=67).
Results: The eteplirsen-treated patients
in Studies 201 and 202 had similar baseline levels and similar decline compared with the
All CINRG and Genotyped CINRG cohorts for the first year. A divergence appeared after
Year 2 that continued to widen over the 4-year period. At Year 4, a difference of
approximately 10% in FVC%p favored eteplirsen treatment in comparison to both CINRG DNHS
cohorts.
Conclusion: The temporal pattern of
divergence of respiratory function in the eteplirsen-treated patients from the natural
history comparators is similar to the pattern of divergence seen for motor function.
Presenting author e-mail:
NKahn@Sarepta.com
Eteplirsen Is Well Tolerated in Men with Mild or Moderate Renal Impairment
Candida Fratazzi, MD; Emily Naughton;
Heidi Krenz
Sarepta Therapeutics, Inc., Cambridge, MA, USA
Introduction: Duchenne muscular dystrophy
(DMD) is a rare, X-linked, fatal, neuromuscular disease caused by DMD
gene mutations that disrupt the dystrophin messenger ribonucleic acid (mRNA) reading
frame and prevent the production of functional dystrophin protein. Eteplirsen is a
phosphorodiamidate morpholino oligomer (PMO) approved by the US Food and Drug
Administration for treatment of DMD patients with mutations amenable to exon 51
skipping. Eteplirsen excludes exon 51 to restore the dystrophin mRNA reading frame and
to enable translation of internally shortened dystrophin protein. PMOs have uncharged
backbones and bind in a sequence-specific manner to RNA targets through Watson-Crick
base pairing. PMOs represent a unique chemistry, structurally and biologically distinct
from other synthetic antisense ribonucleic acid therapeutics, such as phosphorothioates
(PSOs). Toxicities observed with PSOs have not been observed in nonclinical or clinical
studies of eteplirsen. In DMD patients studied, approximately 64% of the total systemic
clearance of eteplirsen 30 mg/kg (approved dose) is via renal excretion.
Aim: To determine the pharmacokinetics
(PK), safety, and tolerability of eteplirsen in men with mild or moderate renal
impairment.
Methods: This single-dose, parallel-group
study enrolled male volunteers with mild (estimated glomerular filtration rate [eGFR]
=60 to <90 mL/min; n=8) or moderate (eGFR =30 to <60 mL/min; n=8) renal
impairment. Participants received intravenous (IV) eteplirsen 30 mg/kg, and postdose PK,
safety, and tolerability results were evaluated and compared with demographically
matched men with normal renal function (n=9).
Results: All enrolled participants
completed the study. Total plasma clearance decreased by 27.5% (mild group) and 60.6%
(moderate group), with proportional reductions in renal clearance (22.7% and 56.6%,
respectively) and higher overall exposure compared with the normal group. The single IV
dose appeared to be well tolerated by all groups. Three participants, one in each group,
reported 6 treatment-related adverse events (AEs). These were dizziness (normal group);
pyrexia (mild group); and pollakiuria, micturition urgency, and 2 events of incontinence
(moderate group). All AEs were mild to moderate, considered nonserious, and
resolved.
Conclusions: Eteplirsen was well tolerated
across renally impaired and normal renal function groups. Exposure to eteplirsen
increased as a function of decrease in renal clearance.
Presenting author e-mail:
CFratazzi@Sarepta.com
Arm cycling in Facioscapulohumeral Dystrophy (FSHD) patients: results of pilot
study
R
Kulshrestha1, N. Emery1,
Marco Arkesteijn,2 T. Willis1
1Robert Jones and Agnes Hunt Orthopaedic Hospital (RJAH) Foundation
Trust
2Sport and Exercise Research Scientist, Aberystwyth
University
Email:
richakulshrestha@nhs.net
Background: FSHD sufferers live a long
life with disability. Symptoms may develop in early childhood and weakness usually
noticeable in the teenage years with 95% of affected individuals manifesting disease by
age 20 years. The disorder impacts on the upper extremity and torso, impacting
negatively on the muscle mass, shoulder mobility and functional tasks. Consequently
chronic weakness of the shoulder and upper limbs negatively impacts independence of
sufferers, prospects of employment and staying at work. At present there is no known
cure and knowledge regarding the mechanisms underpinning FSHD is not sufficient to halt
the progression of the disease via pharmacological interventions or gene therapy.
Surgical interventions are used to improve scapular stabilisation but long term effect
on disease progression is limited.
Aim: This study aims to test our
hypothesis that shoulder muscle weakness and reduced range of movements at upper limb
joints would not affect arm cycling ability of FSHD patients.
Methods: patients were recruited from our
clinical group and FSHD registry. They were assessed for muscle strength, contractures
of upper limb, Oxford shoulder score. Subjects performed 2 minute of upper limb cycling
x 5 at self-selected cadence (total 10 minutes work), with 30 seconds of rest between
arm cycling exercise intervals. Rate of perceived exertion (RPE), cadence and video
analysis was performed throughout the exercise period.
Results: 15 patients participated in the
study; age 18-60 years, 9 males and 6 females. Patients had varying degrees of shoulder
involvement with Oxford Shoulder Scores from 4-48. Participants gradually increased the
cadence, maintained or increased resistance. All participants were able to complete the
exercise programme. RPE was increased marginally by the 5th cycle. No
significant problems were reported after the study. Participants were positive about
this as a home exercise programme.
Conclusion: Static arm cycling offers a
potential tool for upper limb rehabilitation for patients with FSHD in home setting but
its effectiveness needs to be proven.
Additional benefits of Ataluren: 2 case reports
R
Kulshrestha,1 C. Sewry,1 N.
Emery,1 T. Willis1
1Robert Jones and Agnes Hunt Orthopaedic Hospital (RJAH) Foundation
Trust
Email:
richakulshrestha@nhs.net
Background: Dystrophinopathy is a rare,
severe muscle disorder, with nonsense mutations found in 13% of cases. Ataluren was
developed to enable ribosomal read through of premature stop codons in nonsense mutation
genetic disorders. Effectiveness of this drug has been assessed by National Institute of
Clinical Excellence in the UK and the drug is available by managed access agreement
(MAA) for patients after 5 years of age.
Methodology: We describe two patients, 7
and 21 years, on Ataluren. One patient had access to treatment by MAA and another older
patient had treatment through Continued access programme from PTC pharmaceuticals. North
star and physiotherapy assessments pre and post treatment were compared.
Results: Patient 1 has significant
learning difficulties and attends special school. He has point mutation in exon 65
(c.9461T>A; p.Leu3154X). On muscle biopsy dystrophin was absent with labelling of rod
domains and only a rare revertant-like fibres are seen with a C terminal antibody.
Pre-treatment timed rise from floor was 34.8 seconds, 10 meters run was 11.2 seconds and
North Star Ambulatory assessment was 7/34. These responded within 5 months of treatment
on Ataluren to timed rise from floor to 11 seconds, 10m run to 8 seconds and North Star
Ambulatory assessment to 15/34. According to assessors this difference was related to
improvement in understanding of instructions.
Patient 2 has been on Ataluren from the age of 10
years, when he was enrolled in the first clinical trials, and subsequently he has
continued on Ataluren post trials (2016). He has a point mutation (exon 59). He has no
learning difficulties and became non-ambulant at 14 years. He currently is not
ventilated with a FVC at 1.62; his cardiac status; LVEF 46%, FS 26%. His last assessment
shows preservation in upper limb strength (PUL 2.0 21/42) and he currently self-propels
on level ground.
Conclusion and significance: Effectiveness
of Ataluren is proved for motor abilities in patients with DMD in research trials mainly
concentrating on motor outcomes of ambulatory children. These two cases support its
benefit outside what has been researched so far.
Long-term efficacy of ataluren for the treatment of nonsense mutation Duchenne
muscular dystrophy: observational data from the STRIDE Registry
Abdallah Delage,1 Filippo Buccella,2
Isabelle Desguerre,3 Francesco Muntoni,4 Andrés
Nascimento,5 Már Tulinius,6 Salvatore Colucci,7 Allan
Kristensen,7 Claudio Santos,7Dinesh Kumar,8 Panayiota Trifillis,7 Olivia
Zhang,7 and Eugenio Mercuri9
1PTC Therapeutics, Zug, Switzerland; 2Parent Project
Onlus, Rome, Italy; 3Necker-Enfants Malades APHP, Paris V Descartes
University, Paris, France; 4University College London Great Ormond Street
Institute of Child Health, London, UK; 5Hospital Sant Joan de Déu,
Universidad de Barcelona, CIBER, ISCIII, Barcelona, Spain; 6Gothenburg
University, Queen Silvia Children’s Hospital, Gothenburg, Sweden; 7PTC
Therapeutics, South Plainfield, New Jersey, USA;8PTC Therapeutics, Limited,
Guildford, UK; 9Department of Pediatric Neurology, Catholic University,
Rome, Italy.
Presenting author email: dkumar@ptcbio.com
Background: Duchenne muscular dystrophy
(DMD) is a fatal, X-linked disease, characterized by progressive muscle weakness.
Approximately 10–15% of cases of DMD are caused by a nonsense mutation (nmDMD) in the
dystrophin gene, which leads to translation of truncated, non-functional dystrophin.
Ataluren is the first approved therapy to target the underlying cause of nmDMD, enabling
formation of full-length, functional dystrophin.
Aim: In this study, long-term
effectiveness of ataluren (40 mg/kg/day) in routine clinical practice was evaluated in
patients with nonsense mutation Duchenne muscular dystrophy (nmDMD) enrolled in the
international STRIDE Registry (N=213).
Methods: We compared data from registry
participants (with =48 weeks between first and last assessment) with data from
participants in Study 020 (NCT01826487) receiving ataluren (N=115) or placebo (N=115)
for 48 weeks. Data were extracted from the registry on 9 July 2018.
Results: Registry participants had a mean
(standard deviation, SD) age of 9.8(3.7) years at first assessment compared with
8.9(1.8) and 9.0(1.7) years in the ataluren and placebo arms of Study 020, respectively.
Mean exposure to ataluren ranged from 71.8 to 121.6 patient-years depending on the
different outcome measures used. Registry participants experienced smaller 48-week mean
(SD) functional declines than patients receiving ataluren or placebo in Study 020,
respectively, in 6-minute walk distance (n=66, -35.0[69.7], vs n=109, -42.2[84.9], and
n=109, -57.6[98.8] meters), and time to walk/run 10 meters (n=61, 1.6[3.2] vs n=109,
2.3[5.2], and n=110, 3.5[6.4] seconds), stand from supine (n=55, 2.9[5.0] vs n=101,
3.8[6.1], and n=96, 3.9[6.9] seconds), climb 4 stairs (n=47, 1.2[2.2] vs n=105,
2.7[5.3], and n=103, 4.5[7.3] seconds) and descend 4 stairs (n=40, 0.5[1.5] vs n=106,
2.2[5.3], and n=100, 3.9[7.9] seconds). Safety outcomes were consistent with the known
safety profile of ataluren.
Conclusions: Although the registry cohort
consisted of older patients compared with those in Study 020, these patients showed
smaller functional declines, suggesting that ataluren may slow disease progression in
patients with nmDMD in routine clinical practice.
Effect of Ataluren on Age at Loss of Ambulation in nonsense mutation Duchenne
Muscular Dystrophy: Observational Data from the STRIDE Registry
Abdallah Delage,1 Filippo Buccella,2
Isabelle Desguerre,3 Francesco Muntoni,4 Andrés
Nascimento,5 Már Tulinius,6 Salvatore Colucci,7 Allan
Kristensen,7 Claudio Santos,7Dinesh Kumar,8 Panayiota Trifillis,7 Olivia
Zhang,7, and Eugenio Mercuri9
1PTC Therapeutics, Zug, Switzerland; 2Parent Project
Onlus, Rome, Italy; 3Necker-Enfants Malades APHP, Paris V Descartes
University, Paris, France; 4University College London Great Ormond Street
Institute of Child Health, London, UK; 5Hospital Sant Joan de Déu,
Universidad de Barcelona, CIBER, ISCIII, Barcelona, Spain; 6Gothenburg
University, Queen Silvia Children’s Hospital, Gothenburg, Sweden; 7PTC
Therapeutics, South Plainfield, New Jersey, USA; 8PTC Therapeutics,
Limited, Guildford, UK; 9Department of Pediatric Neurology, Catholic
University, Rome, Italy.
Background: Duchenne muscular dystrophy
(DMD) is a fatal, X-linked disease, characterized by progressive muscle weakness. Loss
of ambulation is a major milestone in disease progression. Approximately 10–15% of cases
of DMD are caused by a nonsense mutation (nmDMD) in the dystrophin gene, which leads to
translation of truncated, non-functional dystrophin. Ataluren is the first approved
therapy to target the underlying cause of nmDMD, enabling formation of full-length,
functional dystrophin.
Aim: In this study, age at loss of
ambulation was evaluated in patients with nmDMD taking ataluren for at least 12 months
while enrolled in the international, multi-center STRIDE (Strategic Targeting of
Registries and International Datasets of Excellence) Registry (N=207).
Methods: In this study, age at loss of
ambulation was evaluated in patients with nmDMD taking ataluren for at least 12 months
while enrolled in the international, multi-center STRIDE (Strategic Targeting of
Registries and International Datasets of Excellence) Registry (N=207). Data were
extracted from the registry on 9 July 2018. Kaplan–Meier analyses were used to
investigate age at loss of ambulation.
Results: Mean (SD) age of registry
participants starting ataluren treatment was 9.8 (3.7) and 89.2% were being treated with
corticosteroids in addition to ataluren. At the date of data extraction, registry
participants had a mean (SD) age of 11.6 (3.6) years. Mean (SD) exposure to ataluren
within the registry was 372.6 (211.6) patient-years and 44.6% of patients had been on
ataluren for more than 720 days. Mean (standard error) age at loss of ambulation in
registry participants was 15.5 (0.3) years, and 50% of patients were still ambulatory at
the age of 16.5 years. Safety outcomes were consistent with the known safety profile of
ataluren.
Conclusions: Ataluren may delay loss of
ambulation in patients with nmDMD.
STRIDE: A Patient Registry Study Examining the Use of Ataluren (Translarna™) in
Patients with Nonsense Mutation Muscular Dystrophy (nmDMD)
Francesco Muntoni1,Isabelle Desguerre2,
Michela Guglieri3, Eugenio Mercuri4, Andres Nascimento
Osorio5, Janbernd Kirschner6, Mar Tulinius7 and
Filippo Buccella8, Abdallah Delage9, Gary Elfring10,
Christian Werner9, Dinesh Kumar11,Traci
Schilling10, Panayiota Trifillis10
1University College London Institute of Child Health Great Ormond
Street Hospital 30 Guildford Street Hospital Dubowitz Neuromuscular Unit 1st Floor
London, WC1N 1EH United Kingdom; 2 Hôpital Necker - Enfants Malades, Paris,
France;3The Newcastle upon Tyne Hospitals, NHS Foundation Trust Clinical
Research Facility Level 6, Leazes Wing, Royal Victoria Infirmary Newcastle upon Tyne,
NE1 4LP United Kingdom;
4U.O.Complessa di Neuropsichiatria Infantile Policlinico A.
Gemelli-Università Cattolica Sacro Cuore Largo Gemelli 8 Rome, 00168 Italy;
5Hospital Sant Joan de Deu Unidad de Patología Neuromuscular-Servicio de
Neurología – Universidad de Barcelona- CIBERER. Passeig Sant Joan de Déu, 2,
Barcelona, 08950 Spain. 6Department of Neuropediatrics and Muscle
Disorders, Medical Center – University of Freiburg, Faculty of Medicine, Mathildenstr.
1, 79106 Freiburg, Germany;7Queen Silvia Childrens Hospital Ostra Sjukhuset
Rondvägen 15 Goteborg, SE-416 85 Sweden;8Parent Project Onlus, Rome,
Italy;9PTC Therapeutics Switzerland GmbH, Zug, Switzerland;
10PTC Therapeutics Inc., South Plainfield, NJ, USA; 11PTC
Therapeutics, Limited, Guildford, UK
Presenting author email: dkumar@ptcbio.com
Background: Ataluren (Translarna™) is the
first-approved treatment for nonsense mutation Duchenne muscular dystrophy (nmDMD) in
the European Union, in ambulatory patients aged 5 years and older.
Aim: The STRIDE Registry is an ongoing,
multicenter, observational study aimed at building a patient data repository to provide
real world experience regarding the treatment patterns for ataluren in routine clinical
practice.
Methods: An enrollment of approximately
200 patients was targeted, based in part on the size of the nmDMD population in the EU.
Patients are being followed for at least 5 years from the date of a patient’s
enrollment, or until withdrawal of patient consent, or death, whichever occurs first.
The study is being conducted in the countries in which the drug is commercially
available.
Results: Baseline data (as of 31.01.2018)
from a cohort of 154 patients (98.1% males, n=151) with nmDMD from sites across 12
counties were analyzed in terms of demography and clinical manifestations of nmDMD. In
this cohort, the median age was 10.2 (5.0, 45.4) years; nmDMD was most frequently
diagnosed between the ages of 5-10 years (n=74, 48.1%), and the median age at genetic
confirmation was 5.5 years (0.02, 39.1). The median age of symptomatic detection was 2.5
years (0, 30) with a mean time (SD) between first clinical/biochemical symptoms and
nmDMD diagnosis confirmation (i.e., identification of dystrophin gene mutation) is 3.0
years (2.5). The exclusion of female carriers from the analysis had minimal impact on
median age (10.1 years; min, max: 5, 22.8) and median age at genetic confirmation (5.4
years; min, max: 0.9, 15.6). Additional descriptive statistics from demographic data are
forthcoming.
Conclusions: These data suggest a need for
increased awareness of nmDMD symptoms in order to reduce delays in diagnosis; the later
median age at diagnosis observed here relative to recent results from the global DMD
population (3.4 - 4.3 yrs) suggests that some countries may still face challenges with
genetic testing for nmDMD.
Caveolar dysfunction as a new player in cardiac disease in Duchenne Muscular
Dystrophy
Elena Marrosu, Hong Wang, Eric Johnson,
Charlotte Scott, Kwan Hau, Liwen Zhang, Nalinda Wasala, Dongsheng Duan, Federica
Montanaro
Background: Cardiac failure is a major
contributor to mortality in Duchenne muscular dystrophy (DMD). Current cardiac
management relies on cardio-protective drugs that delay cardiac failure, but do not
prevent it. In cardiac and skeletal muscles, dystrophin is an essential scaffold for a
large glycoprotein complex with structural and signalling roles. Our previous research
shows that dystrophin assembles different glycoprotein complexes in heart and skeletal
muscle, suggesting distinct muscle-specific functions. Consistent with this, recent
studies suggest that the C-terminus of dystrophin and a portion of its central rod
domain are uniquely important for cardiac function. ΔR4-R23/ΔCT micro-dystrophin is a
leading gene therapy construct that lacks the two putative cardio-protective domains and
only partially protects from cardiac disease in the mdx mouse model of DMD. The
molecular basis for the incomplete cardiac rescue provided by ΔR4-R23/ΔCT
micro-dystrophin is currently unknown. This knowledge is important to guide the
optimisation of micro-dystrophin gene therapy constructs to fully prevent development of
cardiac disease.
Aim: To compare the protein complexes
assembled by dystrophin and ΔR4-R23/ΔCT micro-dystrophin in the heart in order to
identify differences in composition that may underlie the incomplete cardiac rescue
offered by ΔR4-R23/ΔCT micro-dystrophin.
Materials: Wild-type, mdx
and mdx mice that express ΔR4-R23/ΔCT micro-dystrophin were used in
these studies. Proteomic analysis was performed on isolated dystrophin and ΔR4-R23/ΔCT
micro-dystrophin protein complexes to compare their protein composition. Western blot
analyses were performed to validate proteomic results and evaluate protein expression
levels in total protein extracts. Heart tissue sections were immunolabelled to compare
protein localisation. Electron microscopy experiments were performed to visualise
ultrastructural differences between genotypes.
Results: We report here a new
cardiac-specific interaction between dystrophin and the cavin protein complex that is
not rescued by ΔR4-R23/ΔCT micro-dystrophin. Cavins play important roles in regulating
the shape, function and dynamics of caveolae (specialised membrane domains implicated in
cardiac contraction and disease). We discovered that the localisation of cavin-1,
cavin-2 and cavin-4 is disrupted in the mdx heart and is not rescued by ΔR4-R23/ΔCT
micro-dystrophin. A screen of different micro- and mini-dystrophins for their ability to
interact with cavin proteins is ongoing.
Conclusion: Our results identify caveolar
dysfunction as a new potential player in cardiac disease in DMD. These findings offer
new insights into the cardiac interactions of dystrophin and are highly relevant to the
optimisation of gene therapy approaches with enhanced protection from cardiac
disease.
Functional-capacity Outcome Measures for Myotonic Dystrophy (OMMYD)
1Welcome Trust Mitochondrial Research Centre, Institute of
Neurosciences, Newcastle University, UK. 2John Walton Muscular Dystrophy
Research Centre, Institute of Genetic Medicine, Newcastle University, UK.
3National Hospital for Neurology and Neurosurgery, University College
Hospitals, London, UK. 4Department of Neurobiology, Care Sciences and
Society, Division of Physiotherapy, Karolinska Institutet, Stockholm, Sweden.
5Center of Research in Myology Sorbonne Université, Institut de Myologie,
Paris, France 6Functional Area Occupational Therapy & Physiotherapy,
Allied Health Professionals Function, Karolinska University Hospital, Stockholm,
Sweden. 7Department of Neuropediatrics and Muscle Disorders, Medical
Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
8Centro Nacional de Análisis Genómico (CNAG-CRG), Center for Genomic
Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.
9Research Institute, The Children’s Hospital of Eastern Ontario, and at
the Division of Neurology, Department of Medicine, Ottawa University, Canada.
Cecilia.jimenez-moreno@ncl.ac.uk
Background: Myotonic Dystrophy type 1
(DM1) is a multisystem disease with high heterogeneity representing an obstacle when
defining outcome measures that can be reliable yet still valid for different phenotypes.
The Outcome Measures for Myotonic Dystrophy (OMMYD) Consortium has proposed a set of
Functional-capacity Outcome Measures highlighted for consideration in clinical trials
for DM1.
Aims: For this study, a cohort of 213
patients enrolled in the natural history study of PhenoDM1 (Myotonic Dystrophy Type 1
Deep Phenotyping to Improve Delivery of Personalized Medicine and Assist in the
Planning, Design and Recruitment of Clinical Trials) was analysed. We aimed to assess:
[1] intra-session (i.e. trial to trial) reliability; and, [2] construct validity (i.e.
associations with disease parameters) of the OMMYD-defined functional-capacity outcome
measures.
Methods: The study visit assessments
included: [1] Standard medical history and demographical data; [2] Strength assessments;
[3] OMMYD-defined functional-capacity outcome measures (six-minute walk test, timed 10 m
walk test, timed 10 m walk/run test, 30 seconds sit and stand test, and 9-hole peg
test); and [4] disease-specific patient-reported outcomes that assess functional
performance (including DM1-ActivC and MDHI subscales).
Results: By comparing selected OMMYD
functional-capacity outcome measures to clinical manifestations of the disease and to
the reported burden of illness, we assessed their sensitiveness to discriminate
different disease phenotypes. Their association to muscle strength and with
participants’ perceived functional-performance provides an insight of these outcomes
construct validity. Differences between sexes were attributed to height differences. All
tests showed a significant difference between scores from the first trial to the second
trial. Thirty seconds sit and stand test showed to discriminate for disease severity and
test repetition assess different disease parameters apart from muscle strength or
capability to walk.
Conclusion: This study established the
feasibility and reliability of these tests in large-scale studies. This data will serve
as reference values from a representative sample of DM1 adults for future clinical
trials. These OMMYD tests can be used as a battery of outcomes or independently as some
have shown overlapping on psychometric features.
A Human iPS Cell-Derived Artificial Skeletal Muscle for DMD Therapy
Development
3The Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute
of Child Health & Great Ormond Street Hospital for Children, London, UK
*Equally contributing
Presenting author:
moustafa.khedr.16@ucl.ac.uk
Corresponding author:
f.s.tedesco@ucl.ac.uk
Background: Duchenne muscular dystrophy
(DMD) is a genetic disorder characterised by progressive muscle wasting and severe
weakness. It occurs in 1 in 5000 boys and is caused by the disruption of dystrophin, a
protein that maintains the integrity of muscle cells and protects the cell membrane from
rupturing during contraction and relaxation cycles. Dystrophin is the
largest human gene, having different isoforms, and patients have various mutations
leading to expression of dysfunctional protein forms. Exon-skipping (using anti-sense
oligonucleotides), gene editing (using CRISPR/Cas9) and read-through technologies are
promising therapies that produce a truncated yet functional protein. However, the
development and optimisation of these therapies and determining their relative
efficiencies, is limited by the lack of an overarching (temporally, economically and
ethically viable) platform.
Aims and methods: We utilise CRISPR/Cas9
gene editing technology to develop dystrophin-detectable human induced pluripotent stem
cells (iPSCs), by inserting a versatile multiple-reporter cassette, that can track
different dystrophin forms’ expression levels temporally and spatially, in real time and
in fixed cells. Following that, iPSCs will be used in tissue engineering an in vitro
human skeletal mini-muscle “organoid”, maintaining the dystrophin-tracking
properties.
Finally, once established, the developed modelling
parameters and outcome measures will be used to assess the efficacy of different
therapies on patient-derived DMD dystrophin-tracking artificial muscles to determine
their ability to restore dystrophin function and ameliorate the dystrophic phenotype in
each line.
Results and conclusion: The designed
cassette has been developed and tested by insertion into skeletal muscle progenitor
cells that have been differentiated and one of the cassette functions has been
confirmed. Multiple lines are currently being developed and potential outcome measures
are being researched.
This platform could accelerate research for therapies,
offer more tailored and potentially personalised treatment, reduce need for muscle
biopsies and provide a reliable method to quantify dystrophin. This reporting system can
also be applicable to other neuromuscular conditions.
Comparison of home-based versus hospital-based spirometry measurements in Duchenne
muscular dystrophy
Gunnar Buyse1 for the DELOS study group, Christian
Rummey2, Thomas Meier2, Mika Leinonen2, Shabir
Hasham2, Catherine Lawrence3Lisa Moore Ramdin3 Thomas
Voit4 and Oscar H Mayer5
1University Hospitals Leuven, Leuven, Belgium 2Santhera
Pharmaceuticals, Pratteln, Switzerland; 3Santhera UK 4UCL Great
Ormond Street Institute of Child Health, London, UK; 5Children’s Hospital
of Philadelphia (CHOP), USA
Background: Respiratory function decline
in Duchenne muscular dystrophy (DMD) is caused by progressive weakening of respiratory
muscles. Standard of care guidelines recommend routine respiratory function monitoring
to guide patient management. We compared respiratory function measurements obtained
using a hand-held device allowing frequent home-based measurements to hospital-based
spirometry measures in patients with DMD taking part in the DELOS trial.
Methods: Respiratory function data were
collected from 64 DMD patients aged 10-18 years. All patients were required to have
established respiratory function decline (peak expiratory flow <80%p) at baseline.
Patients were treated with idebenone (900 mg/day) or placebo. Spirometry was conducted
during hospital visits at baseline and 3 month intervals over 52 weeks. Patients also
measured peak expiratory flow (PEF) weekly at home using the ASMA-1 device
(Vitalograph). Data were analyzed using a mixed model for repeated measures method.
Results: Patients enrolled in the DELOS
study were in an advanced stage of disease, with a mean age of 14.3 years. A majority
(92%) were non-ambulatory and showed signs of significant upper limb weakness at
baseline (59% had a Brooke’s score of 5 or above). The changes in PEF measured at home
(expressed as percent of predicted, PEF%p) compared well with those from hospital-based
measurements, showing a 5.6% (p = 0.002) treatment difference in favor
of idebenone across all weekly visits, compared to a 6.27% difference at last visit
using standard hospital spirometry (p = 0.031). Similarly to the
hospital based data, the weekly ASMA-1 data indicated a stabilization of PEF%p in the
idebenone group versus a consistent decline in the placebo group.
Conclusion: Home-based recording of
respiratory function measures is reliable in pediatric and adolescent patients with DMD,
and may prove useful when used in combination with hospital-based monitoring. These data
further support the efficacy of idebenone in slowing respiratory function decline in
DMD.
Assessing idebenone’s impact on respiratory function in Duchenne muscular
dystrophy: Meta-analysis of two clinical trials
Mika Leinonen1, Christian Rummey1, Shabir
Hasham1, Thomas Voit2, Catherine Lawrence3Lisa Moore Ramdin3 Oscar H Mayer4, and
Gunnar Buyse5 for the DELOS study group
1Santhera Pharmaceuticals, Pratteln, Switzerland; 2UCL
Great Ormond Street Institute of Child Health, London, UK; 3 Santhera UK;
4Children’s Hospital of Philadelphia (CHOP), USA; 5 University
Hospitals Leuven, Leuven, Belgium
Background: Patients with Duchenne
muscular dystrophy (DMD) experience respiratory function decline that accelerates after
they become non-ambulatory, leading to significant disease burden and, in many cases,
death. The ability of idebenone to slow respiratory function decline in patients with
DMD has been investigated in two randomized, placebo-controlled trials (the phase 2
DELPHI trial and phase 3 DELOS trial). DELPHI included patients (aged 8 to 16 years)
irrespective of baseline respiratory function status, while DELOS required patients
(aged 10 to 18 years) to be in the respiratory decline phase. DELPHI allowed the
randomization of both glucocorticoid users and non-users, whilst DELOS included only
glucocorticoid non-users.
Methods: Source data from both trials were
pooled and analyzed for treatment effects on peak expiratory flow and forced vital
capacity, expressed as a percentage of predicted (PEF%p and FVC%p). Change in PEF%p and
FVC%p from baseline to weeks 26 and 52 (end of study) were analyzed using a mixed model
for repeated measures, with study as a random effect.
Results: In total, 76 patients (DELPHI:
12; DELOS: 64) were in the respiratory function decline phase (PEF%p < 80%), of which
72 were not using glucocorticoids. Across all patients, the difference between the
idebenone and placebo groups in PEF%p from baseline to week 52 was 8.0%p (p = 0.003).
When omitting glucocorticoid users, this difference was 7.7%p (p = 0.005). For FVC%p,
the treatment difference was 3.6%p (p = 0.046) in all patients, with a comparable
difference of 3.5%p (p = 0.051) when omitting glucocorticoid users.
Conclusion: Results from a meta-analysis
with data from two randomized, placebo-controlled trials (DELPHI and DELOS) demonstrate
that idebenone slows respiratory function decline in patients with DMD. Additionally,
the magnitude of treatment difference observed was unaffected by concomitant
glucocorticoid use.
Regulation of the dystrophin-associated glycoprotein complex composition by the
metabolic properties of muscle fibres
1college of Medicine, Wasit University, Kut, Iraq; 2UCL
Great Ormond Street Institute of Child Health, Developmental Neurosciences Programme,
London, UK; 3School of Biological Sciences, Reading University, Reading,
UK.
Background: Skeletal muscle is a highly
compliant tissue that undergoes both qualitative and quantitative remodeling in response
to mechanical, electrical and chemical stimuli. The dystrophin-glycoprotein complex
(DGC) links the muscle cytoskeleton to the extracellular matrix and is responsible for
force transduction and for protecting the muscle fibre from contraction induced damage
(Kjaer, 2004). Mutations in any of the proteins constituting the DGC are associated with
muscular dystrophies and congenital myopathies (Wicklund and Kissel, 2014). Expression
of DGC components have been shown to be altered in many myopathies. In contrast we have
very little evidence of whether adaptive changes in muscle impact on DGC expression.
Aims: To investigate the relationship
between muscle fibre phenotype and the DGC.
Methods: Immunohistochemistry, Western
blot, Quantitative PCR, Semi-quantitative measurement of Collagen and Dystrophin
Glycoprotein Complex (DGC) protein expression by immunofluorescence, Sarcolemma
thickness measurement, and Imaging and analysis
Results: We show that the DGC composition
is influenced by the phenotype of the muscle fibre.
Conclusion: Our work shows that the
metabolic property of a muscle fibre is a key determinant in regulating the expression
of DGC proteins at the sarcolemma.
Patient Reported Outcome Measures in Dysferlinopathy – How suitable are
they?
Anna G. Mayhew
PT1, Meredith James1, Helen
Sutherland1, Marni Jacobs2,3, Simone Spuler4, John W.
Day5, Kristi J. Jones6, Diana X. Bharucha-Goebel7,
Emmanuelle Salort-Campana8, Alan Pestronk9, Maggie C.
Walter10, Carmen Paradas11, Tanya Stojkovic12, Madoka
Mori-Yoshimura13, Elena Bravver14, Jordi Diaz
Manera15, Elena Pegoraro 16, Jerry R. Mendell 17, The
Jain COS Consortium, Kate Bushby1, Volker Straub1
1John Walton Muscular Dystrophy Research Centre, Newcastle University
and Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK,
2Center for Translational Science, Division of Biostatistics and Study
Methodology, Children’s National Health System, Washington, DC, USA,
3Pediatrics, Epidemiology and Biostatistics, George Washington University,
Washington, DC, USA, 4Charite Muscle Research Unit, Experimental and
Clinical Research Center, a joint cooperation of the Charité Medical Faculty and the
Max Delbrück Center for Molecular Medicine, Berlin, Germany, 5Department of
Neurology and Neurological Sciences, Stanford University School of Medicine; Stanford,
CA, USA, 6Institute for Neuroscience and Muscle Research, Children’s
Hospital at Westmead, Sydney, Australia, 7Department of Neurology
Children’s National Health System, Washington, DC, USA, 8Neuromuscular and
ALS center, La Timone Hospital, Aix-Marseille Université, Marseille, France,
9Department of Neurology, Washington University School of Medicine, St.
Louis, MO, USA, 10Friedrich-Baur-Institute, Dept. of Neurology,
Ludwig-Maximilians-University of Munich, Germany, 11Neuromuscular Unit,
Department of Neurology, Hospital U. Virgen del Rocío/Instituto de Biomedicina de
Sevilla, Sevilla, Spain, 12Institut de Myologie, AP-HP, G.H.
Pitié-Salpêtrière 47-83, Boulevard de l’Hôpital, Paris, France,
13Department of Neurology, National Center Hospital, National Center of
Neurology and Psychiatry Tokyo, Japan, 14Carolinas Healthcare System
Neurosciences Institute, Charlotte, NC, USA, 15Neuromuscular Disorders
Unit, Neurology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain,
16Department of Neuroscience, University of Padova, Italy,
17Nationwide Children’s Hospital, Columbus, OH, USA
Background: The COS is an international
natural history study of patients with dysferlinopathy. Dysferlinopathy presents with
heterogeneity in muscle weakness, including both distal and proximal phenotypes which
poses significant challenges for developing appropriate clinical trial outcome measures
as well as significant functional and quality of life challenges for patients. 204
patients, at 15 sites in 8 countries were evaluated using four Patient Reported Outcome
Measures (PROMs)– Individualised Quality of Life Measure for Neuromuscular Disease,
ACTIVLIM, International Physical Activity Questionnaire and for non-ambulant patients
the Egan Klassifikation Scale (EK)
Aims: To review the suitability of these
PROMs in this cohort over a period of three years. To summarise change scores over this
time for the four PROMs.
Methods: Patients completed the InQOL,
IPAQ, ACTIVLIM at annual visits over three years. The EK was completed only if the
patient was non-ambulant. INQOL is a quality of life measure that aims to capture the
impact of key muscle disease symptoms (weakness, fatigue, pain, and locking), the impact
on particular areas of life including activities, independence, relationships, and body
image. Participants respond primarily using a 7-point Likert scale and also with some
yes/no responses. The IPAQ assesses physical activity using a set of seven questions and
hours of activity at different levels of intensity. The ACTIVLIM is a Rasch built
measure of activity limitation and using a three-point grading system of impossible,
difficult and easy and the EK was originally designed for Duchenne muscular dystrophy
but assessing key “own functioning” in non-ambulant patients.
Data were analysed using descriptive statistics for
each visit and over the period of the study and for rating scales (ACTIVLIM, EK, INQOL)
modern psychometric analysis using Rasch analysis (RUMM2030 software) was also conducted
to assess item fit, person fit, targeting, dependency and reliability
Results: Not all of the measures appeared
to be suitable measurement tools in this disease specific cohort, this was particularly
true for the INQOL. The ACTIVLIM appeared more robust. Change scores were noted for
several of the measures over time.
IPAQ
EK
ACTIVLIM
INQOL
Baseline
204
48
200
204
12 months
189
50
189
192
24 months
189
60
185
184
36 months
166
55
159
113
Discussion: Further work is required to
adapt the current PROM’s or alternative measures need to be selected.
Anna.Mayhew@newcastle.ac.uk
A comparison of the utility between three muscle strength assessment methods in
Dysferlinopathy
Lindsay Alfano1, Natalie
Miller1, Meredith K. James2; Anna G Mayhew2 ,Robert
Muni-Lofra2, Marni Jacobs3,, Aurelie Canal4, Tina
Duong5, Richard Gee5, Megan Harman6, Scott
Holsten7, Elke Maron8, Bosco Mendez9, Irene Pedrosa
Belmonte10, Chikako Sakamoto11, Claudio Semplicini12,
Catherine Seiner13, Simone Thiele14, Bruno
Vandervelde15, The Jain COS Consortium16, Kate
Bushby2, Volker Straub2, Linda Lowes1.
1Nationwide Children’s Hospital, Columbus, USA; 2Newcastle
University, John Walton Muscular Dystrophy Research Centre, Newcastle Hospitals NHS
Foundation Trust, Newcastle upon Tyne, UK; 3Children’s National Health
System, Washington, DC, USA; 4 Institut de Myologie, Paris, France;
5Stanford University School of Medicine, Stanford, USA;
6Children’s Hospital at Westmead, Sydney, Australia; 7Carolinas
Healthcare System Neurosciences Institute, Charlotte, USA; 8Elan
Physiotherapy, Berlin, Germany; 9Hospital U. Virgen del Rocío/Instituto de
Biomedicina de Sevilla, Department of Rehabilitation, Sevilla, Spain;
10Hospital de la Santa Creu i Sant Pau, Department of Rehabiliation,
Barcelona, Spain; 11National Center of Neurology and Psychiatry Tokyo,
Japan; 12University of Padova, Department of Neuroscience, Padova, Italy;
13Washington University School of Medicine, St Louis, USA;
14Friedrich-Baur-Institute, Munich, Germany; 15Neuromuscular and
ALS center, Marseille, France; 16Jain Foundation, Seattle, USA.
Background: The COS is an international
natural history study of patients with dysferlinopathy. Dysferlinopathy presents with
heterogeneity in muscle weakness, including both distal and proximal phenotypes which
poses significant challenges for developing appropriate clinical trial outcome measures.
203 patients, at 15 sites in 8 countries were evaluated using three methods to measure
strength on a variety of upper and lower limb muscle groups
Aims: This study compared results between
3 methods of muscle strength testing used in the Jain Clinical Outcome Study (COS) for
Dysferlinopathy performed at baseline and year 1 visits.
Methods: Manual muscle testing (MMT) uses
the Medical Research Council scoring system to subjectively grade the amount of
resistance the subject can withstand as the evaluator pushes against the extremity. Hand
held dynamometry (HHD) uses a force transducer in a small device held against the
extremity by the clinical evaluator. Quantitative muscle testing (QMT) uses a force
transducer secured to a wall mounted fixed frame. All sites completed MMT and HHD, while
only 7 of the 15 sites had the QMT equipment available. Subjects also completed
functional tests including as appropriate for their ambulatory status including adapted
North Star Ambulatory Assessment for Dysferlinopathy, 10 metre walk test (10m) and 6
minute walk test (6MWT).
Results: The 3 strength testing methods
were highly & significantly correlated with each other (r = 0.8, P<0.001). All 3
strength testing methods were highly & significantly correlated with functional
tasks (r = 0.7 – 0.8, P<0.001). Age was significantly correlated with performance of
all strength & functional tasks.HHD was the only strength testing method that
demonstrated significant change in summed strength over one year. There was a trend
toward decreased strength in Year 1 measured with QMA, but this was not significant. HHD
was the only modality capturing decline consistently across muscle groups – thus
demonstrating significant change across 1 year in total strength
Conclusion: Strength measurements are
highly correlated with performance of functional tasks in Dysferlinopathy. Hand held
dynamometry is sufficient for testing strength in key muscle groups & is sensitive
to disease progression in 1 year
lindsay.alfano@nationwidechildrens.org
Clinical Outcome Study for Dysferlinopathy: Three years of natural history data for
clinical trial readiness
Meredith K. James P1, Anna G.
Mayhew1 ,Robert Muni-Lofra1, Marni Jacobs2,, Lindsay
Alfano3, Tina Duong4 Richard Gee4, Megan
Harman5, Bruno Vandervelde6, Elke Maron7 Catherine
Seiner8, Bosco Mendez9, Aurelie Canal10, Scott
Holsten11, Simone Thiele12, Chikako Sakamoto13, ,
Irene Pedrosa Belmonte14, Claudio Semplicini15, Linda
Lowes3 The Jain COS Consortium, Kate Bushby1, Volker
Straub1.
1Newcastle University, John Walton Muscular Dystrophy Research
Centre, Newcastle University and Newcastle Hospitals NHS Foundation Trust, Newcastle
upon Tyne, UK; 2 Children’s National Health System, Washington, DC, USA;
3 Nationwide Children’s Hospital, Columbus, USA; 4Stanford
University, School of Medicine, Stanford, USA; 5Children’s Hospital at
Westmead, Sydney, Australia, 6 Neuromuscular and ALS center, Marseille,
France, 7 Elan Physiotherapy, Berlin, Germany, 8Washington
University School of Medicine, St. Louis, USA; 9Hospital U. Virgen del
Rocío/Instituto de Biomedicina de Sevilla, Department of Rehabilitation, Sevilla,
Spain, 10Institut de Myologie, Paris, France; 11Carolinas
Healthcare System Neurosciences Institute, Charlotte, USA; 12
Friedrich-Baur-Institute, Munich, Germany; 13National Center of Neurology
and Psychiatry, Tokyo, Japan;14Hospital de la Santa Creu i Sant Pau,
Department of Rehabilitation, Barcelona, Spain; 15University of Padova,
Department of Neuroscience, Padova, Italy;
Background: The Jain Clinical Outcome
Study (COS) is an international study evaluating patients with genetically confirmed
dysferlinopathy. 203 patients were recruited across 15 sites and in 8 countries.
Dysferlinopathy clinically presents with a spectrum of muscle weakness including both
distal and proximal phenotypes which presents significant challenges for developing
appropriate clinical trial outcome measures.
Aims: COS aims to develop understanding of
disease progression and identify the most relevant outcome measures for this cohort to
facilitate trial readiness in dysferlinopathy. Here we report three years of
longitudinal functional data.
Methods: Patients attended six visits over
three years. Physiotherapy medical and MRI assessments were conducted. Physiotherapy
assessments included manual muscle testing (MMT) hand held dynamometry (HHD) and
functional scales (North Star Assessment for Dysferlinopathy and Performance of Upper
limb), as well as timed tests (rise from floor, 10 metre walk / run, four stair climb
and descend; Timed Up and Go, TUG; Six Minute Walk Distance, 6MWD) and respiratory
function testing (forced vital capacity and mouth inspiratory pressure).
Results: Significant change across all
three years in respiratory function, muscle strength testing, timed function tests, 6MWT
and ACTIVLIM. All timed tests except rise from floor showed significant and consistent
decline across all three years. NSAD, 10m and TUG demonstrated the most significant
change. These results will be examined for differences between gender and ethnicity.
Conclusion: Progression in dysferlinopathy
can be demonstrated using functional outcomes and dynamometry as measured by
Physiotherapists over three years. Results support future study design and help power
future clinical trials.
meredith.james@ncl.ac.uk
A Clinical Outcome Study for Dysferlinopathy: biobanking samples collected through
a collaborative international multisite study
Heather Hilsden1, Dan Cox1,, John W.
Day2; Kristi Jones3; Diana Bharucha-Goebel4; Alan
Pestronk5; Maggie Walter6; Carmen Paradas7; Tanya
Stojkovic8; Elena Bravver9; Jordi Diaz-Manera10;
Elena Pegoraro11; Jerry Mendell12; Jain Consortium; Kate
Bushby1; Volker Straub1
1John Walton Muscular Dystrophy Research Centre, Newcastle University
and Newcastle Hopsitals NHS Foundation Trust, Newcastle upon Tyne, UK; 2
Department of Neurology and Neurological Sciences, Stanford University School of
Medicine; Stanford, CA, USA 3Institute for Neuroscience and Muscle
Research, Children’s Hospital at Westmead, University of Sydney, Australia;
4 Department of Neurology Children’s National Health System, Washington,
DC, USA; 5 Department of Neurology, Washington University School of
Medicine, St. Louis, MO, USA; 6Friedrich-Baur-Institute, Dept. of
Neurology, Ludwig-Maximilians-University of Munich, Germany; 7Neuromuscular
Unit, Department of Neurology, Hospital U. Virgen del Rocío/Instituto de Biomedicina
de Sevilla, Sevilla, Spain;
8Institut de Myologie, AP-HP, G.H. Pitié-Salpêtrière 47-83, Boulevard
de l’Hôpital, Paris, France ; 9Carolinas Healthcare System Neurosciences Institute,
Charlotte, NC, USA
10Neuromuscular Disorders Unit, Neurology Department, Hospital de la
Santa Creu i Sant Pau, Barcelona, Spain; 11 Department of Neuroscience,
University of Padova, Italy
12Nationwide Children’s Hospital, Columbus, OH, USA
Background: The Jain Clinical Outcome
Study (COS) is an international study evaluating patients with genetically confirmed
dysferlinopathy. 203 patients were recruited across 15 sites and in 8 countries. This
three year study was extended for a two year time period until all sites completed year
3 visits. All sites completed year 3 visist, while some also completed year 4 and year 5
visits.
Further to the Clinical Outcome Study data collection
allowing the characterization of the disease (results reported elsewhere), patients also
had the option to donate biobank samples to the MRC Biobank for Rare and Neuromuscular
Diseases curated at Newcastle.
Aims: COS aims to develop understanding of
disease progression and support future scientific research
Methods: Standardised Protocols for
collection, processing and shipment were provided to each site. Blood for DNA and RNA
was collected at one time point only. Plasma and Serum were collected annually. Patients
could opt to provide a skin sample to generate fibroblast cell lines. Urine was
introduced from year 4 at Newcastle only. Samples have been shipped to the Newcastle MRC
Centre Biobank for Rare and Neuromuscular diseases.
Results: 12 sites of 15 sites participated
in the biobanking element.
While 97% patients opted to donate blood samples, only
43% patients opted to donate skin samples for fibroblasts.
Samples currently at Newcastle are as follows: Blood
for RNA n142; DNA n155; Baseline Plasma n147; Year 1 Plasma n143; Year 2 Plasma n134;
Year 3 Plasma n70 (pending final shipments); Baseline Serum n147; Year 1 Serum n143;
Year 2 Serum n135; Year 3 Serum n70 (pending final shipments); Limited year 4 and year 5
samples are also available.
Conclusion: This is a valuable resource
for scientists worldwide to develop the body of knowledge regarding Dysferlinopathies
necessary to support design of future therapeutic targets and clinical trials.
The COS Study has been extended for two further years.
COS2 will also collect urine across all participating sites.
Heather.Hilsden@ncl.ac.uk
Clinical Outcome Study in Dysferlinopathy: Medical comorbidities and polytherapy in
a large population of dysferlinopathy patients
Roberto Fernandez-Torron1,2, Helen
Sutherland1, Jordi Diaz-Manera3; Meredith K
James1; Anna G. Mayhew1; Simone Spuler4; John W.
Day5; Kristi Jones6; Diana Bharucha-Goebel7;
Emmanuelle Salort-Campana8; Alan Pestronk9; Maggie
Walter10; Carmen Paradas11; Tanya Stojkovic12; Madoka
Mori Yoshimura13; Elena Bravver14; Elena Pegoraro15;
Jerry Mendell16; Jain Consortium; Kate Bushby1; Volker
Straub1
1John Walton Muscular Dystrophy Research Centre, Newcastle University
and Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK;
2Neuromuscular Area. Biodonostia Health Research Institute. Neurology
Service. Donostia University Hospital. Donostia-San Sebastian. Spain;
3Neuromuscular Disorders Unit, Neurology Department, Hospital de la Santa
Creu i Sant Pau, Barcelona, Spain; 4Charite Muscle Research Unit,
Experimental and Clinical Research Center, a joint cooperation of the Charité Medical
Faculty and the Max Delbrück Center for Molecular Medicine, Berlin, Germany;
5Department of Neurology and Neurological Sciences, Stanford University
School of Medicine; Stanford, CA, USA; 6Institute for Neuroscience and
Muscle Research, Children’s Hospital at Westmead, University of Sydney, Australia;
7Department of Neurology Children’s National Health System, Washington,
DC, USA; 8Neuromuscular and ALS center, La Timone Hospital, Aix-Marseille
Université, Marseille, France; 9Department of Neurology, Washington University School
of Medicine, St. Louis, MO, USA; 10Friedrich-Baur-Institute, Dept. of
Neurology, Ludwig-Maximilians-University of Munich, Germany;
11Neuromuscular Unit, Department of Neurology, Hospital U. Virgen del
Rocío/Instituto de Biomedicina de Sevilla, Sevilla, Spain; 12Institut de
Myologie, AP-HP, G.H. Pitié-Salpêtrière 47-83, Boulevard de l’Hôpital, Paris, France;
13Department of Neurology, National Center Hospital, National Center of Neurology and
Psychiatry Tokyo, Japan; 14Carolinas Healthcare System Neurosciences
Institute, Charlotte, NC, USA; 15Department of Neuroscience, University of
Padova, Italy; 16Nationwide Children’s Hospital, Columbus, OH, USA
Background: The Jain Clinical Outcome
Study (COS) is the largest international natural history study In patients with
genetically confirmed dysferlinopathy.
203 patients (aged 12-88) have been recruited across 15
sites in 8 countries. All patients were molecularly diagnosed and followed over 3
years.
Aims: To describe the comorbidities in a
large cohort of COS patients at baseline and during follow-up (3 years).
To analyse the drugs and supplements taken before the
screening and during the natural history study.
Methods: Medical history and comorbidities
were collected by specific questionnaires administered by trained nurses or medical
doctors. Medication used were categorised using the British National Formulary.
Results: The most common comorbidities at
baseline were: cardiovascular (33 patients-16%-; 27 of which are hypertension),
endocrine (21 patients-10%-; 10 of which are hypothyroidism) and respiratory (16
patients-7%-; 15 if which are asthma). 5 patients were diagnosed from autoimmune
diseases. During follow-up, hypertension was diagnosed in 6 patients. 141 patients (69%)
were taking drugs or supplements at the beginning of COS (range: 0-10 medications). 65
patients (32%) were on daily vitamins or nutritional supplements, 27 (13%) on
anti-hypertensive, 25 on NSAIDs (12%), 16 (8%) on opioids and 16 (8%) on
antidepressants. During follow-up, anti-hypertensive, antidepressants, opioids and
anti-epileptic prescribed more commonly than other medications. Of note, before
participation in COS and due to misdiagnosis with inflammatory myopathies, 56 patients
(28%) had previously been treated with steroids, 16 (8%) with ivIg and 12 (6%) with
immune suppressants.
Conclusion: Hypertension, asthma and
hypothyroidism are the most common comorbidities in COS. Besides vitamins and
supplements, anti-hypertensive, NSAIDs, opioids and antidepressants are the most
prescribed drugs. 28% of the patients had previously taken steroids due to
misdiagnosis.
We believe this information should be taken into
account when designing interventional clinical trials. This Study has been supported by
the Jain Foundation
Helen.Sutherland@ncl.ac.uk
The UK Myotonic Dystrophy Patient Registry: A Key Tool in the Facilitation of
Clinical Research
Ben
Porter1, Phillip Cammish1,
Chris Turner2, Emma Heslop1, Chiara Marini-Bettolo1
1The John Walton Muscular Dystrophy Research Centre, Institute of
Genetic Medicine, Newcastle University and Newcastle upon Tyne Hospitals NHS
Foundation Trust, Newcastle upon Tyne, UK
2University College Hospital, National Hospital for Neurology and
Neurosurgery, London, UK
Background: The UK Myotonic Dystrophy
Patient Registry (www.dm-registry.org/uk)
is a patient self-enrolling online database collecting clinical and genetic information
about both myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). The
registry was established in May 2012 with support from Muscular Dystrophy UK (MDUK) and
the Myotonic Dystrophy Support Group (MDSG), assisted by the TREAT-NMD Alliance and is
coordinated Newcastle University.
Aims: The registry’s primary aim is to
facilitate and accelerate clinical research in Myotonic Dystrophy type 1 (DM1) and type
2 (DM2). The registry also aims to act as the most comprehensive distributor of
information relating to upcoming academic and non-clinical studies in Myotonic
Dystrophy.
Methods: The registry is used to capture
longitudinal, self-reported data through an online portal available to patients and
clinicians.
Results: Between May 2012 and December
2018, 749 patients have registered. In those who have a clinical diagnosis, 96% have DM1
and 4% have DM2. There is an even distribution of patients registered from both genders
and 62% of patients were between 31 and 60 years old. Patients most frequently reported
their current best motor function as either ambulatory unassisted (61%) or ambulatory
assisted (30%). The most commonly reported symptoms were fatigue (74%) and myotonia
(70%), with 62% of patients reporting both.
Conclusion: The registry has been a
successful tool in assisting in the recruitment to a number of clinical studies since
launching in 2012. It is an example of a novel, online-based, cost-effective, and
patient-driven registry. The continued collection of longitudinal data and ongoing
recruitment of new participants over time will be imperative when assessing the
progression of the condition, and will assist in the design and feasibility of clinical
studies in the future.
Email: ben.porter@ncl.ac.uk
The UK FSHD Patient Registry: A Key Tool in the Facilitation of Clinical
Research
Ben
Porter1, Phillip Cammish1,
Richard Orrell2, Emma Heslop1, Chiara
Marini-Bettolo1
1The John Walton Muscular Dystrophy Research Centre, Institute of
Genetic Medicine, Newcastle University and Newcastle upon Tyne Hospitals NHS
Foundation Trust, Newcastle upon Tyne, UK
2Queen Square Institute of Neurology, University College London,
London, UK
Background: The UK FSHD Patient Registry
(https://www.fshd-registry.org/uk/) is a patient driven,
clinician verified tool designed to support clinical research. The registry shares a
common dataset with many registries worldwide and is able to collect symptomatic
information longitudinally. This online patient driven registry aims to facilitate and
accelerate planning and recruitment of clinical trials. Patient reported outcomes are
entered into a secure online portal that are then combined with clinician verified
genetic details. Core clinical and genetic information has been collected through the
registry with additional data about pain (MPQ-SF) and quality of life (INQoL).
Aims: The registry’s primary aim is to
facilitate and accelerate clinical research in to Facioscapulohumeral Muscular Dystrophy
(FSHD). The registry also aims to act as the most comprehensive distributor of
information relating to upcoming academic and non-clinical studies in FSHD.
Method: The registry is used to capture
longitudinal, self-reported data through an online portal available to patients and
clinicians.
Results: Between May 2012 and December
2018, 916 patients have registered, 87% with a clinical diagnosis of FSHD1. There is an
almost even distribution of patients registered from both genders and 59% of patients
were between 31 and 60 years old. Muscle weakness was widely reported with periscapular
shoulder weakness occurring most frequently (87%) followed by weakness of the hip girdle
(67%) and facial muscles (67%) then foot dorsiflexor (64%). Onset of periscapular
shoulder weakness was reported to occur before the age of 30 in 59% of cases, earlier
than weakness in other areas. The use of a wheelchair was reported to begin most often
after the age of 30 (76%).
Conclusion: A broad spectrum of patients
have registered providing a new insight into the FSHD population in the UK. The Registry
aims to help in the planning and recruitment of research. Sharing a common dataset with
a growing number of FSHD registries around the world will allow the registry to achieve
this locally and internationally. The registry is well placed to inform future clinical
research and help develop of standards of care.
Email: ben.porter@ncl.ac.uk
The Global Registry for COL6-related dystrophies
Dr Alison Blain*, Prof. Volker Straub*
*The John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, International Centre for Life, Newcastle upon Tyne NE1 3BZ.
Alison.Blain@ncl.ac.uk
Background: Mutations in the Collagen 6
genes (COL6A1, COL6A2 and COL6A3) cause a clinically and genetically heterogeneous group
of rare diseases (Bethlem Myopathy, Ullrich Congenital muscular dystrophy (UCMD) and
intermediate phenotypes) which are collectively known as the COL6- related dystrophies
(COL6-RDs) . The newly launched Global Registry for COL6-related dystrophies
(www.collagen6.org), funded by the Collagen VI Alliance through MDUK, allows secure
capture and storage of data from individuals affected with a COL6-RD and from the
medical professionals in charge of their care.
Aims: The primary objectives of the
registry are to:
-Contribute to trial readiness of COL6-related
dystrophies (RDs), allowing identification of genetically well characterised cohorts for
participation in research
studies and clinical trials;
- Add to the understanding of disease natural history
and prevalence;
- Assist doctors and other health professionals by
providing them with up-to-date information on managing COL6-RDs, to help them deliver
better standards of care for their patients;
- Stimulate industry interest in the COL6-RDs by
demonstrating the availability of clinician validated data to support future trials and
post marketing surveillance;
- Provide individuals with COL6-RD with up to date
summaries of relevant current research and news via the website and newsletters.
Methods: Affected individuals can access
information sheets about the registry, provide consent to being part of the registry and
submit their data online. As part of the consent process, affected individuals consent
to their clinicians entering data on their behalf and medical professionals are provided
with a user account to allow them to enter data online. Requests for anonymous data from
the registry are approved by the registry steering committee (containing professional
and patient representation), who have overall oversight of the registry governance.
Results: Here we describe the development
of the registry, plans for its future development and an analysis of the data contained
within the registry thus far.
Conclusion: The Global Registry for
COL6-related dystrophies is a new resource for the COL6-RD community which has potential
to stimulate, accelerate and support clinical and basic research in this group of
diseases.
Peripheral Neuropathy
Preliminary falls and functional balance data from the BALTiC study: A feasibility
analysis of home based BALance Training
in people with Charcot-Marie-Tooth
disease
1MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology,
London UK
2Faculty of Health, Social Care and Education, Kingston University
and St Georges University of London
Background: Charcot-Marie-Tooth disease
(CMT) is the most common inherited neuromuscular disorder. Features include a slowly
progressive, length dependent sensorimotor neuropathy. People with CMT report a high
incidence of falls, though few groups to date have looked at exercise based
interventions to improve balance in this population.
Aims: This study explored the feasibility
and effect of delivering a home based training programme using multi-sensory
rehabilitation and proximal strength training.
Methods: In this randomised controlled
feasibility trial, 14 participants (m=21.4%) with CMT1A were randomised to either a 12
week home based exercise programme of multi-sensory balance exercises and proximal
strengthening with a falls management session or a falls management session only.
Participants self-recorded falls and activity for a planned 20 weeks. During the
intervention, balance and strengthening exercises were prescribed in the home
environment. Outcome measures included balance performance in static and dynamic
posturography, functional balance tests, lower limb strength tests plus subjective
measures of balance confidence and function. Brief qualitative interviews were also
undertaken to capture participants’ experience of taking part.
Results: No significant differences in
disease severity existed between the two groups. Training was well tolerated with high
participation levels. The intervention group had greater delays with completing the
study in the aimed for 20 weeks because of work commitments of a small number
(Intervention mean=32wks, SD±10.6 Control mean=24wks SD±2.6). Total reported falls were
not significantly different (Intervention mean= 7 SD±5.2; Control mean= 4 SD±4;
P=0.32).
Functional measures of balance showed larger
improvement effect sizes, favouring the intervention group (Berg Balance Score, Hedge’s
G = 1.14; BESTest, Hedge’s G 1.04, 10MTW, Hedge’s G = 0.97, Functional Gait Assessment =
0.87). Centre of pressure variables showed moderate improvements in visual dependency
(COP velocity with feet apart eyes closed, Hedge’s G =0.48). Balance confidence showed
small effect changes.
Conclusions: This RCT was safe and
feasible. Functional measures show promise in this small and varied group with possible
improvements in visual dependency. Now that feasibility has been demonstrated and,
further exploration of efficacy will need to be undertaken on a larger sample.
Cross sectional study on CK levels in CMT and related disorders
Mariola
Skorupinska1, Menelaos
Pipis1, Alexander M. Rossor1, Matilde Laurá1, Mary M.
Reilly1
1MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology,
London UK
Background: Creatine kinase (CK) exists as
different isoenzymes and is influenced by a number of factors including gender, race,
muscle bulk and physical activity. An elevated CK may indicate acute muscle injury such
as acute myositis or rhabdomyolysis or chronic muscle damage as is the case with chronic
denervation in CMT. Aside from chronic denervation which occurs early in
motor-predominant subtypes of CMT and later in other forms such as HSN, other factors
including the duration of the disease and medications may influence CK levels.
Aim: To assess the cross-sectional
correlation of CK with the CMTES, ulnar CMAP and molecular diagnosis in CMT and related
disorders.
Methods: We retrieved CK values at
specific time-points for 161 patients and correlated them with the corresponding CMTES
and ulnar CMAP scores. Furthermore, we correlated the biochemical data of CK corrected
for CMTES across all molecular diagnoses.
Results: We evaluated the CK in 63 females
and 98 male patients. 73% (118) of evaluated patients had various subtypes of CMT (CMT1,
CMT2, CMTi) 15% HMN (24), 8% HSN (13) and 4% HNPP (6). In our cohort the CK range was
39-2205. 48% (77) of patients had an elevated CK level and the majority were male.
Abnormal CK levels were found in 40% of CMT1 patients, 55% of CMT2, 40% of CMTi, 67% of
HMN and 38% of HSN. There was no difference in the average level of CK between the
different groups of CMT subtypes. Patients with HMN, were the most likely to have
elevated rather than normal CK levels and there was a trend of near-normal CK levels
with higher CMTES. Impairment as measured by CMT Examination Score (CMTES) was on
average 10.81 (± 5.23) (range 0– 21). Average ulnar CMAP scores at the corresponding
time point was 5.01 (range between 0-9.6).
Conclusion: Based on this small cohort
size, there was no statistically significant correlation between the CK level and CMTES
or ulnar CMAPs. A CMT cohort with larger numbers would be able to assess more accurately
assess if there is any correlation between CK and disease severity during the early
stages of various CMT subtypes.
A novel homozygous mutation extending the PMP22 protein by 9 amino acid associated
with an isolated severe sensory ataxia
Alberto Zambon1, Matthew Pitt2, Mary M
Reilly3, Francesco Muntoni1*
1Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health & Great Ormond Street Hospital, London, UK.
2Department of Clinical Neurophysiology, Great Ormond Street Hospital
for Children NHS Foundation Trust, Great Ormond Street, London, UK.
3MRC Centre for Neuromuscular Diseases, National Hospital for
Neurology and Neurosurgery and UCL Institute of Neurology, Queen Square, London WC1N
3AR, UK.
Background: Peripheral myelin protein 22
(PMP22) related neuropathies account for over 50% of inherited peripheral neuropathies,
the so-called Charcot-Marie-Tooth. An increase or decrease in PMP22 gene copy number
results in distinct phenotypes, ranging from CMT1A (Duplication), hereditary neuropathy
with liability to pressure palsies (HNPP; single deletion) and CMT1E (Point mutations).
Of note, the underlying pathological mechanisms are still poorly understood and
homozygous mutations of PMP22 are very rare.
Aims: To present a patient with a new
homozygous mutation in the PMP22 gene and a peculiar phenotype.
Patient: We have studied an 8-year-old
girl who presented before the age of 1 year with severe locomotor delay, hypotonia and
absent deep tendon reflexes. She never acquired independent ambulation and requires
support also for standing in view of her severe sensory ataxia. Strikingly, her muscle
power and muscle bulk are within normal limits in all segments and small sensory fibers
are clinically spared.
Results: Nerve conduction studies showed
an extremely severe sensorimotor demyelinating neuropathy (Conduction
velocities<5m/s). Genetic analysis revealed a homozygous sequence change in the PMP22
gene causing the loss of termination codon (c.483A>G; p.*161Trpext*10). This variant
is predicted to extend the protein by an addition of 9 amino acids and could potentially
lead to loss-of-PMP22 function. Neurophysiological studies of the 2 heterozygous parents
identified clear features supporting a diagnosis of HNPP in them.
Conclusion: PMP22-deficient human models
are as rare as important to decipher the physiological function of the PMP22 protein
in vivo. The predominance of large fiber sensory involvement in this
and other rare similar cases suggests a pivotal role played by PMP22 in the
embryogenesis of dorsal root ganglia in humans.
Plasma Neurofilament Light Chain Concentration compared with I-RODS in Stable
Patients with Inflammatory Neuropathies on regular Intravenous Immunoglobulin
Mahima Kapoor1, Aisling Carr2,
Michael P Lunn2, Alex Rossor1, Professor
Mary M Reilly1
1MRC Centre for Neuromuscular Diseases, UCL Institute of
Neurology
2National Hospital for Neurology and Neurosurgery, London,
UK
michaellunn@nhs.net
Background: Blood neurofilament light
chain (NfL) concentration using ultrasensitive single-molecule array (Simoa) assay has
been explored in numerous neurological conditions. In acquired neuropathies, the
stability of plasma NfL concentration in patients on regular intravenous immunoglobulin
(IVIg) measured using ultrasensitive single-molecule array (Simoa) assay is unknown.
Aim: To perform a pilot, longitudinal
study that assesses the reliability and validity of a Simoa assay measuring plasma NfL
concentration in stable patients with inflammatory neuropathies (CIDP and MMN) on IVIg
and assess for potential correlation with the inflammatory Rasch-built overall
disability scale (I-RODS).
Methods: Blood samples and I-RODS were
collected over two-time points in 20 clinically stable patients, on a regular IVIg
regimen. Plasma NfL concentration was measured using an in-house developed Simoa
assay.
Results: There were 14 patients with CIDP
and 6 with MMN. 35% of patients were females, mean (SD) age of 56.6 (9.48) years
receiving an average dose of 2.42 (2.25)g/kg/month. There was no significant difference
in NfL concentrations or I-RODS scores between the two time-points (median= 11.38pg/mL
vs. 9.97pg/mL, T= 47, p= 0.46) and (median= 63 vs. 63, T= 7.5, p= 1). NfL levels did not
significantly differ between diagnoses (p= 0.91), or gender (p= 0.51). NfL results did
not correlate with change in I-RODS (t= 0.098, p= 0.76).
Conclusion: These results suggest that the
Simoa assay is stable in a range of IVIg doses and reinforce I-RODS as a subjective
marker of clinical stability. The lack of correlation between NfL results and I-RODS
needs to be further evaluated in larger and more diverse groups. These results suggest
that adequately treated inflammatory neuropathy does not have a significant, measurable
signal of axonal damage, compared to normal control levels from other studies.
Intravenous Immunoglobulin Treatment and Risk of thromboembolic events
Kapoor M2, Spillane J1, Englezou
C1, Sarri-Gonzalez S1, Bell R3, Rossor A2,
Manji H2, Lunn MP1, Carr AS1,
Reilly MM2
1National Hospital of Neurology and Neurosurgery (NHNN), Queen
Square, London, UK.
2MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology,
London, UK
3Department of Cardiology, UCLH, London, UK.
aisling.carr@nhs.net
Background: The potential risk of arterial
thromboembolic events (aTEE) with the use of IVIg has been investigated in numerous
groups of patients with conflicting results. Our unit has previously conducted a
retrospective cohort study of 111 inflammatory neuropathy patients receiving regular
IVIg and 333 neurology outpatients over a 30-month period that showed a higher incidence
of arterial events in patients exposed to IVIg.
Aim: In neurology outpatients, evaluate
individual risk factors (including IVIg exposure as a novel risk factor) and create a
predictive model for risk of arterial TEE at two and a half years.
Methods: We performed multivariate
logistic regression to assess the effect of individual risk factors. Kaplan-Meier
analysis was used to estimate the arterial TEE cumulative incidence for patients for
significant variables in combination and independently.
Results: Data on 445 neurology outpatients
including 112 neuromuscular patients receiving IVIg during this period was
retrospectively collected between January 2014 and July 2016. These groups were matched
on all cardiovascular risk factors except for smoking and diabetes. History of any IVIg
exposure, hypertension and diabetes mellitus were significant contributors to aTEE
outcomes, (odds ratio 3.3, 3.8 and 3.6, respectively, p<0.05). At two and a half
years, Kaplan-Meier analysis showed cumulative probability of arterial TEE of 50% if
patients had all three risk factors, compared to patients who had none of the risk
factors; 15% if patients had both IVIg and hypertension, and 53% if patients had both
IVIg and diabetes, compared to patients who had neither of these risk factors.
Conclusions: This review quantifies the
contribution of IVIg and traditional cardiovascular risk factors in aTEE in neurology
outpatients. It also provides a predictive model for future aTEE that will needs
validation. Having a predictive model may be the first step to identifying ways to
mitigate this risk given two of the three risk factors are modifiable.
Applications of unbiased datasets and machine learning for discovery of novel
dHMN-causative variants
Matthew Jennings1*, Alexander
Smith2, Rita Horvath2, Alan Robinson2+
1Department of Clinical Neurosciences, University of
Cambridge
2MRC Mitochondrial Biology Unit, University of Cambridge.
* mjj47@cam.ac.uk
Background: Despite advances in genomic
technology, for many distal hereditary motor neuropathy (dHMN) patients, a single
causative gene cannot be identified. The predictive capacity of combined annotation
dataset for predicting relevant variants has been shown before using the Mitominer tool
created within the MRC Mitochondrial Biology Unit. This is hindered, however, by the
lack of a complete and robust proteome of the peripheral nerve or even the axon.
Aims: We utilize publicly available
unbiased experimental datasets to highlight genesets enriched for axonal function and
peripheral nerve biology, alongside individual variant characteristics to determine the
qualities of causative genes and develop a machine learning tool which can assess the
likelihood of any single variant causing dHMN.
Methods: Experimental data relating to
genes either causative or not causative for dHMN, identified using ClinVar and ExAC,
allowed us to identify patterns of data associated with genes causing axonal neuropathy.
This data was then used to train a random forest machine learning predictive model,
optimized for maximal separating causative variant from solved exome data.
Results: We find that there are several
characteristics which distinguished neuropathy-causing genes, but that only certain
experimental datasets were useful. We do find however that other public data not
specifically related to motor neuropathies is also useful in the identification of dHMN
genes. Machine learning methods effectively stratified the relevance of variants within
patient exome data and were able to highlight variants of interest with unsolved
cases.
Conclusions: We developed an approach to
the challenge of recognising novel variants causing dHMN by identifying the
characteristics of the genes currently associated with the condition. Furthermore, we
have developed a machine learning model using these to distinguish between a set of
potentially causative variants. Additional non-biased datasets relating to the
peripheral motor neuron can be added to the model we have developed and will improve its
accuracy, and the generation of these should be encouraged in future. Our bioinformatic
tool applies a non-biased approach to the identification of novel dHMN variants
appropriate for the next generation sequencing era.
Genetic investigation of inherited neuropathy in families from Middle East using
next generation sequencing
Alaa M. Khan, MSc, Tipu
Sultan, MD, Yamna Kriouile, MD, Menelaos
Pipis, MD, Jana Vandrovcova, PhD, Stephanie
Efthymiou, MSc, Vincenzo Salpietro, MD, Mary M. Reilly, MD,
FRCP, FRCPI, Henry Houlden, MD, PhD.
MRC Centre for Neuromuscular Disease and Department of Molecular Neuroscience,
UCL, Institute of Neurology. Queen Square, London WC1N 3BG, UK. Children’s Hospital
and Institute of Child Health, Lahore, Pakistan. Service de pédiatrie II; Hôpital des
enfants, Rabat, Morocco.
Background: Inherited peripheral
neuropathies are a group of clinically and genetically heterogeneous disorders, that
encompass Charcot-Marie-Tooth disease (CMT), distal hereditary motor neuropathy (dHMN),
hereditary sensory neuropathy (HSN), hereditary sensory and autonomic neuropathy (HSAN)
and hereditary neuropathy with a liability to pressure palsy (HNPP). Moreover, inherited
peripheral neuropathies can be part of a more complex neurological syndrome. The advent
of next generation sequencing has allowed the discovery of new disease-causing genes,
however, 50% of patients with CMT2 remain without a diagnosis and reaching one remains a
challenge due to the heterogeneity of the condition.
Aim: To explore the genetic background of
undiagnosed inherited neuropathies in families from the Middle East using next
generation sequencing.
Patients and methods: We recruited 11
families within the SYNaPS study, mostly consanguineous, with a clinical diagnosis of an
inherited peripheral neuropathy. These families mostly originate from the Greater Middle
East. Whole exome sequencing was performed. Variants were filtered using our
bioinformatics system to only include rare homozygous and compound heterozygous variant
with MAF<0.01 in publically available databases. Segregation analysis and
confirmation by Sanger sequencing of candidate mutations is currently undertaken. In
selective cases with novel phenotypic features, further functional studies will be
performed.
Results: Neuropathy was the predominant
feature in 6 families (100% consaguoinous) and 5 families presented with a neuropathy as
a feature of a wider with multisystem condition. Likely pathogenic mutations were
identified in 7 families (63.6%), with 4 variants being novel. All diagnosed cases were
autosomal recessive (AR) type and variants were found in the following genes:
SH3TC2, MTMR2, NEFL, GAN, LAMA2, POLG, and WNK1.
Conclusion: A genetic diagnosis has been
obtained in most of families with an autosomal recessive inheritance pattern. Phenotype
and genotype correlation was undertaken and unusual phenotypic features in some
conditions have been identified.
Email: skgtamq@ucl.ac.uk
Frequent central nervous system, pachymeningeal and plexus MRI changes in POEMS
syndrome
1MRC Centre for Neuromuscular Diseases, National Hospital for
Neurology and Neurosurgery, London, UK; 2Department of Molecular
Neuroscience, UCL Institute of Neurology, London, UK; 3Lysholm Department
of Neuroradiology, National Hospital for Neurology and Neurosurgery, London, UK;
4Department of Haematology, Cancer Division, University College London
Hospitals NHS Foundation Trust, London, UK.
Background: Polyneuropathy, organomegaly,
endocrinopathy, monoclonal gammopathy, skin changes (POEMS) syndrome is a rare
multisystem disease associated with a plasma-cell dyscrasia. Although pachymeningeal
involvement has rarely been described, the extent of central nervous system (CNS)
involvement has not yet been extensively investigated.
Aim: To identify the characteristics and
frequency of CNS involvement in POEMS syndrome.
Methods: We retrospectively evaluated CNS
MRI findings from Europe’s largest single-centre cohort of POEMS syndrome patients. Of
seventy-seven patients who fit diagnostic criteria for POEMS syndrome, 41 had MRI brain
and 29 had MRI spine examinations. A control group of 33 patients with chronic
inflammatory demyelinating polyneuropathy (CIDP) was used as a comparator as this is the
major differential diagnosis. Of these CIDP patients, 12 underwent both MRI brain and
spine, 7 had MRI brain only and 14 had MRI spine.
Results: In 41 POEMS patients with MRI
brain studies, we identified frequent smooth, diffuse meningeal thickening of the
cerebral convexities and falx (n=29, 71%), of which 4 had meningeal collections.
Seventeen (41%) had vascular abnormalities including white matter disease, of which 4
had established infarcts. Of 29 patients with MRI spine, 17 (59%) had thickening of the
brachial and lumbosacral plexus. Conversely in 19 CIDP patients with MRI brain, none had
meningeal thickening (p < 0.0001), however 8 (42%) had vascular abnormalities (p =
0.85). Of 26 patients with MRI spine, 9 (35%) had brachial or lumbosacral plexus
thickening (p = 0.06).
Conclusions: In contrast to CIDP, POEMS
patients frequently have pachymeningeal thickening. Vascular abnormalities and plexus
thickening were common in both POEMS and CIDP. We propose that pachymeningitis should be
incorporated into the diagnostic criteria for POEMS syndrome, which serves to
distinguish features characteristic to POEMS syndrome which are not seen in alternative
conditions within the differential diagnosis.
Optimising Response Assessment Following High-Dose Chemotherapy and Autologous
Transplant for POEMS Syndrome
Presenting author: S Keddie
Tomkins O1, Keddie S2, Lunn MP2,
D’Sa1
1Department of Haematology, University College London Hospitals NHS
Trust, London NW1 2PG
2Centre for Neuromuscular Disease, National Hospital for Neurology
and Neurosurgery, Queen Square, London WC1N 3BG
Background: High dose melphalan autologous
stem cell transplantation (ASCT) has emerged as an effective therapy for patients with
systemic POEMS syndrome (polyneuropathy, organomegaly,
endocrinopathy, monoclonal-protein and skin
changes).
Aim: To assess outcome measures following
ASCT in POEMS syndrome and identify potential prognostication markers for disease
progression.
Methods: We reviewed 42 patients who
underwent melphalan ASCT over the last 18 years to measure outcomes following ASCT and
optimise treatment response. Overall survival was 92.9%, one year survival was 94.6%,
and progression free survival rate 80.1% (mean follow up 62.6 months, range 4-226
months). Three patients (16.7%) had engraftment syndrome, with no significant difference
observed according to the mobilisation agent used. Average serum vascular endothelial
growth factor levels improved from 4,959pg/ml (95% CI 3,895-6,190) to 489.5pg/ml (95%CI
416-1,132) at six months and 330pg/ml (95% CI 313-869) at one year. Significant
neurological improvement was seen in all but two patients after ASCT, with median
pre-transplant Overall Neuropathy Limitation Scale score of 6 improving to a
post-transplant score of 2 (p<0.01). Seven percent of patients were bed bound before
ASCT, but none were on most recent follow up. Nerve conduction studies demonstrated
continued improvement even three years after ASCT. Formal haematological response was
evaluable in 33/42 patients. No patient who achieved complete haematological response
has relapsed by most recent follow up, whereas of those with less than complete response
had a 31.6% relapse rate (p=0.027).
Conclusion: Long-term outcome is
favourable in patients treated with ASCT for POEMS syndrome. VEGF, considered to be a
surrogate biomarker for disease activity significantly improves following treatment, and
patients who achieve complete haematological response have significantly better
progression free survival than those who don’t. Neurological improvement following ASCT
is often ongoing for several years.
The Clinical Spectrum of MORC2 associated CMT2
Carolynne Doherty1, Menelaos
Pipis1, Andrea Cortese1, Alexander Rossor1, Adnan
Manzur2, Francesco Muntoni2, Mary M Reilly1
1Queen Square Centre for Neuromuscular Diseases, UCL Institute of
Neurology, Queen Square, London, WC1N 3BG
2Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health &Great Ormond Street Hospital Trust, London, UK
email: c.doherty@ucl.ac.uk
Background: The MORC2 gene encodes a
member of the Microrchidia protein superfamily. It is a DNA-dependent ATPase that
relaxes chromatin to facilitate DNA repair. Mutations in MORC2 associated with axonal
Charcot Marie Tooth Disease were first described by Sevilla and Colleagues in 2016 and
designated as CMT2Z.
Aims: We describe the clinical and
neurophysiological features in three families identified though whole exome or mini
exome sequencing at our centre who have MORC2 associated CMT2. We present a detailed
discussion on the phenotypic spectrum described in the literature.
Patients: A now 36 year old man first
walked at 18 months, and could only just run at age 15, presented at age 25 with
evolving painful distal tingling and hand cramping and “locking.” Detailed phenotyping
and neurophysiological evaluation revealed axonal sensory and motor neuropathy with
retinal dystrophy, pyramidal and subtle cerebellar signs, and higher mental function
impairment (IQ 80). Following extensive evaluation including metabolic evaluation,
targeted genetic testing and CMT2/intermediate panels, whole exome sequencing identified
a p.R252W mutation in MORC2. His child carries the same mutation, evaluation at age 5
demonstrated delayed gross motor and language skills, behavioural lability, and
sensorimotor axonal neuropathy. Subsequently, a 27 year old female was found to have the
same mutation, describing childhood onset of intellectual disability and delayed motor
milestones, hearing impairment, intoed walking and upper limb involvement in her teens.
She required a wheelchair at age 22. A further patient, now age 29 had delayed motor
milestones, early foot deformity and scoliosis and patchy upper limb symptoms, has
required a wheelchair when outdoors since age 27. She was identified to carry the E236G
mutation. This patient’s child had abnormal gait and falls when first walking at 15
months and on clinical examination had brisk lower limb reflexes with ankle dorsiflexion
weakness with marked asymmetrical varus deformity. At age 4 there is marked speech and
language delay and segregation of the mutation has been confirmed.
Conclusion: Detailed phenotyping seeking
associated features such as hearing impairment, retinal dystrophy, ataxia, pyramidal
features and neurodevelopmental impairment is of vital importance in the evaluation of
peripheral neuropathy and pursuit of the diagnosis.
Pentanucleotide repeat expansion causes CANVAS and Late-Onset Sensory
Ataxia
Andrea Cortese1, Roberto
Simone2, Rosin Sullivan1,$, Jana Vandrovcova1,$, Huma
Tariq1, Yau Way Yan1, Jack Humphrey1, Jaunmuktane
Zana2, Sivakumar Prasanth1, Ilyas, Muhammad1, Polke
James3, Eloise Tribollet1, Pedro J Tomaselli4, Grazia
Devigili5, Ilaria Callegari6, Maurizio Versino6,
Vincenzo Salpietro1, Stephanie Efthymiou1, Diego
Kaski1, Nick W Wood1, Nadja S Andrade7, Elena
Buglo8, Adriana Rebelo8, Alexander M Rossor1, Adolfo
Bronstein2, Pietro Fratta1, Wilson J Marques3,
Stephan Zuchner8, Mary M Reilly1,#, Henry
Houlden1,#
1Department of Neuromuscular Disease and; 2Department of
Clinical and Movement Neurosciences; 3Neurogenetics Laboratory, UCL
Institute of Neurology and The National Hospital for Neurology, Queen Square, London,
WC1N 3BG, UK; 4Department of Neurology, School of Medicine of Ribeirão
Preto, University of São Paulo, Ribeirão Preto, Brazil; 5UO Neurologia I,
Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Milano; 6IRCCS Mondino
Foundation, Pavia, Italy; 7Department of Psychiatry and Behavioural
Sciences, Center for Therapeutic Innovation; 8Dr. John T. Macdonald
Foundation Department of Human Genetics and John P. Hussman Institute for Human
Genomics, University of Miami Miller School of Medicine, Miami, USA.
$equal contribution
#these authors jointly directed this project
Background: Late-onset ataxia is a common
reason for neurological consultation, but its cause often remains idiopathic. Cerebellar
dysfunction, but also proprioceptive or vestibular impairment, can lead to ataxia. When
in combination, this more severe type of ataxia is termed cerebellar ataxia, neuropathy,
vestibular areflexia syndrome (CANVAS). Both sporadic and familial cases of CANVAS have
been reported, suggesting the possibility of a recessive transmission of the disease
Aim: to identify the genetic cause of
CANVAS and late-onset sensory ataxia
Methods: non-parametric linkage analysis
and genome sequencing, functional studies
Results: we identified a recessive
pentanucleotide repeat expansion as the cause of CANVAS and a common cause of late-onset
sensory ataxia. The recessive repeat expansion showed full segregation in 23 cases from
11 families. Additionally, 33 (22%) out of 150 sporadic cases with late-onset ataxia
carried the recessive repeat expansion. The percentage raised to 62% in patients with
sensory neuronopathy and cerebellar involvement and 92% in full-blown CANVAS disease.
The expansion resides in the polyA tail of an Alu element and differs in terms of both
size and nucleotide sequence from the reference allele. Notably, the pentanucleotide
repeat expansion does not affect expression of the repeat hosting gene at mRNA and
protein levels in patient fibroblasts, lymphoblasts, muscle and brain tissue suggesting
that there is no overt loss of function.
Conclusions: these data, together with the
observation of an allelic carrier frequency of the expanded repeat of 0.7% in the
European population, suggests that this biallelic pentanucleotide repeat expansion
represents a frequent cause of late-onset ataxia, with clinical similarities and disease
frequency to that of Friedreich’s ataxia.
Contact email:andrea.cortese@ucl.ac.uk
Motor Nerve Disorders
miRNAs in the CSF of SMA patients as prognostic biomarkers of the potential
response to Spinraza treatment
*Bruno Doreste1, Mariacristina Scoto1,
Irina Zaharieva1, Haiyan Zhou2,3 and Francesco
Muntoni1,3
1The Dubowitz Neuromuscular Centre, Developmental Neurosciences
Programme, Great Ormond Street Institute of Child Health, University College London;
2Genetics and Genomic Programme, Great Ormond Street Institute of Child
Health, University College London; 3NIHR Great Ormond Street Hospital
Biomedical Research Centre, London, UK
Background: Spinal Muscular Atrophy is the
most common genetic cause of infant mortality, resulting from homozygous deletion in the
Survival Motor Neuron gene 1 (SMN1). Spinraza
(Nusinersen) is an antisense-oligonucleotide (AON) drug, which is the first FDA approved
treatment for SMA. Although the majority of patients show a significant clinical
improvement in response to Spinraza, some patients show minimal or no improvement.
Aims: To determine if micro-RNAs (miRNAs)
in the cerebrospinal fluid (CSF) can be developed as prognostic biomarkers to predict
the clinical outcomes of type I SMA patients in response to Spinraza treatment.
Methods: For discovery profiling, miRNA RT
panels containing 752 miRs were performed on miRNAs extracted from the CSF samples of
type I SMA patients at pre- (n=10) and 2 months (n=10) post- Spinraza treatment. 13
significantly differentially expressed miRNAs were identified (p<0.05) for further
validation. Candidate miRNAs were validated using customized quantitative real-time PCR
panels in CSF samples from 8 additional type I SMA patients, at pre-, 2 months post- and
6 months post- Spinraza treatment. The levels of miRNAs at pre-treatment were used in a
stepwise multiple linear regression, with the percentage change in CHOP-INTEND motor
scores at 10 months post-treatment, a time point when a clear clinical response is
expected, set as the dependent variable.
Results: The levels of 3 candidate miRNAs
(CM) prior to Spinraza treatment were significant predictors (CM1: β=-0.694, p<
1x10-3; CM2: β=0.518, p=0.002, CM3: β=-0.273; p=0.013) for improvement of
the motor ability of the patients, measured as the percentage change in CHOP-INTEND
motor scores at 10 months post-treatment to pre-treatment.
Conclusions: These data show that CSF
miRNAs could be employed as prognostic biomarkers to predict SMA patients’ responses to
Spinraza treatment. Further research is under way to increase the robustness of this
prognostic panel by increasing sample size, identifying further prognostic miRNAs, and
include longitudinal data at different time-points during treatment.
Determination of tissue-water T2 of fat infiltrated upper and lower limb
skeletal muscle with MRI in amyotrophic lateral sclerosis, Kennedy’s disease and
Duchenne muscular dystrophy
Nick
Zafeiropoulos1,2, Uros
Klickovic1, Luca Zampedri1,2, Jasper Morrow1,2,
Matthew Evans1,2, Christopher Sinclair1,2, Stephen
Wastling1,2, Enrico De Vita6, Valeria Ricotti4,
Robert Janiczek3, Paul M Matthews5, Linda
Greensmith1,2, Francesco Muntoni4, Mary M Reilly1,2,
Michael G Hanna1,2, Tarek Yousry1,2, Pietro Fratta1,2
and John S Thornton1,2
1UCL Queen Square Institute of Neurology, University College London,
London, United Kingdom, 2MRC Centre for Neuromuscular Diseases, UCL Queen
Square Institute of Neurology, London, United Kingdom, 3GlaxoSmithKline,
London, United Kingdom, 4Dubowitz Neuromuscular Centre, UCL Institute of
Child Health, London, United Kingdom, 5Imperial College London, London,
United Kingdom, Department of Biomedical Engineering and UK Dementia Research
Institute, 6School of Biomedical Engineering and Imaging Sciences, King’s
College London, London, United Kingdom
Background & Aims: The aim of this
work was to develop novel optimised quantitative magnetic resonance imaging (MRI)
biomarkers to detect and monitor neuromuscular diseases. The primary objective was to
estimate skeletal muscle-water spin-spin relaxation time (T2m), a challenging
endeavour in fat infiltrated muscles common in patients with these conditions.
Methods: A multi-component
slice-profile-compensated extended phase graph (sEPG) model for multi-echo
Carr-Purcell-Meiboom-Gill (CPMG) spin-echo sequence signals was implemented, with the
fat signal modelled as two sEPG components with fixed parameters, and the remaining
unknown parameters (B1 field factor, T2m, fat fraction
(ffa), global amplitude and Rician noise standard deviation) determined by
maximum likelihood estimation. Through software simulations the performance of the
method was compared with conventional least-squares parameter estimation, and the
commonly assumed multi-exponential signal model. The algorithm was used to analyse
clinical muscle study data including patient groups with amyotrophic lateral sclerosis
(ALS), Kennedy’s disease (KD) and Duchenne muscular dystrophy (DMD) – and their
longitudinal follow up scans – and matched healthy controls. The processing pipeline to
generate parameter estimate maps incorporated quality control according to quality of
fit criteria and physical meaningfulness.
Results: Stable fitting was generally
obtained, with little T2m variation between muscles in healthy controls. In
ALS and KD median T2m were significantly elevated in varied patterns, but
showed a trend to decrease in DMD, where other T2m distribution histogram
metrics such as the skewness and full width at quarter maximum differed significantly
from those observed in healthy volunteers. Maps of estimated ffa were
qualitatively consistent with independently acquired Dixon fat fraction maps. Comparison
of T2m maps with short TI inversion recovery (STIR) MRI signal intensity
distributions suggested that conventional STIR contrast may depend on factors additional
to T2m.
Conclusion: The data quality available
from conventional clinical scanner CPMG protocols places limitations on the achievable
accuracy and precision of multi-component transverse-relaxation decay parameter
estimates. Nevertheless, the developed methods provide reproducible measures which
change with disease, and which may distinguish different pathological processes.
Mortality in patients with spinal muscular atrophy over the last 10 years in the
Northeast of the UK
The John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle University, Newcastle upon Tyne, UK
(Sabine.Specht@ncl.ac.uk)
Background: Infantile onset SMA is the
most common genetic cause of death in infants (Kolb et al Ann Neurol 2017). Since the
introduction of Standards of Care (Wang et al Consensus Statement for Standard of Care
in Spinal Muscular Atrophy 2007), a more proactive approach to care for SMA resulted in
improvements in the natural history of the disease. However, for SMA I infants the
approach has been in most cases palliative until more recently with the introduction of
novel treatments like antisense nucleotide and gene therapy.
Aims: In view of this dramatic change in
the care of SMA patients, from a palliative approach to causal therapies, an audit was
conducted to identify the mortality of SMA patients in the Northeast population over the
last 10 years.
Results: In our cohort of neuromuscular
patients, 77 were diagnosed with SMA 0, I, II or III. Currently 53 SMA patients are
followed up in our service; of these 4 SMA I children are receiving intrathecal
injection of Nursinersen. Twenty-four SMA patients died of the disease. Of this latter
group, 2 patients had SMA 0 and died within their first month of life. Seventeen
suffered from SMA I, 2 patients from SMA II and 3 from SMA III respectively. Four
patients died while receiving Nursinersen treatment, at the age of 6, 7, 20 and 26
months after their second or third intrathecal injection. The causes of death were
respiratory arrest, hypoxic ischaemic brain injury secondary to out of hospital
cardio-respiratory arrest and pneumonia. Two of those patients were followed up out of
area after having initiated Nursinersen at our site. Prior to the introduction of
Nursinersen, all 13 patients with SMA I died at the mean age of 8 months. The age of the
deceased patients with SMA II varied greatly between 3 years and 56 years. As for
SMAIII, patients died aged on an average of 61 years.
Conclusions: Over the past 10 years 25%
SMA 0-I patients died at an average age of 8.4 months (age range 0 – 20), 6% SMA II -
III patients died at mean age of 47 years (age range 3- 75), cardiorespiratory failure
being the most common cause of death in all cases.
Development of a Rasch optimised functional outcome measure for people with Spinal
and Bulbar Muscular Atrophy
G Ramdharry*, H Devine*, L Zampredi*, G.
Soraru**, G Querin**, S Fenu***, D. Pareyson***, P. Fratta*
Department of Neuromuscular Diseases, UCL, London, UK*; University of Padua,
Italy**; Fondazione IRCCS Istituto Neurologico C.Besta, Milan, Italy***
Background: Spinal and Bulbar Muscular
Atrophy (SBMA) or Kennedy’s disease, is a rare neurodegenerative disease characterised
by progressive weakness in the limb and bulbar muscles due to loss of lower motor
neurons. In SBMA there are currently several functional rating scales in use. The
revised amyotrophic lateral sclerosis functional rating scale (ALSFRS-R), the spinal and
bulbar muscular atrophy functional rating scale (SBMAFRS) and the adult myopathy
assessment tool (AMAT). The SBMAFRS was developed by clinicians from the ALSFRS for
people with SBMA.
Aims: The aim was to combine items from
three commonly used functional scales and optimise it specific to people with SBMA.
Methods: Three functional rating scales
commonly used for people with SBMA were individually analysed. Item performance was
evaluated using Rasch analysis. Individually, the items of the scales did not cover the
spread of the ability of individual persons. The individual items for the three scales
were combined and repeated items were deleted. Additional items were discarded that did
not fit the model or were not deemed clinically relevant to the clinical team. Items
were rescored where there were disordered thresholds, and evaluation of item dependency
and uni-dimensionality took place.
Results: 140 individual observations were
entered into the analysis. The resulting scale consisted of 23 items and 17 categories
were rescored. There was a good overall fit to the Rasch model (X2 = 53.18,
p=0.22). Reliability indices were 0.76 and item fit residuals were 0.02. Person location
mean was high compared to the item location mean. Dependency was observed between
stepping and speech; sit to stand, stairs and rise from supine. The clinical team
justified retaining the items as they are functionally distinct. The scale was not
uni-dimensional on testing (15.7% significant t-tests), but there was no clear
delineation in items between the positively loaded and negatively loaded items.
Conclusion: We have combined items from
three functional scales to create a tailored tool for SBMA. There are some issues of
dimensionality and dependency, but the performance of the scale has been optimised to
create a more clinically relevant tool specific to people with SBMA, with improved item
performance.
Is Protein Intake Associated With Fat Free Mass In Type I Spinal Muscular
Atrophy?
G. Baranello1, R. S. De
Amicis2, A. Leone2, A. Foppiani2, A.
Battezzati2, C. Mastella3, M. Bassano2, E.
Giaquinto4, S. Bertoli2.
1The Dubowitz Neuromuscular Centre, University College London Great
Ormond Street Institute of Child Health & Great Ormond Street Hospital, London, UK
and Developmental Neurology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta,
Milan, Italy
2International Center for the Assessment of Nutritional Status
(ICANS), Department of Food Environmental and Nutritional Sciences (DeFENS),
University of Milan, Italy
3SAPRE-UONPIA, Fondazione IRCCS Ca’ Granda Ospedale Maggiore
Policlinico, Milan, Italy
4Dietetic and Nutrition Center, M. Bufalini Hospital, Cesena,
Italy
Background: Spinal Muscular Atrophy Type I
(SMA1) is a severe autosomal recessive neuromuscular disease characterized by
degeneration of alpha motor neurons in the spinal cord with subsequent muscular atrophy
and increased fat free mass (FFM). Amount of energy and dietary protein intake play a
key role in maintenance of FFM, but optimal protein needs in SMA still remain
unclear.
Aim: we investigated the association
between FFM and daily food protein intake (PI) in a SMA1 sample with an optimal energy
intake according to sex, age and energy metabolism.
Methods: 14 children with SMA1 (57%M; mean
age:6 months, range 4-13) were recruited among a large sample involved in an ongoing
longitudinal study, only if they presented an energy intake +/- 20% of energy
expenditure assessed by indirect calorimetry. WHO growth charts were used as reference
values for BMI. FFM was investigated by DXA and FFM/length ratio (g/cm) was calculated.
Mean energy and nutrients’ intakes by 3-days food dietary records were compared with
age-based dietary recommendations. Descriptive results are expressed as median values
with interquartile ranges (IQR).
Results: BMI Z-Score was -2.5
(IQR=-4.1–-1.7). FFM was 63% (IQR=62–69%) and FFM/length ratio was 63g/cm
(IQR=59–66g/cm). 100% of patients had an energy intake in line with measured energy
expenditure; only 43% satisfied the carbohydrates’ recommendations, while 71% consumed
more fat than recommendations. Proteins/kg BW were 1.9g/kg (IQR=1.4–2.1g/kg): +46% of
recommendations (IQR=5–+83%). PI/kg BW (r=0.293, p=0.308) and adequacy index (r=0.486,
p=0.078) were not correlated with FFM and FFM/length ratio.
Conclusion: SMA1 children with adequate
daily food intake consumed more lipids and proteins and less carbohydrates than what
recommended. Higher protein intake was not related to FFM and FFM/length rato; amount of
PI in SMA1 children does not seem to contribute to the improvement of FFM. Further
studies are needed to optimize protein requirements in SMA1 children.
Respiratory trajectories in SMA type 2 and non-ambulant type 3 paediatric patients
within the SMAREACH UK network
Federica Trucco1,2, Deborah
Ridout3, Mariacristina Scoto1, Francesco Muntoni1 on
behalf of the SMAREACH UK network
1Dubowitz Neuromuscular Centre, UCL GOS Institute of Child Health,
London
2DINOGMI, University of Genoa, Genoa, Italy
3Population, Policy and Practice Programme, UCL GOS Institute of
Child Health, London
UCL Great Ormond Street Institute of Child Health Institute of Child Health, 30
Guilford Street, WC1N 1EH, London
Background: The availability of new
therapeutic options for SMA2 and 3 has led to modified clinical phenotypes. However, the
natural history of respiratory involvement in these conditions has not been fully
outlined so far. We hypothesized that the progression of respiratory and motor function
would decline differently among SMA2 and SMA3 non-ambulant patients.
Aims: Main aim is to identify the yearly
decline of respiratory function and its correlation with motor function.
Patients and Methods: Retrospective
analysis of SMA2 and non-ambulant SMA3 paediatric patients (age<18years) consented
for SMAREACH UK natural history study. Nine-year data (Jun2010-Sep2018) were collected
from study forms. Patients with at least 2 years follow-up data were included. We
excluded patients in any interventional clinical trial. Anthropometrics, clinical data
(establishment of Non-Invasive ventilation (NIV), cough assist) were collected. Motor
function scores such as Hammersmith functional motor scale (HFMS) and Revised
performance of upper limb (RULM) were collected at each visit. Forced vital capacity
absolute, (FVC) and % predicted (FVC %pred.) were collected from spirometry.
Results: Out of 314 SMA2 and 3 patients,
153 met the inclusion criteria and had a full dataset available. 118/153 were SMA2,
16/153 were non-ambulant SMA3, 19/153 unknown. Median age at first visit was 5.6 (IQR
3.4-9.8) and 11.6 (IQR 4.9-15.8) years for SMA2 and 3 respectively. 44/134 (33%) had
started NIV, main reason being recurrent low tract respiratory infections followed by
hypoventilation detected at sleep study (12 vs 9). 39/134 (29%) had started cough
assist. FVC %pred. yearly rate of decline was 3.2% in SMA2 patients and 4.0% in combined
SMA2 and 3. In SMA2 patients we observed a steeper reduction of FVC %pred. up to 10
years of age, followed by a slower yet regular decline. Conversely, in SMA3 patients FVC
%pred. started declining after the age of 10. In SMA2 HFMS at baseline correlated with
FVC absolute (r=0.8, p=0.01) while both HFMS (r=0.40 p=0.22) and RULM (r=0.4, p=0.0.14)
correlated with FVC% pred.
Conclusion. In SMA2 and 3, the respiratory
function expressed as lung volumes, declines progressively from an early age and is
correlated with motor function.
Feeding difficulties in children and adolescents with SMA type II
Renske Wadman1,2,3, Mariacristina Scoto1,4,
Lucia Schottlaender1,5, Nadia Imbrigiotta1, Federica
Trucco1,4, Chiara Brusa1,4, Tracey Davis4, Marion
Main4, Pinki Munot4, Anna Sarkozy4, Adnan
Manzur4, Francesco Muntoni1,4
1Dubowitz neuromuscular centre, Great Ormond Street Institute of
Child Health, University College London, UK
2Department of Neurology and Neurosurgery, University Medical Center
Utrecht, The Netherlands
3Brain Center Rudolph Magnus, University Utrecht, The
Netherlands
4Dubowitz neuromuscular centre, Great Ormond Street Hospital for
Children, London, UK
5Department of molecular neuroscience, Institute of Neurology,
University College London, UK
Corresponding email address:
r.wadman@ucl.ac.uk
Background: Spinal muscular atrophy(SMA)
is a progressive motor neuron disorder caused by deletion of the SMN1
gene. The most prominent feature is muscle weakness of axial and limb muscles. Scoliosis
and respiratory problems are common comorbidities in SMA types I and II as a reflection
of paraspinal and respiratory muscle weakness. Weakness of bulbar muscles and muscles of
the gastrointestinal tract have been described to a lesser extent, mostly in severe SMA
(type I). Disease course of feeding difficulties in intermediate SMA (type II) are
relatively unknown. The implementation of disease-modifying therapies will change motor
function and survival in SMA, but effects on non-motor co-morbidities are unknown. The
natural course of feeding difficulties needs to be explored in order to have a reference
for treatment effects on these symptoms.
Aims: Identify feeding difficulties and
their disease course in treatment-naïve SMA type II patients.
Patients and methods: We included all SMA
type II patients from the GOSH SMA database. Data was retrieved through retrospective
chart review. Growth curves including BMI were analysed adjusted for age and gender.
Results: We included 47 patients with a
mean age of 10years (range 2-19yrs). Seven (72%) had a relatively mild SMA type II
phenotype and achieved the ability to stand or walk with support in addition to sitting.
Mean follow up time was 6years(range 1-15yrs). 31 of 47 Patients (66%) were underweight.
Reported feeding difficulties included unsafe swallowing, (severe) weight loss or both.
Feeding difficulties were present in 48% of patients. Fourteen patients(30%) needed
gastrostomy because of severe weight loss(40%), swallowing difficulties(40%) or
both(20%). Six out of 15 patients had recurrent pulmonary infections with improvement
after gastrostomy suggestive of recurrent (silent) aspiration.
Conclusion: Feeding difficulties were
present in 48% of patients with SMA type II, especially with more severe weakness.
However, enteral feeding was needed in only 30% of patients and was indicated when
severe weight loss or swallowing difficulties with or without (silent) aspiration were
present.
Survival of motor neuron (SMN) protein levels before and after treatment with
risdiplam (RG7916) in patients with Type 1 to 3 spinal muscular atrophy (SMA) compared
with healthy subjects
Heidemarie Kletzla*, Christian
Czecha, Yumi Clearya, Stefan Sturma, Andreas
Günthera, Giovanni Baranellob, Eugenio Mercuric,
Laurent Servaisd, John W. Daye, Nicolas Deconinckf,
Andrea Kleing, Basil Darrash, Riccardo Massonb,
Janbernd Kirschneri, Nathalie Goemansj, Maria Carmela
Perac, Claudia A. Chiribogak, Dirk Fischerl,
Ksenija Gornia, Omar Khwajaa
aRoche Pharmaceutical Research and Early Development, Roche
Innovation Center Basel, Basel, Switzerland
bCarlo Besta Neurological Research Institute Foundation,
Developmental Neurology Unit, Milan, Italy
cPaediatric Neurology and Nemo Center, Catholic University and
Policlinico Gemelli, Rome, Italy
dInstitute of Myology, Paris, France; Reference Center for
Neuromuscular Disease, Centre Hospitalier Régional de La Citadelle, Liège,
Belgium
eDepartment of Neurology, Stanford University, Palo Alto, CA,
USA
fQueen Fabiola Children’s University Hospital, Université Libre de
Bruxelles, Brussels, Belgium; Neuromuscular Reference Center UZ Ghent, Ghent,
Belgium
hBoston Children’s Hospital, Harvard Medical School, Boston, MA,
USA
iDepartment of Neuropediatrics and Muscle Disorders, Medical
Center-University of Freiburg, Freiburg, Germany
jNeuromuscular Reference Centre, Department of Paediatrics and Child
Neurology, University Hospitals Leuven, Leuven, Belgium
kDepartment of Neurology, Columbia University Medical Center, New
York, NY, USA
lDivision of Neuropediatrics, University Children’s Hospital Basel,
University of Basel, Basel, Switzerland
email: heidemarie.kletzl@roche.com
Background: Spinal muscular atrophy (SMA)
is characterised by motor neuron loss and muscle atrophy, due to reduced levels of
survival of motor neuron (SMN) protein from loss of function of the SMN1 gene. While
SMN1 produces full-length SMN protein, a second gene, SMN2, produces only low levels of
functional SMN protein. Risdiplam (RG7916; RO7034067) is an investigational, orally
administered, centrally and peripherally distributed small molecule that modulates SMN2
pre-mRNA splicing to increase SMN protein levels.
Aim: To report on the SMN protein levels
measured in studies of risdiplam in patients with Type 1 SMA (FIREFISH - NCT02913482)
and patients with Type 2 and 3 SMA (SUNFISH - NCT02908685; JEWELFISH - NCT03032172).
Methods: At abstract submission, SMN
protein data are available from 63 patients with Type 2 and 3 SMA and 21 patients with
Type 1 SMA, at baseline prior to treatment and after treatment with risdiplam. SMN
protein levels in 49 healthy subjects have been collected in two other studies. This is
the first detailed comparison of SMN protein levels across SMA types and healthy
individuals, between copy numbers, across a wide age range (3.3 months to 52 years in
patients with SMA), and in longitudinal data for patients receiving risdiplam versus
patients on placebo. The same procedures and assays were used for SMN protein sample
collection and analysis, enabling a robust comparison.
Results: In SUNFISH and JEWELFISH, SMN
protein increased in a dose-dependent manner upon treatment with risdiplam, with a
median 2.5-fold increase (range 1.5–3.5) at the highest dose. In FIREFISH, an individual
SMN protein increase of up to 6.5-fold (range 1.6–6.5) was observed at the highest
dose.
Conclusion: This increase in SMN protein
is expected to lead to significant clinical benefit, based on the comparison of SMN
protein levels in different SMA severity types and healthy subjects.
Developing a bulbar motor neuron protocol to study Spinal and Bulbar Muscular
Atrophy
Devine HE1, Malik
B1, Greensmith L1 and Patani R 1,2
1Sobell Department of Neuromuscular Disease, UCLIoN; 2The
Francis Crick Institute
Background: Spinal and Bulbar Muscular
Atrophy (SBMA) is a rare progressive motor neuron disease caused by an X-linked,
expanded polyglutamine repeat in the androgen receptor gene. It affects male patients
who present with wasting and weakness of facial, bulbar and limb muscles in their fourth
to sixth decade Spinal motor neurons (MNs) have been studied in mouse models of SBMA,
pathological specimens and increasingly in MNs derived from patient induced pluripotent
stem cells. However, there are currently no established protocols for studying bulbar
MNs.
Aims: To develop a protocol to generate
bulbar MNs from SBMA patient-derived iPSCs.
Methods: SBMA IPSCs (SB1, SB3, SB5 and
SB6) were kindly provided from the lab of Dr Kurt Fischbeck at the National Institute
for Neurological Disorders and Stroke (USA) through an MTA. Three lines of patient
derived and three lines of control iPSCs were plated to 100% confluency and then
differentiated into neuroepithelium using small molecules (dorsomorphin, SB431542 and
CHIRR) in N2B27 media. At day 7 the cells were patterned using 5 developmentally
rationalised experimental conditions. At day 14 MN precursors were treated with 0.1µ M
purmorphamine for a further 4 days. The precursors were harvested at day 18.
Results: RNA was extracted from the cells
and qPCR used to identify the experimental conditions which most closely demonstrated
the HOX gene expression pattern found in bulbar MNs. The closest experimental conditions
were fine-tuned to optimise the protocol. They will be retested for HOX gene expression
pattern. These precursors will undergo terminal differentiation using Compound E. At day
7 of terminal differentiation the cells will be fixed and immunocytochemistry will look
for evidence of Phox2B expression which defines bulbar MNs.
Conclusions: These bulbar MNs will be used
to further explore the pathogenic mechanisms which underlie motor neuron diseases.
Defining the patterning cues for bulbar v spinal MNs will lay the foundation for
regionally specified astrocytes for further study.
1John Walton Muscular Dystrophy Research Centre, Newcastle
University
2Newcastle upon Tyne NHS Trust Foundation Hospitals
*Presenting author:
lindsay.murphy@ncl.ac.uk
Background: The UK SMA Registry collects
clinical and genetic information from individuals with spinal muscular atrophy (SMA).
Launched in 2008, the registry holds patient-entered data on over 500 SMA patients
living within the UK and Ireland. SMA is a recessive, progressive neuromuscular disorder
with a prevalence of approximately 1–2 per 100,000 persons.
Aims: The purpose of the registry is to
aid the rapid identification of eligible patients for clinical studies. It disseminates
SMA-relevant information to participants; provides a source of information to academics,
industry and healthcare professionals; and supports the SMA community.
Methods: Registration is patient-initiated
through a secure online portal. Participants give their informed consent and are invited
to complete a questionnaire about their condition, including: genetic confirmation and
SMN2 copy number; clinical diagnosis; SMA classification; current and best motor
function; wheelchair use; scoliosis surgery; gastric/nasal feeding; ventilation status;
family history.
Results: Currently, 512 participants are
registered with the UK SMA Registry, with an age range of six months to 84 years. The
greatest number of participants report a diagnosis of SMA type 2 (40%), followed by
individuals with SMA type 3 (31%) and with SMA type 1 (12%) (17% unspecified).
Considering motor function, 31% percent of individuals are unable to support themselves
in a seated position, 37% are able to sit unsupported and 23% are able to walk (9%
unspecified). Sixty-three percent of participants either always or sometimes use a
wheelchair and 13% never use a wheelchair (24% unspecified).The majority of registry
participants do not use a ventilation aid. Occasional use of non-invasive ventilation is
reported by 16% and permanent use, by 1% of participants. Use of invasive ventilation is
lower, with reported occasional use by 1% and permanent use, by 1% of participants (12%
unspecified for each).
Conclusion: The UK SMA Registry is a
valuable tool for the collection of patient data which informs academics, healthcare
professionals and industry. It represents a trial-ready cohort of individuals and
supports the SMA patient community.
The impact of spinal surgery on respiratory and motor function and weight gain in
patients with SMA II and non-ambulant III
Brusa C1, De Graaf
J1, Manzur A1, Main M1, Milev E1, Iodice
M1, Ramsey D1, Tucker S2, Ember T2,
Nadarajah R2, Muntoni F1, Scoto M1
1Dubowitz Neuromuscular Unit, Great Ormond Street Institute of Child
Health, University College London, UK; 2 Orthopaedic and Spinal Surgery
Unit, Great Ormond Street Hospital, London, UK.
Background: Scoliosis is a significant
orthopaedic complication in Spinal Muscular Atrophy (SMA). Bracing is palliative and
surgical intervention frequently required. Studies in small cohorts have reported the
impact of spinal surgery on respiratory function. No data are available on motor
function and weight.
Aim: To better understand the impact of
spinal surgery on respiratory and motor function and weight in patients with SMA II and
non-ambulant III.
Methods: We retrospectively reviewed the
notes of SMA patients who underwent spinal surgery at Great Ormond Street Hospital (last
10 years). Data were collected up to 5 years both before and after the procedure as
Forced Vital Capacity (FVC) %, Hammersmith Functional Motor Scale (HFMS), Revised Upper
Limb Module (RULM), and weight trajectories (UK-WHO growth charts). Cobb angle values
and post-operative pain were also documented.
Results: The notes of 33 patients (26 SMA
II, 7 SMA III) were reviewed. 24 underwent spinal fusion, 4 traditional growing rods
(GR, followed by final growth spinal fusion in 2), 3 magnetic GR. Mean age at surgery
was 10.9 years (range 5.4 – 16.7), mean pre-operative Cobb angle 68 degrees (range 35 –
97). Within the first year 69% showed FVC % decline (mean -11.4, range -1 – -26), 31%
showed FVC % improvement (mean +10.5, range +1 - +20). Motor scores showed progressive
worsening especially in gross motor function with relative spared upper limbs function.
15 patients negatively deviated from previous growth curve in the first year after
surgery; 5 presented significant weight loss (>5% of total weight) successfully
treated with food supplements and/or gastrostomy.
Conclusions: Most patients presented
respiratory function decline early after surgery. A drop in motor function was noticed
which was not reported before. Significant weight loss was detected in a proportion of
patients suggesting this aspect needs careful management. Further studies are planned to
address post-surgical pain.
Challenges in the implementation of nusinersen treatment for SMA in the UK
Anna Peel, Gail Eglon, Robert Muni-Lofra, Anna Mayhew,
Volker Straub, Chiara Marini-Bettolo
Chiara.marini-bettolo@ncl.ac.uk
The John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle University and Newcastle upon Tyne Hospitals NHS Foundation Trust,
Newcastle upon Tyne, UK
Spinal muscular atrophy (SMA) is a rare inherited
neuromuscular condition and until recently no drug treatment was available. Nusinersen
is an antisense oligonucleotide and is the first drug approved by FDA and EMA for all
types of SMA.
Following the positive outcome of an interim analysis
of the Phase 3 ENDEAR study in SMA type 1, Ionis Pharmaceuticals and Biogen announced
the early termination of the ENDEAR study and that all patients on the study would be
transitioned onto an Open Label Extension (OLE) study given the positive results. In
addition, the company opened an Expanded Access Program (EAP) to offer access to the
drug, free of charge, to all patients with SMA1.
This exciting news for the SMA community has, however,
been accompanied by major frustrations due to delays in the implementation of both the
OLE and EAP and more recently delays in the overall NICE approval process of
nusinersen.
The main challenges have been around allocating NHS
resources for the EAP as the cost of delivering the drug was not met by the company.
Unfortunately, the whole process took more than six months to see the first child being
treated in the UK as part of the EAP. Initially, access was allowed only to local
patients registered at the sites authorised to deliver the EAP, raising concerns about
rationale of inclusion criteria and equity to accessing treatment. Since then additional
sites have been able to deliver this new treatment and all eligible SMA1 children in the
UK had access to the treatment.
The EAP closed to all new SMA patients in November 2018
and whilst most EU countries are administering the treatment in clinical practice, in
England we are still going through a NICE appraisal process which is causing delays in
accessing the treatment and raises serious concerns around equity to accessing the
treatment.
AVXS-101 Gene-Replacement Therapy (GRT) for Spinal Muscular Atrophy (SMA): From
Bench to Bedside
Petra Kaufmann1, Kevin
Foust1, Allan Kaspar1, Brian Kaspar1, Jerry
Mendell2–4
1AveXis, Inc., Bannockburn, IL, USA; 2Center for Gene
Therapy, Nationwide Children’s Hospital, Columbus, OH, USA; 3Department of
Pediatrics, Ohio State University, Columbus, OH, USA; 4Department of
Neurology, Ohio State University, Columbus, OH, USA
Background: Onasemnogene abeparvovec
(AVXS-101) is an investigational, one-time GRT that treats the genetic root cause of
SMA, a progressive neurological disease. AVXS-101 delivers the survival motor neuron
gene (SMN) via a self-complementary adeno-associated serotype 9 viral
vector (scAAV9) that crosses the blood-brain barrier. AVXS-101 is designed for immediate
and sustained expression of SMN protein in non-dividing neurons, allowing for rapid
onset and durable therapeutic effect.
Aims: Report AVXS-101 GRT development for
SMA.
Methods: SMA mice
(SMN2+/+,SMNΔ7+/+,Smn-/-)
received intravenous scAAV9-SMN or scAAV9-GFP at P1;
survival and motor function were assessed. Non-human primates (NHPs) received
intravenous scAAV9-GFP; transduced cell types were assessed. In the phase 1 trial
(NCT02122952), symptomatic SMA1 patients received a one-time AVXS-101 infusion at low
(n=3) or proposed therapeutic dose (n=12). Safety (primary objective), event-free
survival (no death/permanent ventilation), sitting unassisted (secondary objectives),
CHOP-INTEND, and other milestones were assessed.
Results: In SMA mice,
scAAV9-SMN improved survival (>200 versus 15 days in controls),
and increased motor function (90% had righting ability at P13 versus ~20% in controls).
In NHPs, scAAV9-GFP efficiently targeted motor neurons throughout the
CNS. In the phase 1 trial, all patients survived, event free, at 24 months. In the
therapeutic dose cohort, 11/12 patients reached CHOP-INTEND =40; 11 sat unassisted =5s,
10 for =10s, 9 for =30s. Two patients crawled, stood, and walked. In the long-term
follow-up study (LTFU), 2 more patients sat =30s and 2 stood with support. No patient
received nusinersen during the 24-month study period. Four patients had an asymptomatic
transient rise in serum aminotransferase. The oldest patient is 59.2 months of age with
53.3 months of follow-up post–AVXS-101 therapy (as of September 27, 2018).
Conclusion: AVXS-101 demonstrated
transformational event-free survival, motor function, and milestone improvements in
symptomatic SMA1 infants. Long-term safety is being monitored for 15 years (LTFU). Phase
3 trials in SMA1 patients in the US and Europe are ongoing. Additional trials
investigate AVXS-101 in presymptomatic SMA, and in older patients using intrathecal
administration. This experience with SMA is hoped to inform other programs that build on
the AveXis platform to bring much-needed gene therapy to patients with rare
diseases.
FIREFISH Part 1: Early clinical results following an increase of survival of motor
neuron protein (SMN) in infants with Type 1 spinal muscular atrophy (SMA) treated with
risdiplam (RG7916)
Giovanni Baranelloa*, Laurent
Servaisb, John W. Dayc, Nicolas Deconinckd,
Eugenio Mercurie, Andrea Kleinf, Basil Darrasg,
Riccardo Massona, Heidemarie Kletzlh, Yumi Clearyh,
Muna El-Khairii, Timothy Seabrookh, Christian Czechh,
Marianne Gerberh, Pia Somugompelyh, Kristina Gelblinh,
Ksenija Gornih and Omar Khwajah
aThe Dubowitz Neuromuscular Centre, University College London Great
Ormond Street Institute of Child Health & Great Ormond Street Hospital, London, UK
and Developmental Neurology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta,
Milan, Italy
bInstitute of Myology, Paris, France; Reference Center for
Neuromuscular Disease, Centre Hospitalier Régional de La Citadelle, Liège,
Belgium
cDepartment of Neurology, Stanford University, Palo Alto, CA,
USA
dQueen Fabiola Children’s University Hospital, Université Libre de
Bruxelles, Brussels, Belgium; Neuromuscular Reference Center UZ Ghent, Ghent,
Belgium
ePaediatric Neurology and Nemo Center, Catholic University and
Policlinico Gemelli, Rome, Italy
Background: Spinal muscular atrophy (SMA)
is characterised by motor neuron loss and muscle atrophy, due to reduced levels of
survival of motor neuron (SMN) protein from loss of function of the
SMN1 gene. While SMN1 produces full-length SMN
protein, a second gene, SMN2, produces low levels of functional SMN
protein. Risdiplam (RG7916; RO7034067) is an investigational, orally administered,
centrally and peripherally distributed small molecule that modulates
SMN2 pre-mRNA splicing to increase SMN protein levels.
Aim: To report on the FIREFISH Part 1
dose-finding study (NCT02913482), an ongoing, multicentre, open-label, two-part,
seamless study of risdiplam in infants aged 1–7 months with Type 1 SMA and two
SMN2 gene copies.
Methods: Part 1 is exploratory and
principally assesses the safety, tolerability, pharmacokinetics and pharmacodynamics of
different risdiplam dose levels (enrolment complete). Confirmatory Part 2 (n=40)
assesses safety and efficacy of risdiplam, with a primary endpoint of the proportion of
infants sitting without support for 5 seconds after 12 months.
Results: Part 1 interim analysis presents
a dose-dependent increase in SMN protein levels in whole blood, with an up to 6.5-fold
increase vs. baseline after 4 weeks of treatment at the highest dose of risdiplam
(2.0–6.5-fold). To date (data-cut 07/09/18), no safety-related stopping rules have been
met, and none of the following events have been reported: loss of ability to swallow,
tracheostomy, or permanent ventilation. Part 1 motor milestone, safety, and survival
data for infants that have been treated for a minimum of 6 months will be presented.
Conclusion: The up to 6.5-fold increase in
SMN protein observed in Part 1 is expected to lead to clinical efficacy based on the
differences in SMN protein levels between SMA severity types (e.g., Type 2 vs. Type 1
with differences of ~2-fold). Part 2 of the FIREFISH study is ongoing.
MRI detection of human motor unit fasciculation in Amyotrophic Lateral
Sclerosis
Roger G Whittaker1, Paola Porcari2, Luis P
Braz3, Timothy L Williams3, Ian S. Schofield3,
Andrew M Blamire4.
1Institute of Neuroscience, Newcastle University.
2 Institute of Cellular Medicine & Centre for in vivo imaging,
Newcastle University.
3Institute of Genetic Medicine & Centre for in vivo imaging,
Newcastle University.
4Directorate of Clinical Neurosciences, Royal Victoria Infirmary,
Newcastle upon Tyne.
Background: Patients with Amyotrophic
Lateral Sclerosis (ALS) typically wait 12 months between symptom onset and receiving a
definitive diagnosis. This delay prevents the early instigation of life-prolonging
therapies, and hampers timely recruitment into clinical trials. There is therefore a
strong need to develop improved diagnostic technologies for ALS. A hallmark of ALS is
the presence of fasciculation; random and irregular muscle twitching produced by
involuntary activation of individual skeletal motor units. The micro-movement of muscle
fibres during fasciculation offers a target for new diagnostic approaches.
Aims: To evaluate the potential of a novel
diffusion weighted MRI protocol to detect fasciculation in patients with ALS.
Methods: We have developed a novel
diffusion weighted MRI protocol which is sensitive to micrometer-scale movement of
skeletal muscle and which can detect activity of motor units [1] – referred to as Motor
Unit MRI (MUMRI). The legs of 4 patients with confirmed ALS and 6 healthy controls were
scanned using the MUMRI method, acquiring time series of scans in both legs at rest over
multiple 3 minute epochs. The frequency and spatial distribution of spontaneous motor
unit activity was assessed.
Results: Patients with ALS showed a
significantly higher rate of spontaneous motor unit activation at rest than controls
(mean 99.1 per minute, range 25.7-161 in patients versus 7.7 per minute, range 4.3-9.7
in controls, p<0.05, Students t-test). The percentage muscle cross sectional area in
which fasciculation was detected was significantly higher in patients than controls
(15.9%, SD 2.8 vs 2.9%, SD 1.6).
Conclusion: Our results are the first
demonstration of the ability of MR imaging to detect fasciculation in patients with ALS.
This technique offers unprecedented insights into human skeletal muscle physiology and
pathophysiology. MUMRI can potentially be extended to whole body assessment and could
provide a rapid, pain free and sensitive means of diagnosing and monitoring patients
with ALS as well as other neuromuscular disorders.
References: 1: Porcari et al, Proc ISMRM,
p330, Paris 2018.
Neuromuscular Junction Disorders and Channelopathies
Investigating muscle phenotype change with age in monogenic disease may provide new
insights into the normal ageing process
K. J. Suetterlin1, R.
Mannikko1, J.
Dick1, H.
Bostock1, M. Grounds2, A.
Sayer3, S.V. Tan1,4, E.
Matthews1, L.
Greensmith1, M.G.
Hanna1
1MRC Centre for Neuromuscular Diseases, UCL Queen Square Institute of
Neurology, UK
2School of Human Sciences, University of Western Australia, Perth,
Western Australia
3NIHR Newcastle Biomedical Research Centre, Newcastle upon Tyne
Hospitals NHS Foundation Trust and Newcastle University, Newcastle upon Tyne,
UK
4Guy’s and St Thomas’ NHS Foundation Trust, London, UK
karen.suetterlin@ucl.ac.uk
Background: It is often reported that the
severity and frequency of acute attacks of paralysis in people with both hyperkalaemic
and hypokalaemic periodic paralysis starts to reduce around age forty. Forty is also the
age around which optimal motor performance in master athletes starts to decline and
above which pathological changes e.g. up to 5 COX negative fibres on muscle biopsy, are
accepted as within normal limits for age.
Aims:
To determine
if the reduction in periodic paralysis attack severity with age is conserved
across species.
To investigate whether the reduction
in periodic paralysis attack severity with age is a result of normal muscle ageing
or ageing in the presence of single ion channel
dysfunction.
Methods: To minimise genetic and
environmental confounders and standardise the paralytic attack, male hyperkalaemic
periodic paralysis (HyperPP) mutant mice were compared to their wild-type brothers. All
animals were housed in the same environment. A paralytic attack was induced on soleus
muscle ex vivo in a tissue chamber maintained at 30 degrees Celsius
using a solution containing 10mM potassium and 1.3mM Calcium as previously described.
The results from young adult (13 to 26 weeks, n=5 WT, n=5 Hyper PP), middle-aged (43 to
70 weeks, n=4 WT, n= 5 Hyper PP) and old (>95 weeks, n=5 WT, n=8 Hyper PP) mice were
compared.
Results: Young and middle-aged soleus lost
significantly more force than old soleus. This was true for both HyperPP (one-way ANOVA
p=0.007) and wild-type (one-way ANOVA p=0.009) animals. This was not simply due to a
reduction in absolute force as maintenance of force also occurred in old soleus that had
greater or equal baseline tetanic force to young soleus. Tetanic force for 2 out of the
5 old wild-type soleus muscles was increased throughout the period of hyperkalaemia.
This was never observed in young or middle-aged wild-type, or young, middle-aged or old
HyperPP soleus.
Conclusions: A reduction in periodic
paralysis attack severity with age appears to be conserved across species. Our data
suggests this phenomenon is the result of ‘normal’ muscle ageing rather than the chronic
consequence of single ion channel dysfunction.
Differences in Muscle Phenotype Severity between Humans and Mice with Monogenic
Disorders may help Identify Novel Therapeutic Pathways
K.J Suetterlin1, H.
Bostock1, R. Mannikko1, J. Dick1, E.
Matthews1, L. Greensmith1, M.G. Hanna1, S.V.
Tan1,2
1MRC Centre for Neuromuscular Diseases, UCL Queen Square Institute of
Neurology, UK
2Guy’s and St Thomas’ NHS Foundation Trust, London, UK
Background: The four transgenic mouse
models of periodic paralysis recapitulate many features of the human disease. They have
similar pathology, the same gender difference in phenotype severity and muscle weakness
is consistently induced on exposure to extremes of potassium. However, none of the mouse
models have ever been observed to have a spontaneous attack of weakness and the
potassium concentration needed to induce an attack is more extreme than that reported
for humans.
Aims:
To identify differences
in normal mouse and human muscle excitability and explore these differences using
selective pharmacological blockade.
To test whether
the identified differences protect against spontaneous attacks of weakness in mice
with periodic paralysis.
Methods: Muscle velocity recovery cycles
(MVRCs) can be used to indirectly measure muscle excitability in vivo.
In human muscle there are two phases of increased conduction velocity. The first, known
as early supernormality, has a correlate in nerve and reflects passive decay of charge
that has accumulated in the sarcolemma. The second, known as late supernormality, is
specific to muscle and reflects activity-induced potassium accumulation in the
t-tubules. MVRCs have not previously been reported in mice. MVRCs were recorded under
anaesthesia in Wild-type C57BlJ6 mouse Tibialis Anterior (TA), using QTRAC software, as
described for humans. Intraperitoneal injection of 9-Anthracene-carboxylic-acid was used
to block ClC-1 channels and micromolar ouabain to selectively block the t-tubule Na/K
pump. MVRCs were recorded pre- and post-pharmacology in the same animal.
Results: In contrast to human MVRCs, mouse
MVRCs had no late supernormality. Blockade of ClC-1 induced late supernormality in mouse
TA (p=0.002, t-test with Welch correction). Blockade of the t-tubule Na/K pump induced
late supernormality in 3 of 14 TAs exposed to ouabain compared to 0 of 66 not exposed to
ouabain (p= 0.007, two-tailed Fisher exact test).
Conclusions: MVRCs can be recorded in
mice. Our data suggest that, compared to humans, mice have more effective t-tubule
potassium buffering and clearance. This is due, at least in part, to an increased ClC-1
conductance and t-tubule Na/K pump activity. The next step will be to determine if this
protects against spontaneous attacks of weakness in mice with periodic paralysis.
Anderson Tawil Syndrome: expanding the phenotype and assessing cardiac risk
Vinojini Vivekanandam, Karen Suetterlin, Natalie James,
Sarah Holmes, Damian Kozyra, Louise Germain, Iwona Skorupinska, Richa Sud,
Konstantinos Savvitis, Jatin Pattni, Dorren Fialho, Michael G Hanna, Emma
Matthews
MRC Centre for Neuromuscular Diseases, Queens Square Institute of
Neurology.
Background: Anderson Tawil Syndrome (ATS)
is a rare neuromuscular channelopathy traditionally characterised by periodic paralysis,
cardiac arrhythmias and dysmorphic features. Mutations in the KCJN2 gene are associated
with ATS. Accurate and early diagnosis is important in facilitating treatment of
episodic paralysis and preventing potentially life-threatening cardiac events.
Aims: To fully characterise the phenotype
in a carefully stratified cohort including cognitive deficits and cardiac risk.
Patients: At the Nationally Commissioned
Highly Specialised Service for Channelopathies we have one of the largest cohorts of
patients with ATS in the world. Patients with a genetically confirmed diagnosis were
consented and enrolled into our Channelopathy cohort study. Clinical and family history,
examination findings and investigations including neurophysiology, psychometric, cardiac
and radiological assessments were reviewed.
Results: 66 patients were identified with
KCJN2 mutations. Comprehensive clinical information has so far been collected for 18.
Cardiac symptoms were prominent. Two thirds (11) had daily or very frequent
palpitations. 27% (5) reported shortness of breath. Serious cardiac complications
occurred in 4 patients (ICD insertion, left ventricular dysfunction/cardiomyopathy). 39%
(7) reported pain which has previously not been appreciated in ATS. At least 44.4%(8)
patients had fasciculations. 22%(4) patients had minimal decrement (<48%) on Long
Exercise Tests (LET) despite having episodic weakness. Lower limb Muscle MRI was
abnormal in 6 patients. Daytime somnolence was reported, prompting sleep studies.
Neuropsychometric testing suggests slowed processing speed. While dysmorphic features
exist, these can be subtle. Short stature is not ubiquitous, with a height range of
147-180.34cm. Heterogeneity within families was commonly seen.
Conclusions: The phenotypic spectrum of
ATS is broader than currently appreciated including frequent pain, fasciculations and
daytime somnolence suggestive of nocturnal hypoventilation. Fasciculations may suggest
additional lower motor neurone pathology. A negative LET is common and should not deter
from pursuing genetic testing. Cardiac symptoms are common and complications beyond
ventricular ectopics require screening for. Data analysis is ongoing.
Applied Neuromuscular-Junction Facility to investigate structure and function of
mammalian neuromuscular junction
Veronika Boczonadi1*, Clarke
Slater1, Rita Horvath2, Hanns Lochmuller3, Roger
Whittaker1 Alex Laude1, Doug Turnbull4
1Institute of Neuroscience, Bio-Imaging unit, Newcastle University,
Framlington Place Newcastle upon Tyne, NE2 4HH, UK
(veronika.boczonadi@ncl.ac.uk);
2University of Cambridge, Department of Clinical Neuroscience;
3Department of Genetics, CHEO Research Institute, University of Ottawa, K1H
8L1 Ottawa, Canada
4Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2
4HH
Background: The Neuromuscular junction
(NMJ) is the specialized chemical synapse that mediates the transmission of action
potentials in motor axons to skeletal muscle fibers. The NMJ is the best characterized
cholinergic synapse and its study for many years has provided much of our general
knowledge of synapse structure, function and development. While an increasing number of
molecular defects are known to cause impaired neuromuscular transmission, in very few
cases has the impact of these molecular defects on the structure and function of the
NMJs been characterized in detail, much less understood. Partly as a result of this lack
of knowledge, available treatments for diseases of the NMJ are limited in number and
efficacy.
Aims: To address these concerns we aimed
to establish the Applied NMJ Facility to study NMJ structure and function.
Methods: Here, we are initially using
murine nerve-muscle preparations for electrical recording combined with high-resolution
laser confocal microscopy (LSM880) allowing accurate investigation of NMJ functionality
at a single synapse resolution. It is our intention to extend our structural and
functional NMJ studies to human biopsy samples later on this year.
Result: Our facility can provide detailed
information about synaptic transmission at the NMJ, using both extra- and intra-cellular
electrical recording, as well as high resolution imaging of NMJ morphology, membrane
recycling, mitochondrial membrane potential and Ca2+ dynamics.
Conclusion: This is a broadly applicable
technique which can be adopted to investigate alterations of NMJ activity. Furthermore
it will facilitate the development and assessment of new therapies in mouse models of
neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and
myasthenic syndromes.
Exploring novel treatments for disorders that feature defects of the neuromuscular
junction structure
Mirela D. Panea1, MSc, Judith
Cossins1, DPhil, David Beeson1, PhD
1Weatherall Institute of Molecular Medicine, Nuffield Department of
Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
Congenital myasthenic syndromes (CMS) are a group of
inherited disorders that affect the signal transmission at the neuromuscular junction
(NMJ) and share the clinical feature of fatigable muscle weakness. In
DOK7-CMS compromised neurotransmission is due to small and
destabilised NMJs. DOK7 is a cytoplasmic-adaptor protein that amplifies the signalling
from muscle-specific receptor tyrosine kinase (MuSK), that is responsible for the
formation and stabilisation of the NMJ. It has recently been shown that large amounts of
DOK7 protein in muscles generate enlarged NMJ in mice, which are very efficient in
signal transmission and have no reported detrimental effects.
This study aims to identify small molecules that can
specifically upregulate the amount of DOK7 in muscles.
First, the level of upregulation needed to generate the
enlarged NMJ is undefined. We used a retroviral vector harbouring a human
DOK7 expression cassette to infect wild-type C2C12 and subsequently
titrate levels of DOK7 needed to generate enlarged AChR clusters. A 4-5 fold increase in
DOK7 protein levels proved to be enough to mimic the generation of enlarged synapses
in vitro.
Subsequently, approximatively 5000 small molecules from
a library of muscle-specific compounds were used in screening experiments. Initial
screenings were performed in triplicates on a C2C12 reporter cell line designed to
contain a 1-Kb sequence of the DOK7 promoter fused to the Luciferase-reporter.
Confirmatory results were performed for potential candidate upregulating compounds on a
similar EGFP reporter cell line. Five potential hits that show significant upregulation
after being tested on both reporter cell lines, are being further investigated. They
will provide the basis for a more detailed analysis of the effects of DOK7-upregulation
in cell culture biological assays and ultimately for in vivo
testing.
Email address:
mirela.panea@ndcn.ox.ac.uk
Salbutamol modulates postsynaptic specialisation at the neuromuscular junction in a
mouse model of ColQ myasthenic syndrome
aJohn Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle University, Newcastle Upon Tyne, UK
bInstitute of Neuroscience, Newcastle University, Newcastle Upon
Tyne, UK
cDepartment of Clinical Neurosciences, University of Cambridge,
Cambridge, UK
dDepartment of Neuropediatrics and Muscle Disorders, Medical Center –
University of Freiburg, Faculty of Medicine, Freiburg, Germany
eCentro Nacional de Análisis Genómico (CNAG-CRG), Center for Genomic
Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona,
Catalonia, Spain
fChildren’s Hospital of Eastern Ontario Research Institute,
University of Ottawa, Ottawa, Canada and Division of Neurology, Department of
Medicine, The Ottawa Hospital, Ottawa, Canada
Correspondence: grace.mcmacken@ncl.ac.uk
Background: The β-adrenergic agonists
salbutamol and ephedrine have proven to be an effective therapy for human disorders of
the neuromuscular junction (NMJ), in particular many subsets of congenital myasthenic
syndromes (CMS). However, the mechanisms underlying this clinical benefit is
unknown.
Aims: In order to explore the effect of
salbutamol in CMS, we investigated the effect of salbutamol treatment on the NMJ in the
ColQ deficient mouse, a model of end-plate acetylcholinesterase deficiency.
Methods: ColQ-/- mice received
7 weeks of daily salbutamol injection, and the effect on muscle strength and NMJ
morphology was analysed.
Results: Salbutamol to a gradual
improvement in muscle strength in ColQ-/- mice. In addition, the NMJs of
salbutamol treated mice showed significant improvements in several postsynaptic
morphological defects, including acetylcholine receptor density and area, but did not
affect nerve terminal area or axon diameter. Salbutamol had no measurable effect on
muscle fibre size or fibre type proportion.
Conclusion: These results suggest that
β-adrenergic agonists lead to clinical benefit in CMS by inducing long-term structural
changes at the NMJ, and that these effects are primarily at the postsynaptic
membrane.
Congenital myasthenic syndrome due to a mutation in a nuclear membrane
protein
Judith Cossins1, Pedro
Rodriguez Cruz1,2, Richard Webster1, Susan Maxwell1,
Jacqueline Palace2, Ji-Yeon Shin3, David Beeson1
1Neurosciences Group, Weatherall Institute of Molecular Medicine,
University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
2Nuffield Department of Clinical Neurosciences, John Radcliffe
Hospital, University of Oxford, Oxford OX3 9DU, UK.
3Department of Cell and Developmental Biology, University of
Michigan, Ann Arbor, United States
Email: Judith.cossins@imm.ox.ac.uk
Background: Next generation sequencing has
led to the identification of an increasing number of unexpected gene mutations affecting
synaptic transmission at the neuromuscular junction. Here we have identified a family
with a novel congenital myasthenic syndrome (CMS) with mutations in a nuclear envelope
protein. We describe both the characterisation of this disorder and our investigation of
the underlying pathogenic mechanism.
Aim: To learn how a ubiquitously expressed
nuclear envelope protein mutation can cause a disorder where the dominant symptoms are
due to defective neuromuscular transmission.
Results: The index case and his brother
have mild fatiguable muscle weakness in shoulder abductors, finger extensors and ankle
dorsiflexors, and mild wasting of deltoid and medial gastrocnemius. They showed
decrement of 40% and 26% on RNS at 3Hz respectively. Whole exome sequencing identified a
homozygous single nucleotide deletion c.127delC; p.P43fs in TOR1AIP1.
The gene encodes nuclear envelope protein LAP1 (lamin-associated protein 1), of which at
least two isoforms (LAP1B and shorter LAP1C) exist. The variant, which is predicted to
ablate the expression of LAP1B but not LAP1C, was shown by Sanger sequencing to
segregate with disease. Mutations in TOR1AIP1 are rare and an effect on
neuromuscular transmission has not previously been reported. Trapezius biopsy showed
mild variation in muscle fibre size, and no detectable expression of LAP1 in muscle
nuclei. Electron microscopy revealed abnormal herniated nuclei. A mouse model for the
disorder was obtained in which LAP1 is ablated in striated muscle. Mice were initially
strong, with normal EMG and diaphragm spontaneous and evoked endplate potentials. They
became weak from 6-7 weeks of age and by 12 weeks of age showed up to 15% decrement at
20Hz RNS. Diaphragm spontaneous and evoked endplate potentials became prolonged, and EDL
and soleus endplates were very fragmented with some sprouting and denervation. Synaptic
AChR levels were unchanged in the model mouse diaphragm compared with wild type
(125I-BuTx), but extra-synaptic AChRs increased 3-fold. MuSK expression was
decreased. H&E staining showed a large variation in fibre size with central nuclei.
Electron microscopy showed muscle fibre morphology ranging from normal to very
disorganised and atrophied, and with abnormal endplates structure.
Conclusion: Our data is consistent with denervation and muscle
degeneration and regeneration, with ongoing remodelling of the NMJs, and impaired
neuromuscular transmission caused by defective synaptic structure.
Mitochondrial Disease
The phenotypic and genotypic spectrum of MT-ATP6-related
mitochondrial disease
Yi Shiau Ng1, Gráinne S.
Gorman1, Mika H. Martikainen1,2, Alasdair Blain1,
Enrico Bugiardini3, Apphia Bunting4, Andrew M.
Schaefer1, Charlotte L. Alston1, Emma L. Blakely1,
Sunil Sharma1, Imelda Hughes5, Albert Lim1, Christian
DeGoede6, Meriel McEntagart7, Stefan Spinty8, Iain
Horrocks9, Mark Roberts10, David Gosal10, Cathy E.
Woodward3, Patrick F. Chinnery11, Rita Horvath1,
Victoria Nesbitt12, Carl Fratter13, Joanna Poulton4,
Michael G. Hanna3, Robert D.S. Pitceathly3, Robert W.
Taylor1, Doug M. Turnbull1, Robert McFarland1
Email: yi.ng@ncl.ac.uk
1Wellcome Centre for Mitochondrial Research, Newcastle University,
Newcastle upon Tyne, UK
2Faculty of Medicine, University of Turku, and Division of Clinical
Neurosciences, Turku University Hospital, Turku, Finland
3MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology
and National Hospital for Neurology and Neurosurgery, London, UK
4Nuffield Department of Obstetrics and Gynaecology, University of
Oxford, Oxford, UK.
5Royal Manchester Children’s Hospital, Central Manchester University
Hospitals NHS Foundation Trust, Manchester, UK.
6Department of Paediatric Neurology, Royal Preston Hospital, Preston,
UK.
7South West Thames Regional Genetics Service, St. George’s Hospital,
London, UK.
8Alder Hey Children’s NHS Foundation Trust, Liverpool, UK.
9Greater Glasgow and Clyde NHS Yorkhill Hospital, Glasgow,
UK.
10Greater Manchester Neuroscience Centre, Salford Royal NHS
Foundation Trust, Manchester Academic Health Science Centre, Salford, UK.
11Department of Clinical Neuroscience & Medical Research Council
Mitochondrial Biology Unit, School of Clinical Medicine, University of Cambridge,
Cambridge, UK
12Department of Paediatrics, The Children’s Hospital, Oxford,
UK.
13Oxford Medical Genetics Laboratories, Oxford University Hospitals
NHS Foundation Trust, Oxford, UK.
Background: Mutations in
MT-ATP6 are associated with maternally-inherited Leigh syndrome (LS)
and neurogenic weakness, ataxia and retinitis pigmentosa (NARP). Early studies suggested
a strong correlation between mutant heteroplasmy level and disease severity in two
common pathogenic variants, m.8993T>C/G.
Aims: To determine the phenotypic and
genotypic spectrum of MT-ATP6-related mitochondrial disease.
Methods: A retrospective, observational
cohort study (January 2009 – October 2018) of individuals referred to the three national
mitochondrial centres in Newcastle upon Tyne, London and Oxford that comprise the NHS
Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children.
Results: We identified 88 clinically
affected individuals (median current age 26.5 years, range 0.75–74 years, interquartile
range: 33.3 years) and 37 asymptomatic family members from 60 pedigrees. Fifteen
patients (17%) died, and four patients were lost to follow up during the study period.
The common clinical features were cerebellar ataxia (83%), peripheral neuropathy (74%),
and cognitive dysfunction (65%). Thirty-six per cent of patients manifested with Leigh
syndrome (LS), and only 7 patients exhibited a pure NARP syndrome. The median age of
onset was younger in patients with LS compared to those presenting with other clinical
phenotypes (1.5 vs 15 years, p<0.001). Four adult patients developed unexpected,
subacute brainstem dysfunction. Nine pathogenic variants were identified in our patient
cohort, and five of them accounted for 91% of all cases. The maternal transmission of
the pathogenic MT-ATP6 variants was established in 68 patients (77%),
and the mutation likely arose de novo in three patients (3%). Logistic
regression modelling revealed a correlation between the risk of clinical manifestation
and mutant heteroplasmy level in five common mutations; the chance of being
clinically-affected increasing significantly when the mutant load exceeded 50%.
Conclusion: This is the most extensive
observational study of MT-ATP6-related mitochondrial disease to date.
Our data highlight that sequencing of the MT-ATP6 gene should be
included in the diagnostic workup of patients with both cerebellar ataxia and
neuropathy. Clinicians should be aware that brainstem crisis can occur in adult patients
without previously manifesting LS. Moreover, the expression threshold for the mutant
mtDNA heteroplasmy associated with common, pathogenic MT-ATP6 variants
have important implications for genetic counselling and reproductive options.
FBXL4 related mitochondrial disease is associated with pyruvate
dehydrogenase deficiency
Carl Fratter1, Kate
Sergeant1, Ruth Brown1, Joanna Poulton2, James
Davison3, Emma Footitt3, Usha Kini4, Thomas
Cullup5, Shamima Rahman3,6, Garry Brown1
1Oxford Medical Genetics Laboratories, Oxford University Hospitals
NHS Foundation Trust, Oxford
2Nuffield Department of Women’s and Reproductive Health, University
of Oxford, Oxford
3Department of Metabolic Medicine, Great Ormond Street Hospital for
Children NHS Foundation Trust, London
4Oxford Centre for Genomic Medicine, Oxford University Hospitals NHS
Foundation Trust, Oxford
5Regional Molecular Genetics Laboratory, Great Ormond Street Hospital
for Children NHS Foundation Trust, London
6UCL Great Ormond Street Institute of Child Health, London
Background:FBXL4 related mitochondrial disease or FBXL4 deficiency is a severe
autosomal recessive mitochondrial disorder, typically characterised by neonatal or
infantile onset lactic acidosis, failure to thrive, muscular hypotonia, and variable
additional features. FBXL4 deficiency was first described as a disorder of mitochondrial
DNA (mtDNA) maintenance associated with mtDNA depletion and combined mitochondrial
respiratory chain deficiency. FBXL4 protein is localised to mitochondria but its
function remains unknown.
Aims: To identify patients with FBXL4
deficiency and assess the associated biochemical defects.
Patients and Results:FBXL4 sequencing of 85 patients with suspected mtDNA depletion syndrome
in Oxford did not identify any cases of FBXL4 deficiency. Analysis of other patients
with suspected mitochondrial encephalomyopathy presenting neonatally or in infancy
identified 5 unrelated patients homozygous or compound heterozygous for pathogenic /
likely pathogenic variants in FBXL4. All 5 patients were found to have
pyruvate dehydrogenase deficiency in fibroblasts (0.23-0.57 nmol/mg protein/min; normal
range 0.6-0.9), whereas there was no evidence of mitochondrial respiratory chain
deficiency in muscle (results available for 4/5 patients).
Conclusion: We propose that PDH deficiency is a common consequence
of loss of functional FBXL4 protein and has a major contribution to the disease
phenotype. If substantiated, this finding may have important treatment implications for
patients with FBXL4 deficiency.
Email: carl.fratter@ouh.nhs.uk
Exploring the Impact of mtDNA Mutations on the Metabolic and Epigenetic Profiles of
hIPSC-Derived Myogenic Cells
Benjamin O’Callaghan1* &
Monika Madej1, Michael G. Hanna1, Jennifer Morgan2,
Henry Houlden1
1MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology,
London, 2Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute
of Child Health, London
*Email: zcbthj1@ucl.ac.uk
Background: A number of chromatin
modifying enzymes are reliant on intermediate products of metabolism, underscoring a
focal point that links a cell’s metabolic state with its transcriptional profile.
Metabolic impairments caused by mitochondrial DNA (mtDNA) mutations have the potential
to impact the epigenetic landscape and might represent an unexplored pathomechanism
contributing to the disease that warrants further investigation.
Aims: To develop a cell model for
exploring novel pathomechanisms contributing to mitochondrial disease in skeletal
muscle, including the impact mtDNA mutations have on enzymatic chromatin
modifications.
Methods: In this study we utilised human
induced pluripotent stem cell (hIPSC) technology to establish an in
vitro model of mitochondrial disease. hIPSC clones have been generated from
patients harbouring the heteroplasmic m.8344A>G mt-tRNALys and
m.3243A>G mt-tRNALeu(UUR) mutations most commonly associated with
myoclonic epilepsy with ragged red fibres (MERRF) and Mitochondrial encephalomyopathy,
lactic acidosis, and stroke-like episodes (MELAS) syndromes respectively. By taking
advantage of the random segregation of mutant mtDNA in patient fibroblast populations,
we obtained hIPSC lines with high heteroplasmy for disease modelling alongside isogenic
control lines with low/undetectable levels.
Patient-specific hIPSC lines were then differentiated
into disease-relevant myogenic cell types by recapitulating developmental signalling
gradients that occur during myogenesis. Differentiated cells express key myogenic
regulatory factors (MyoD, Myogenin) and form multinucleated
MyHC+/Titin+ myotubes whilst retaining PAX7+
satellite-like cells.
Results: The m.3243A>G mutation appears
to have a detrimental effect on overall myogenicity. Myotubes with high m.3243A>G
load also show impairments in mitochondrial function and fail to initiate expression of
certain MyHC isoforms which might indicate an impairment in myotube maturation. ChIP-Seq
libraries are currently being subjected for genome-wide sequencing in order to assess
locus-specific changes in metabolically sensitive epigenetic marks that might underlie
observed impairments. This in vitro model provides new insight into
pathomechanisms of mitochondrial disease in skeletal muscle. Epigenetic modifications
and/or expression of specific loci that are affected by mtDNA mutations are being tested
as new targets for novel therapeutic interventions. Ben O’Callaghan is supported by a
PhD studentship from the MRC Centre for Neuromuscular Diseases.
Mutations in POLRMT impair mitochondrial transcription and are
associated with a spectrum of mitochondrial disease clinical presentations
Monika Oláhová1*, Bradley
Peter2, Hector Diaz2, Zsolt Szilagyi2, Ewen W.
Sommerville1, Emma L. Blakely1, Jack Collier1, Viktor
Stránecký3, Hana Hartmannová3, Anthony J. Bleyer3,4,
Kim L. McBride5, Sasigarn A. Bowden6, Zuzana
Korandová7, Alena Pecinová7, Hans-Hilger Ropers8, Kimia
Kahrizi9, Hossein Najmabadi9, Mark Tarnopolsky10,
Lauren I. Brady11, Nicole Weaver12, Carlos E.
Prada12,13,14, Tomáš Mrácek7, Stanislav Kmoch3,
Gráinne Gorman1, Maria Falkenberg2, Claes Gustafsson2,
Robert W. Taylor1
1Wellcome Centre for Mitochondrial Research, Newcastle University,
Newcastle upon Tyne, UK
2Department of Medical Biochemistry and Cell Biology, University of
Gothenburg, Sweden
3Research Unit for Rare Diseases, Department of Pediatrics and
Adolescent Medicine, First Faculty of Medicine, Charles University, Prague, Czech
Republic
4Section on Nephrology, Wake Forest School of Medicine,
Winston-Salem, USA
5Center for Cardiovascular and Pulmonary Research, Department of
Pediatrics, Nationwide Children’s Hospital, The Ohio State University College of
Medicine, Columbus, USA
6Division of Endocrinology, Nationwide Children’s Hospital, The Ohio
State University College of Medicine, Columbus, USA
7Department of Bioenergetics, Institute of Physiology of the Czech
Academy of Sciences, Prague, Czech Republic
8Max Planck Institute for Molecular Genetics, Berlin,
Germany
9Genetics Research Center, University of Social Welfare and
Rehabilitation Sciences, Tehran, Iran
10Department of Pediatrics, Division of Neuromuscular and
Neurometabolic Diseases, McMaster University, Hamilton, Canada
11Department of Pediatrics, McMaster University Children’s Hospital,
Hamilton, Canada
12Division of Human Genetics, Cincinnati Children’s Hospital Medical
Center, Cincinnati, OH, USA
13Department of Pediatrics, University of Cincinnati College of
Medicine, Cincinnati, OH, USA
14Department of Pediatrics, Cardiovascular Foundation of Colombia,
Floridablanca, Colombia
*monika.olahova@ncl.ac.uk
Background: The vast majority of
mitochondrial disorders result from mutations in components of the nuclear-encoded
mitochondrial DNA (mtDNA) maintenance machinery and oxidative phosphorylation (OXPHOS)
subunits. The role of the mtDNA transcription machinery in mitochondrial disease,
however, remains relatively unknown. The mitochondrial RNA polymerase (POLRMT) is the
sole RNA polymerase in mitochondria and is responsible for the transcription of the
mitochondrial genome.
Aim: To characterise the clinical and
molecular nature of novel POLRMT variants that underlie the
mitochondrial disease-associated phenotype present in five unrelated individuals.
Patients and Methods: Using whole-exome
sequencing, we identified novel recessive and dominant POLRMT variants
in five individuals presenting with a variety of clinical problems, ranging from global
developmental delay, hypotonia and growth defects in childhood to late onset progressive
external ophthalmoplegia (PEO). Where investigated, these defects were accompanied by
either a mosaic cytochrome c oxidase deficiency in skeletal muscle
and/or multiple respiratory chain enzyme deficiencies. Mitochondrial mRNA and OXPHOS
protein levels were assessed in mutant POLRMT fibroblasts (3 of 5
patients). In addition, recombinant mutant POLRMT proteins were generated in order to
determine the effect of POLRMT variants on mitochondrial
transcriptional activity in vitro.
Results: Functional characterisation of
patient fibroblasts revealed a defect in mitochondrial mRNA synthesis, although no mtDNA
deletions or copy number abnormalities were identified. Mild decreases in the levels of
both OXPHOS subunits and fully-assembled complexes were observed in
vivo, whilst functional in vitro characterisation of the
investigated recombinant POLRMT variants revealed that patient
mutations exhibited variable, but deleterious effects on mitochondrial
transcription.
Conclusion: Our results demonstrate for
the first time, that pathogenic variants in the POLRMT gene can cause a
spectrum of clinical phenotypes ranging from childhood-onset developmental delay to
late-onset PEO and emphasise the importance of defective mitochondrial transcription as
a disease mechanism.
Mild disease spectrum and trajectory in MTFMT-related Leigh
syndrome
Yi Shiau Ng,1 Hannah
Hayhurst,1 Irenaeus F. M. de Coo,2 Dorota
Piekutowska-Abramczuk,3 Charlotte L. Alston,1 Sunil
Sharma,1 Kyle Thompson,1 Rocio Rius,4, 5 Langping
He,1 Sila Hopton,1 Rafal Ploski,3 Elzbieta
Ciara,3 Nicole J. Lake, 4, 5 Alison G. Compton,4, 5
Martin B. Delatycki,4, 5 Aad Verrips,6 Penelope E.
Bonnen,7 Simon Jones,8 Andrew A. Morris,9 David
Shakespeare,10 John Christodoulou,4,5 Dorota
Wesol-Kucharska,11 Dariusz Rokicki,11 Hubert J. M.
Smeets,12 Ewa Pronicka,3, 11 David R. Thorburn,4, 5
Gráinne S. Gorman,1 Robert McFarland,1 Robert W.
Taylor1
Email: yi.ng@ncl.ac.uk
1Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom
2Department of Neurology, Erasmus Medical Centre, Rotterdam,
Netherlands
3Department of Medical Genetics, Warsaw Medical University, Warsaw,
Poland
4Murdoch Children’s Research Institute and Victorian Clinical
Genetics Services, Royal Children’s Hospital, Parkville, Victoria 3052,
Australia
5Department of Paediatrics, University of Melbourne, Melbourne,
Victoria 3052, Australia
6Department of Neurology, Canisius Wilhelmina Hospital, Nijmegen, The
Netherlands
7Department of Molecular and Human Genetics, Baylor College of
Medicine, Houston, Texas, USA
8Manchester Centre for Genomic Medicine, Central Manchester
University Hospitals NHS Foundation Trust, St Mary’s Hospital, Manchester, United
Kingdom
9Central Manchester University Hospitals NHS Foundation Trust,
Manchester Academic Health Science Centre, Manchester M13 9WL, UK.
10Neuro-rehabilitation unit, Royal Preston Hospital, Preston, United
Kingdom
11Department of Paediatrics, Nutrition and Metabolic Diseases, The
Children’s Memorial Health Institute, 04-730 Warsaw, Poland
12Department of Clinical Genomics, Maastricht University, The
Netherlands
Background: Mitochondrial methionyl-tRNA
formyltransferase (MTFMT) is required for the initiation of translation and elongation
of mitochondrial protein synthesis. Pathogenic variants in MTFMT have
been associated with Leigh syndrome (LS) and mitochondrial multiple respiratory chain
deficiencies.
Aims: To elucidate the spectrum of
clinical, neuroradiological and molecular genetic findings of patients with bi-allelic
pathogenic MTFMT variants.
Methods: Retrospective cohort study
combining new cases and previously published cases.
Results: In this multi-centre study, we
identified eight new patients with pathogenic MTFMT variants and 30
other previously reported cases. The median age of presentation was 14 months (range:
birth to 17 years, interquartile range (IQR) 4.5 years), with developmental delay (59%)
and motor symptoms (47%) being the most common presenting clinical features. Raised
serum lactate was evident in 83% of patients (range 2.7 – 14.3 mmol/L, normal < 2.2),
and a structural cardiac abnormality was observed in 12 patients. MRI head findings
included symmetrical basal ganglia changes (62%), periventricular and subcortical white
matter abnormalities (55%), and brainstem lesions (48%). Six patients (18%) had neither
basal ganglia nor brainstem signal abnormalities. Twenty-five of 34 patients (74%) were
alive at the time of their last clinical follow up (median 6.8 years, range: 14
months-31 years, IQR: 14.5 years); 29% of patients survived into adulthood (=18 years).
Isolated complex I and combined respiratory chain deficiencies were identified in 31%
and 59% of the cases, respectively. Reduction of mitochondrial complex I and complex IV
subunits was identified in all patient fibroblasts analysed (n=13). Sixteen pathogenic
MTFMT variants were identified, of which c.626C>T represents a
founder mutation in the European population.
Conclusion: Patients that harbour
pathogenic variants in MTFMT have a milder clinical phenotype and
disease progression compared to those patients with LS caused by other nuclear-driven
mitochondrial defects. Evaluation of patient fibroblasts may preclude the need for a
muscle biopsy to prove causality of novel variants.
Molecular mechanisms of mitochondrial disease: pathological and genetic studies in
Mendelian disorders of mtDNA maintenance
Diana Lehmann1, Georgia, E. Campbell2,
Helen A.L. Tuppen2, Conor Lawless2, Hannah S. Rosa2,
Mariana C. Rocha2, Amy K. Reeve2,4, Thomas Nicholls2,
Stephan Zierz3, Robert W. Taylor2, Doug M. Turnbull2,4,
Amy E. Vincent2,4
1Department of Neurology, University of Ulm, Ulm, Germany
2Wellcome Centre for Mitochondrial Research, Newcastle University,
Newcastle-upon-Tyne, UK
3Department of Neurology, University of Halle-Wittenberg,
Halle/Saale, Germany
4Centre for Ageing and Vitality, Newcastle University,
Newcastle-upon-Tyne, UK
Email: amy.vincent@ncl.ac.uk
Background: Mitochondrial DNA (mtDNA)
deletions are an important pathological mechanism in adults with mtDNA maintenance
disorders. These mtDNA deletions clonally expand within post-mitotic cells (neurons and
muscle) causing mitochondrial respiratory chain deficiency.
Aim: To attempt to correlate mitochondrial
genetics and respiratory chain deficiency at a single cell level and assess whether
deleted mtDNA species have a replicative advantage.
Methods: We used immunofluorescence to
quantify mitochondrial respiratory chain deficiency in muscle biopsies from patients
with mtDNA maintenance disorders (n=16). Using the same tissue section from a subset of
patients (n=6), we performed laser microdissection and single cell genetic analysis to
investigate the relationship between mtDNA genetics and respiratory chain deficiency.
For a further 3 patients then used single molecule PCR in combination with real time PCR
to determine if a replicative advantage explains the accumulation of mtDNA
deletions.
Results: Quadruple immunofluorescence
demonstrated no obvious differences between patients with dominant or recessive
mutations, with the majority of patients showing some fibres with isolated CI deficiency
and some fibres with both CI and CIV deficiency. Genetic analysis demonstrated major arc
deletions to be more common and showed a clear correlation between deletion level and
respiratory chain deficiency. We find that 62.8% of respiratory chain deficient muscle
fibres contained a single deletion, 34.6% two deletions and 2.6% three deletions. In
cells with multiple mtDNA deletions we find that 56.7%the smallest mtDNA deletion
(Largest mtDNA molecule) clonally expands to the highest level.
Conclusion: There is no genotype based
pattern of respiratory chain deficiency. We demonstrate a clear correlation between the
level of mtDNA deletion and extent of respiratory chain deficiency within a single cell.
Single molecule PCR in combination with real time PCR demonstrates that there appears to
be no replicative advantage for smaller mtDNA molecules.
Understanding multi-dimensional respiratory chain deficiency phenotypes in single
skeletal muscle fibres
Charlotte Warren1,2, Conor
Lawless1, David McDonald3, David Deehan4, Robert W.
Taylor1,5, Andrew Filby3, Doug M. Turnbull1,2, Amy E.
Vincent1,2
1Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom,
2Centre for Ageing and Vitality, Newcastle University, Newcastle upon
Tyne, United Kingdom,
3Flow Cytometry Core Facility, Newcastle Biomedicine, Newcastle
University, Newcastle upon Tyne, United Kingdom,
4Institute of Cellular Medicine, Newcastle University, Newcastle upon
Tyne, United Kingdom,
5National Health Service Highly Specialised Mitochondrial Diagnostic
Laboratory, Newcastle upon Tyne Hospitals, National Health Service Foundation Trust,
Newcastle upon Tyne, United Kingdom
Email: c.warren@ncl.ac.uk
Background: Mitochondrial diseases are
heterogeneous diseases that can arise due to mutations in either the nuclear or
mitochondrial genomes. These conditions are further complicated by the polypoid nature
of mitochondrial DNA (mtDNA), which means that mtDNA mutations can be heteroplasmic and
lead to a mosaic pattern of deficiency within tissues such as skeletal muscle.
Limitations in techniques to date have allowed either highly multiplexed analysis of
protein expression or spatially resolved analysis of the expression of a small number of
proteins.
Aim: To optimise and use imaging mass
cytometry for the assessment of mitochondrial proteins in skeletal muscle of patients
with mitochondrial disease.
Methods: We used imaging mass cytometry to
investigate changes in respiratory chain complexes I-V (CI-V) and mitochondrial mass
markers. Skeletal muscle was assessed from patients with nuclear encoded CI mutations
(n=2), single, large-scale mtDNA deletions (n=2), the m.3243A>G mtDNA point mutation
(n=2) and three other tRNA variants (n=3).
Results: Imaging mass cytometry
demonstrated isolated and severe CI deficiency in 100% of muscle fibres in patients with
nuclear encoded CI mutations. In comparison mosaic patterns of CI, CIII and CIV
respiratory chain deficiency was observed for patients with mtDNA variants. Most
interestingly, in tRNA patients we find that fibres with CI deficiency have a
compensatory increase in CII and CV.
Conclusion: Imaging mass cytometry allows
correlation of a large number of mitochondrial proteins and thus in-depth
characterisation of respiratory chain deficiency. Furthermore, it will allow us to
correlate respiratory chain deficiency with a range of cell signalling and other
mitochondrial markers.
A novel multiplex chromogenic immunoassay for evaluating mitochondrial respiratory
chain complex I and complex IV defects in diagnostic muscle biopsies
D. Chambers1 , A.
Kumar2, L. Feng1, I. Hargreaves3 , A. Lam4
, A. Manzur4 , F. Muntoni4, C. Sewry4, J.
Poulton5, R. Phadke1
1UCL Institute of Neurology, London, UK;
2National Hospital UCLH, London, UK;
3John Moores University, Liverpool, UK;
4Great Ormond Street Hospital, London, UK;
5University of Oxford, Oxford, UK
Background: The investigation of
clinically suspected mitochondrial disease (mtD) includes performing a skeletal muscle
biopsy for biochemical/histochemical assessment of mitochondrial respiratory chain (RC)
defects. COX-SDH histochemistry detects RC-complex IV (CIV) defects, but RC-complex I
(CI) defects cannot be detected histochemically. CI/CIV defects are common in mtD.
Immunohistochemical evaluation of RC-complex defects relies on reduced amount of the
assembled complex associated with catalytic deficiency, detectable with RC
subunit-specific monoclonal antibodies.
Aims: Our aim was to design a dual
chromogenic immunoassay (DCI) for evaluating CI/CIV defects in diagnostic muscle
biopsies to complement the existing histochemical assays.
Methods or Patients or Materials: In the
DCI optimised protocol, primary antibodies (Abcam), TOMM20 (mitochondrial mass), NDUFB8
(CI) and MTCO1 (CIV) were co-incubated (TOMM20+CI and TOMM20+CIV), and then TOMM20
developed to yellow and the other marker to teal (Discovery/Ventana Systems) with
co-localising antibodies visualising as green. The DCI and COXSDH assays were performed
in serial frozen sections. 23 biopsies were assessed: 15 with genetically confirmed mtD
(mtDNA rearrangements/point mutations/depletion), 4 with high clinical/histological
suspicion of mtD, and 4 unaffected controls. % COX and CI/CIV-deficient fibres were
counted in two random fascicles, with high concordance amongst % COX-negative and
CI/CIV-deficient fibres.
Results: Control sections stained as a
mosaic dark green (type I fibres) and light green (type II fibres) pattern. Completely
CI/CIV-deficient fibres stained yellow, and partly CI/CIV-deficient fibres stained
yellow-green, and were easily detectable due to good visual colour contrast. The DCI
detected more CI-deficient fibres in 7/19 cases and more CIV-deficient fibres in 5/19
cases compared to COX-negative fibres (average 6%). Most COX-negative fibres had dual
CI+CIV defects with DCI. Segmental and partial CI/CIV defects were detectable. Equivocal
COX-SDH stained fibres were often strongly CI/CIV-immunodeficient.
Conclusion: In conclusion, our multiplex
DCI reliably detects CI/CIV defects comparable in sensitivity to the COX-SDH
histochemical assay, is easy to evaluate due to a good visual contrast between CI/CIV
positive and negative fibres and can be easily co-opted to routine diagnostic work.
Studies are underway to develop a quadruple chromogenic immunoassay for digital
evaluation of CI/CIV defects.
A novel MT-TG mutation associated with adult-onset multisystem
mitochondrial disease
O.V. Poolea*, A.
Horgaa*, S.A.Hardyb, Enrico Bugiardinia,
C.Woodwardc, I.P. Hargreavesd, J.L. Holtone, R.
Quinlivana, R.W. Taylorb, M.G. Hannaa and R.D.S.
Pitceathlya
*joint first authors
aMRC Centre for Neuromuscular Diseases, UCL Queen Square Institute of
Neurology and National Hospital for Neurology and Neurosurgery, London, UK;
bWellcome Centre for Mitochondrial Research, Newcastle University,
Newcastle upon Tyne, UK
cDepartment of Neurogenetics, UCL Institute of Neurology and The
National Hospital for Neurology and Neurosurgery, London, UK;
dNeurometabolic unit, The National Hospital for Neurology and
Neurosurgery, London, UK;
eDivision of Neuropathology, UCL Queen Square Institute of Neurology,
Queen Square, London, UK
Background: Collectively, mitochondrial
disorders are among the most common inherited neurological disorders. The prevalence of
adult mitochondrial disease caused by mitochondrial DNA (mtDNA) mutations is around
1:5,000.
Aims: To describe the clinicopathological
and biochemical profile of an adult harbouring a novel mutation in
MT-TG.
Patient: A 39 year old female presented
with sensorineural hearing loss since her late teens, visual impairment with bilateral
cataracts, retinal dystrophy and subsequent bilateral retinal detachments in her 20s,
hypothyroidism in her 30s, secondary amenorrhoea, and short stature on examination.
There was no family history of neurological or neuromuscular disease. Blood lactate was
elevated and histochemical staining revealed frequent ragged red and cytochrome
c oxidase (COX) deficient fibres in muscle tissue. Spectrophotometric
analysis of mitochondrial respiratory chain activity revealed reduced activity of
complexes I and IV prompting mitochondrial DNA genetic analysis.
Results: Sequencing of the entire
mitochondrial genome revealed the novel missense mutation m.10038G>A in
MT-TG, encoding tRNA glycine, at a heteroplasmy level of 92% in
muscle, 40% in urine and 15% in blood. Single fibre segregation studies confirmed a
higher mutation load in COX deficient fibres (n=27, 95.3%) as compared with COX positive
fibres (n=26, 78.9%; p=0.0005).
Conclusion: We describe a novel pathogenic
mutation in MT-TG. The m.10038G nucleotide is highly conserved and the
m.10038G>A mutation was present at variable heteroplasmic levels in different
tissues. Histochemical and biochemical evidence of impaired mitochondrial protein
synthesis was indicated in muscle tissue, and single fibre studies confirmed the
biochemical defect segregated with high mutant levels. As such, it is highly likely the
m.10038G>A is pathogenic. The majority of mtDNA mutations occur in the 22
mtDNA-encoded tRNA genes. However, despite over 270 reported tRNA mutations, only five
reside within MT-TG. The cause of the variability in prevalence of
mutations among tRNA gene remains unknown.
The role of mtDNA heteroplasmy, copy number, age and nuclear factors in the
clinical heterogeneity associated with the m.3243A>G mutation
Sarah J Pickett1*, John P
Grady1, Yi Shiau Ng1, Charlotte L Alston1, Emma L
Blakely1, Steven A Hardy1, Catherine L Feeney1,
Alexandra A Bright1, Andrew M Schaefer1, Richard J Q
McNally2, Ian J Wilson3, Heather J Cordell3, Gráinne
S Gorman1, Robert W Taylor1, Doug M Turnbull1, Robert
McFarland1
1Wellcome Centre for Mitochondrial Research, Newcastle University,
Newcastle upon Tyne, UK
2Institute of Health and Society, Newcastle University, Newcastle
upon Tyne, UK
3Institute of Genetic Medicine, Newcastle University, Newcastle upon
Tyne, UK
*
sarah.pickett@ncl.ac.uk
Background: Mitochondrial disease
associated with m.3243A>G, the most common pathogenic mitochondrial DNA (mtDNA)
mutation, is clinically heterogeneous. People who harbour m.3243A>G can present with
a range of clinical features that progress at variable rates, making an accurate
prognosis difficult.
Aim: To describe and understand the cause
of m.3243A>G-associated heterogeneity.
Patients and Methods: We examined the
phenotypic profile of 238 m.3243A>G carriers from the Mitochondrial Disease Patient
Cohort UK using the Newcastle Mitochondrial Disease Adult Scale (NMDAS) and evaluated
which commonly assayed tissue (blood, urine, skeletal muscle) represents the
m.3243A>G mutation load and mtDNA copy number most associated with disease burden. We
then modelled the role of heteroplasmy level, age and additive nuclear genetic factors
in the development of specific phenotypes within 46 pedigrees from the cohort.
Results: Blood heteroplasmy declines by
~2.3%/year and males have ~20% higher m.3243A>G mutation load in urine; we present
formulas to adjust for these effects. Age and m.3243A>G heteroplasmy level in all
three tissues are associated with disease burden (R2 range=0.18-0.27,
P<0.001), with blood heteroplasmy (corrected and uncorrected) being most strongly
associated. A greater proportion of the variation in disease burden is explained if
mtDNA copy number in skeletal muscle is included (R2=0.40, P<0.001).
Common phenotypic features include hearing impairment, psychiatric involvement and
ataxia, however, age and heteroplasmy levels are poor predictors of severity for
individual phenotypes (pseudo-R2=0.17). We found high to moderate
heritability estimates for psychiatric involvement, cognition, ataxia, migraine and
hearing impairment (h2 range=0.40-0.76, P<0.05).
Conclusion: Our results indicate that
m.3243A>G heteroplasmy, skeletal muscle mtDNA copy number and age explain some of the
variation in m.3243A>G-related disease burden, however, nuclear genetic factors also
influence clinical outcomes. This study paves the way for future work identifying these
nuclear modifiers.
Engaging with patients with mitochondrial disease to make them better decision
makers
Julie Murphy, Lyndsey Butterworth
Wellcome Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle
University, United Kingdom
Background: The Wellcome Centre for
Mitochondrial Research, Newcastle supports world class research into mitochondrial
disease. Our Centre also strives to be world class at involving patients and the public
with our research, thereby allow us to develop an effective research strategy for the
benefits of patients.
Aim: Our existing and future aims are: To
engage with patients throughout the UK about our research, helping them to become better
decision makers; to build a community of empowered expert patients and carers who are
involved in developing and implementing a research and engagement strategy for our
mutual benefit and to build capacity and engagement expertise within our staff to create
a listening culture in our scientific community that is responsive to patient needs.
Methods: We have incorporated patients’
and carers’ views in the strategic direction of the Centre; developed our engagement
conferences and patient/carer focus groups; delivered an international multistage design
event with the purpose of involving people living with mitochondrial disease in the
design and development of ideas that could transform their lives.
Results: Members of the Wellcome Centre
for Mitochondrial Research continue to be involved with annual patient engagement
conferences, regular focus groups discussing policy and research issues and the design
of a patient/public focused website. We have devised and delivered a very successful
not-for-profit venture, the Myto project, where researchers from Newcastle University’s
Open Lab teamed up with the Wellcome Centre for Mitochondrial Research and TU Eindhoven
in order to hold three design events across the UK, Netherlands and Italy. These design
events sought to involve people living with and caring for mitochondrial disease in the
creation and development of digital tools to enrich their lives and spread important
messages about mitochondrial disease.
Conclusion: Through engagement we continue
to stimulate national and international debate on issues affecting our patients, and
create strategic partnerships with patient and public groups. This allows us to ensure
research advances make a meaningful difference to the lives of patients with
mitochondrial disease.
Clinical research activity within the Wellcome Centre for Mitochondrial
Research
Clare Jordison, Laura Brown, Professor
Robert McFarland, Professor Robert Taylor, Professor Sir Doug Turnbull, Grainne
Gorman
The Wellcome Centre for Mitochondrial Research, Newcastle University
Clare.jordison@ncl.ac.uk,
Laura.brown8@ncl.ac.uk
Background: The Wellcome Centre for
Mitochondrial Research (WCMR) aims to transform the lives of patients with mitochondrial
disease. Utilising the expertise of clinical and laboratory team members, we work
closely with patients to improve their lives and the lives of future generations
affected by mitochondrial disease.
Aims: We seek to generate substantial
improvements in the health and wellbeing of patients with mitochondrial disease. Our
goal is to offer every patient the opportunity to take part in studies and clinical
trials that may ultimately lead to new therapeutic strategies.
Methods: Within the Centre, close
proximity between the clinical and laboratory team provides an ideal environment for
maximising translational research opportunities.
Results: Currently we have one commercial
phase 1 and one commercial phase 2/3 CTIMP in set-up and have two further phase 1 CTIMPs
in feasibility. We are currently delivering a phase 3 combined CTIMP/device trial,
working with a commercial Sponsor. We are also in the process of setting up an MRC
funded drug repurposing study which will be the largest study ever undertaken in
participants with mitochondrial myopathy and which was designed in collaboration with
patients. In addition to CTIMP studies, we are delivering, preparing to deliver or have
recently completed, 10 observational and interventional studies. These include follow-up
of children born via Mitochondrial Donation, ‘deep phenotyping’ studies and a number of
studies aiming to identify optimal outcome measures for future studies. We also continue
to collect data and tissue for the Wellcome Centre for Mitochondrial Research Patient
Cohort and Newcastle Mitochondrial Research Biobank which provide an invaluable resource
for internal and external researchers.
Conclusion: We work closely with research
sponsors, including The Newcastle upon Tyne Hospitals NHS Foundation Trust, with our
academic and commercial collaborators and also with other support organisations (e.g.
UKCRC Clinical Trials Units) to obtain the necessary regulatory approvals and ensure
that our research is conducted to the highest possible standards.
Preventing the transmission of mitochondrial DNA disease utilising preimplantation
genetic diagnosis
Emma L. Blakely1,2*, Lauren
Medwell2, Suzanne C.E.H. Sallevelt3, Sophie Monnot4,
Charlotte L. Alston1,2, Louise A. Hyslop5,6, Samantha K.
Byerley5, Lyndsey Craven2, Alasdair Blain2, Sarah J.
Pickett2, Gráinne S. Gorman1,2, Catherine Feeney1,2,
Doug M. Turnbull1,2, Jane Stewart5,6, Aimee D.C.
Paulussen3, Jos Dreesen3, Nelly Frydman7, Jean-Paul
Bonnefont4, Hubert J.M. Smeets3, Julie Steffann4,
Mary Herbert5,6, Robert W. Taylor1,2, Robert
McFarland1,2
1NHS Highly Specialised Service for Rare Mitochondrial Disorders,
Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK
2Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, UK
3Department of Clinical Genetics, Maastricht University Medical
Centre, Maastricht, The Netherlands
4Service de Génétique, Groupe hospitalier Necker Enfants Malades,
Assistance Publique – Hôpitaux de Paris, Paris, France
5Newcastle Fertility Centre, Biomedicine West Wing, Newcastle upon
Tyne, UK
6Wellcome Centre for Mitochondrial Research, Institute of Genetic
Medicine, Newcastle University, UK
7AP-HP, Biologie de la reproduction, Université Paris-Sud, Université
Paris-Saclay, Hôpital Antoine-Béclère, Clamart, France
*Emma.watson@nuth.nhs.uk
Background: Pathogenic mitochondrial (mt)
DNA variants are responsible for a broad spectrum of chronic, multisystem presentations
that can be present at birth or develop later in life. mtDNA disease is progressive,
causing debilitating physical, developmental, and cognitive disabilities and with no
effective cure. Given mtDNA is strictly maternally-inherited, the ability to prevent the
transmission of mtDNA disease is possible using pre-implantation genetic diagnosis (PGD)
and more recently, mitochondrial donation.
Aims:
To determine
success rates for mtDNA PGD
To ascertain the
segregation patterns of individual pathogenic mtDNA variants and how
representative a single cell biopsy (8 cell stage) is of the entire
embryo
To generate prediction models for the likelihood
of PGD success for important pathogenic mtDNA variants
Patients: Data were collected from 80 PGD
cycles, representing 43 patients and 12 pathogenic mtDNA variants, generated by three
international, licenced centres offering mtDNA PGD.
Results: The live birth success rate for
mtDNA PGD was 15.2% per PGD cycle. The majority of pathogenic variants displayed a wide
range of heteroplasmy within individual embryos. However, the m.8993T>G and
m.9176T>C pathogenic variants resulted in a binomial distribution of heteroplasmy
levels within individual embryos. A high mutation load of the pathogenic m.3243A>G
and m.8344A>G variants in patients showed significant association with a high level
of heteroplasmy of the pathogenic variant in the patient’s embryos. Subsequent
laboratory investigations of disaggregated blastocyst cells, following unsuccessful PGD,
confirmed that blastomere biopsy at the eight cell stage is representative of the total
mutation load in the embryo. Prediction models were generated and demonstrate that for
the majority of pathogenic mtDNA variants, as the maternal heteroplasmy level increases
the success rate for PGD decreases, due to a lack of embryos with a low mutation
load.
Conclusion: Our data show that mtDNA PGD
can successfully prevent the transmission of mtDNA disease, however for many pathogenic
mtDNA variants, a high level of maternal heteroplasmy confers a low likelihood of
achieving embryos with a suitably low mutation load to warrant implantation. Therefore
for some patients who harbour high levels of a heteroplasmic pathogenic mtDNA variant,
Mitochondrial Donation may represent a more suitable alternative to PGD to prevent the
transmission of mtDNA disease.
Small molecules counter-select deleterious mitochondrial DNA variants by inhibiting
their replication in human cells
D. Ives1*, M. Mennuni2*,
B. Pantic2*, CL.
Nezich3, RDS. Pitceathly4, R. McFarland5, RW.
Taylor5, MG. Hanna4,6, L. Vergani7, IJ.
Holt2,8, A. Spinazzola2,6
* Equal contribution
a.spinazzola@ucl.ac.uk;
b.pantic@ucl.ac.uk
1MRC Laboratory, Mill Hill, London, UK
2Department of Clinical and Movement Neurosciences, UCL Queen Square
Institute of Neurology, Royal Free Campus, London, UK
3MRC Mitochondrial Biology Unit, Cambridge, UK
4Department of Neuromuscular Diseases, UCL Queen Square Institute of
Neurology, London, UK
5Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, The Medical School, Newcastle upon Tyne,
UK
6MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology
and National Hospital for Neurology and Neurosurgery, Queen Square, London,
UK
7Neurosciences Department, University of Padova, Padova,
Italy
8Biodonostia Health Research Institute, San Sebastián, Spain and
IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
Background: Pathological variants of human
mitochondrial DNA (mtDNA) were first reported over 30 years ago; yet treatments are
still lacking for this devastating group of disorders. Deleterious mutations often
affect some, but not all, of the many copies of the mtDNAs -a state known as
heteroplasmy- and, typically, disease manifests only if the proportion of the mutant
molecules exceeds a threshold. Thus, even a modest increase in the proportion of
wild-type mtDNA should be curative. The nucleus ultimately determines the heteroplasmy
level of mtDNA variants, but the molecular mechanisms underpinning the selection for or
against deleterious mtDNAs are still poorly understood.
Aims: To determine the cellular processes
affecting the selection of mtDNA variants, and to use this information to identify small
molecules that favour the propagation of wild-type mtDNA.
Methods: We used both unbiased and
targeted approaches to study human cells carrying the most common pathological mtDNA,
m.3243G. Then, having identified specific features of cells that are capable of
selecting wild-type over mutant mtDNA, we screened for compounds that target the
relevant pathways and processes to determine whether any decreased the level of mutant
mtDNA.
Results: Our studies of cells that do, or
do not, select wild-type mtDNA have led to the discovery of two small molecules that
induce the selection of wild-type mtDNA in somatic cells, and restore the mitochondrial
respiratory capacity without inducing cell death. One of these compounds has been
administered to human subjects previously and so can potentially be rapidly translated
to the clinic. Analysis of the pathways involved in the process has enabled us to begin
dissecting the underlying mechanisms of actions of the small molecules, which involve
the inhibition of the replication of the DNA molecules as function of the mitochondrial
fitness.
Conclusion: There is an urgent unmet
clinical need for therapies to treat patients with mtDNA-related disorders. The
identification of small molecules that favour the selection of wild-type mtDNA
represents an important advance. Moreover, since the compounds rectify the genetic
defect itself by decreasing the number of copies of the mutated gene, they, can
potentially, not only arrest disease progression, but also reverse it. An experimental
medicine study will evaluate the safety and efficacy of one of the compounds in patients
with m.3243G mtDNA.
A homozygous two exon deletion in UQCRH: matching mouse and
human phenotypes
Kyle Thompson1*,
Silvia Vidali2,3, 4, Lore Becker5, Jill Urquhart6, Jan
Rozman5, Charlotte Sanders1, Elisabeth Jamson6,
Catherine Breen6, Birgit Rathkolb5, Patricia da
Silva-Buttkus5, Susan Marschall5, Oana V Amarie5,
Juan Antonio Aguilar-Pimentel5, Julia Calzada-Wack5, Yi-Li
Cho5, Lillian Garrett5, Sabine M Hölter5, Tanja
Klein-Rodewald5, Philipp Mayer-Kuckuk5, Irina
Treise5, Annemarie Zimprich5, Kristine Gampe5, Stefanie
Leuchtenberger5, Kristina Pfannes5, Claudia Stöger5,
Holger Maier5, Jochen Graw7, Wolfgang Wurst7, Kai
Höfig8, René G Feichtinger3, Ulrich Gärtner9, Marten
Szibor10, Ilka Wittig11, Johannes A Mayr3, William G
Newman6, Helmut Fuchs5, Robert W Taylor1, Valerie
Gailus-Durner5, Holger Prokisch2,4, Martin Hrabe de
Angelis5;
1Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, The Medical School, Newcastle upon Tyne, United Kingdom;
2Institute of Human Genetics, Technische Universität München, Munich,
Germany; 3Department of Pediatrics, Paracelsus Medical University,
Salzburg, Austria; 4Institute of Human Genetics#;
5German Mouse Clinic#; 6Manchester Centre for Genomic
Medicine, University of Manchester, United Kingdom; 7Institute of
Developmental Genetics#; 8Research Unit Molecular Immune
Regulation#; 9Institute for Anatomy and Cell Biology,
Justus-Liebig-University of Giessen, Germany; 10Faculty of Medicine and
Life Science, University of Tampere, Finland; 11Functional Proteomics,
Faculty of Medicine, Goethe University Frankfurt, Germany; #Helmholtz
Zentrum München, German Research Center for Enviromental Health, Neuherberg,
Germany.
*kyle.thompson@ncl.ac.uk
Background: The ubiquinol:cytochrome
c oxidoreductase hinge protein (UQCRH) is a very small subunit of
complex III (CIII) of the oxidative phosphorylation (OXPHOS) system. UQCRH connects the
subunits cytochrome c with the cytochrome c1. Thus, it
plays an important role in the assembly of CIII. Mitochondrial disease presentations
linked to UQCRH are not yet described, although somatic
UQCRH variants have been linked to different types of cancer.
Aims: To confirm the pathogenicity of a
homozygous deletion of exons 2 and 3 in UQCRH identified by whole exome
sequencing and autozygosity mapping and to compare the human phenotype to the phenotype
of a mouse model with the equivalent deletion of Uqcrh.
Patients and Methods: We report two male
first cousins from a consanguineous family with recurrent episodes of severe
ketoacidosis, excess blood ammonia and hypoglycaemia and signs of encephalopathy. Brain
MRI’s showed no abnormality and between episodes the health of the two patients was
entirely normal. A mouse model was created by deletion of exons 2 and 3 of
Uqcrh and screening of the mouse phenotypes was performed.
Results: In patient fibroblasts,
steady-state levels of CIII subunit UQCRC2 were decreased and Blue-Native PAGE
assessment showed a slightly smaller assembly of CIII compared to controls. Patient
fibroblasts also demonstrated low CIII enzymatic activity and decreased maximal
respiration by Seahorse analysis. The murine presentation was more severe, with
progressive functional impairment and premature early adult death. Enzymatic activity
and protein expression of OXPHOS complexes were investigated in mouse tissue revealing
significant decreases in CIII activity in heart, brain and liver. mRNA expression
analysis in mouse liver embryonic fibroblasts (MEFs) revealed decreased expression of
the Uqcrh transcript, more severe in the homozygous animals compared to
heterozygotes. Complexome profiling of heart samples from wild-type and homozygous mice
revealed decreased CIII expression and altered assembly profiles in homozygotes, in
agreement with the human patient data.
Conclusion: We describe the first patients
with bi-allelic mutations in UQCRH, with an identical mutation in the
mouse mimicking several phenotypic aspects. Notably, bi-allelic variants in
UQCRC2 and UQCRB, two other subunits of complex III,
result in a similar episodic clinical presentation.
Bladder dysfunction in patients with mitochondrial disease
Catherine Feeney, RGN
MClinRes1,2, Ashwin Sachdeva, PhD1,2, Alistair
Blain1 Alexandra Bright,1,2 Janice Gebbie,1,2,
Christopher Harding, MD2, Robert McFarland, MD1,2,Grainne Gorman,
MD1,2, Doug Turnbull, MD,1,2
1Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, United Kingdom
2Newcastle Hospitals NHS Foundation Trust, Queen Victoria Road
Newcastle-upon-Tyne
Background: Mitochondrial diseases often
present with a spectrum of clinical features with multi-organ involvement. Since
mitochondrial diseases are often characterised by a loss of smooth muscle function, it
is plausible to hypothesise that mitochondrial dysfunction may contribute to bladder
dysfunction.
Aim: To determine the presence of bladder
dysfunction in patients with mitochondrial disease.
Methods: In this observational cohort
study of patients with genetically confirmed mitochondrial disease, we assessed bladder
function using three tools: a validated questionnaire measuring severity scores; bladder
diary measuring nocturnal polyuria index and bladder scan measuring bladder voiding
efficiency.
Results: Between 31st October 2017 and
30th June 2018, 65 consecutive patients aged 18years or over, with a genetic diagnosis
of mitochondrial disease without cognitive impairment were recruited. Twenty percent of
patients manifested with bladder dysfunction as defined by one of: ICIQ-LUTS*
Questionnaire severity score of >60%; NPI* >35% or BVE *<70%, necessitating
referral for further urological investigations. ICIQ-LUTS questionnaire severity scores
demonstrated no patients reported severe LUTS, based upon on 60% severity threshold.
Bladder diary revealed 40% of the cohort reported nocturia (≥=2x/night) whilst 13.2%
were noted to have nocturnal polyuria. Finally, 18% of the cohort were found to have
high post-void residual volumes (bladder voiding efficiency < 70%).
Conclusion: Our findings suggest bladder
dysfunction is under recognised in this patient group and support the need for clinical
urological assessment. A combination screening approach is best suited, as seen in other
chronic disease settings. Further work is required to devise a composite score to
evaluate LUTS and extend this study to a larger patient group.
* ICIQ-LUTS = International Consultation on
Incontinence Modular Questionnaire – Lower Urinary Tract Symptoms
*NPI =Nocturnal Polyuria Index
*BE = Bladder Voiding Efficiency
Guidelines for physiotherapy in mitochondrial disease
Jane Newman, PhD1,2, Cecilia
Jimenez-Moreno PhD,1 Sarah Holmes, M.Clin.Res 3
1Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, United Kingdom
2Biomedical Research Unit. Newcastle University
3Centre for Neuromuscular Disease, National Hospital for Neurology
and Neurosurgery, University College Hospital London, United Kingdom
Background: The physical manifestations of
Mitochondrial disease are extremely varied. Muscle symptoms spanning from fatigue,
muscle pain and weakness and neurological symptoms can be as varied as ataxia or
dystonia. Research investigating the efficacy of physiotherapy interventions in people
with Mitochondrial disease is lacking. However, many symptoms and impairments seen in
people with Mitochondrial disease are also seen in other disease populations. Therefore
this guidance includes references from research evidence for other neurological and
neuromuscular populations alongside research including people with mitochondrial
disease.
Aim: To provide indications for referral
to physiotherapy; to highlight the importance of physiotherapy for people with
mitochondrial disease and provide examples of the relevant outcome measures for use
within the clinical setting.
Methods: This expert consensus opinion was
written and developed by physiotherapists based within the specialist mitochondrial
centres in Newcastle, London and Oxford. The guidance was also reviewed by members of
the multi-disciplinary team based within these centres, colleagues from acute and
community services and patient groups. Where possible this guidance is supported by
existing evidence-based knowledge.
Results: This guidance is intended to be
of interest to the following people or organisations; healthcare professionals; people
with Mitochondrial disease and their carers; patient support groups; commissioning
organisations; service providers and researchers.
Conclusion: In view of the diverse
presentations of mitochondrial disease, we recommend that all patients diagnosed with a
mitochondrial disorder should be able to access a specialist physiotherapist. This will
enable patients to receive advice about their condition and be referred for on-going
physiotherapy in their locality as required. Local physiotherapy services are encouraged
to obtain support in the management of this rare condition from the physiotherapists
that work within the three mitochondrial disease centres for more specialised
advice.
PROSPER 2B: Prospective Observational
Study of PatiEnts
with mitochondrial depletion syndrome, RRM2B
Jane Newman1,2, Renae
Stefanetti1, Cecilia Jimenez-Moreno1 , Albert Lim1,
Yi Shiau Ng1, Catherine Feeney1, Alison Davison1,
Andrew Schaefer1, Youcef Mehellou3, Caterina Garone4,
Doug M Turnbull1, Robert W Taylor1, Robert McFarland1,
Gráinne S Gorman1.
1Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, United Kingdom
2NIHR Newcastle Biomedical Research Centre. Campus for Aging and
Vitality. Newcastle University
3School of Pharmacy and Pharmaceutical Sciences. Cardiff
University.
4MRC Mitochondrial Biology Unit. Cambridge University.
Background: Mitochondrial diseases are an
important group of inherited neuro-metabolic disorders that invariably exhibit
multi-organ involvement, are relentlessly progressive, and result in high disease burden
and premature death. To date, over 70 patients have been reported in the literature
manifesting with a spectrum of RRM2B-related mitochondrial disease.
While pharmacological agents are emerging for this mitochondrial disease little is known
about its natural history.
Aim: To deeply phenotype patients
harbouring recessive mutations in RRM2B over 12 months to enable the
development of disease-specific, patient-centric outcome measures for future
studies.
Methods: Patients (pediatric and adults)
with a genetically proven diagnosis of recessive RRM2B-related mitochondrial disease
enrolled in the Medical Research Council Mitochondrial Disease Patient Cohort Study in
Newcastle, Oxford and London will be invited to participate.
Participants will then be asked to attend Newcastle to complete the
following clinical assessments at study start and end:
Demographics and anthropometrics. Physical examination and Vital
signs.
Disease burden
(NMDAS).
Diurnal variation in skeletal muscle and
liver glycogen by in vivo 13C and 1H magnetic resonance
spectroscopy.
Assessment of bulbar
function.
Pulmonary
Function
Muscle Strength and Motor
Function
Functional
capacity
Physical activity
monitoring
Bowel
Dysfunction
Symptom Severity
Interim visits will also be performed at a venue
convenient to the participant. These visits will include assessments that are deemed
feasible.
Results: The study is scheduled to start
February 2019. The results from the study will analysed to assess to the feasibility and
validity of assessments for use in future clinical trials.
Conclusion: This study will establish
relevant clinical biomarkers for future clinical trials in RRM2B. The interrogation of
feasible, valid and sensitive battery of clinical markers is imperative when new
pharmacological treatments are in the pipeline.
High-throughput screening to discover mitochondrial therapeutics
Oliver Russell1, Shane
Bell1, Gareth Ettridge1, Polly Usher1, Iffath
Ghouri1, Robert N. Lightowlers1, Doug M.
Turnbull1,2
1Wellcome Centre for Mitochondrial Research, Newcastle University,
Newcastle-upon-Tyne, UK
2Centre for Ageing and Vitality, Newcastle University,
Newcastle-upon-Tyne, UK
Email: oliver.russell@ncl.ac.uk
Background: Mitochondrial dysfunction is a
key pathogenesis in numerous neuromuscular disorders. There are currently no effective
curative treatments available to patients with mitochondrial dysfunction.
Aim: To develop a range of high-throughput
assays to discover new treatments for mitochondrial dysfunction
Methods: We have designed and adapted
several assays to enable the application of high-throughput fluorescent imaging
techniques to screen thousands of small molecules and natural products. Due to the
multi-faceted nature of mitochondria our assays are able to improvements in various
aspects of mitochondrial function including mitochondrial biogenesis, mitochondrial
turnover, mitochondrial REDOX status and oxidative phosphorylation rate.
Results: We validated our assays using
previously described modifiers of mitochondrial function. Here we show that the effect
of these control compounds is reproducible even in 384 well format. Once verified, we
used a selection of the assays to screen compound libraries and show that not only can
we detect compounds which improve mitochondrial function, but that they are more
efficacious than previously reported compounds.
Conclusion: Development of high-throughput
assays is key to discovering new treatments for mitochondrial dysfunction. The time
invested to develop these assays is already paying dividends with the discovery of
several promising new potential mitochondrial therapeutics.
Cardiac manifestations of mitochondrial disease in children and adolescents
Albert Lim, Yi Shiau Ng, Emma L Blakely,
Charlotte L Alston, Alex Bright, Cecilia Jimenez-Moreno, Jane Newman, Catherine Feeney,
Andrew Schaefer, Robert W Taylor, Grainne S Gorman, Doug M Turnbull, Robert
McFarland
Wellcome Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle
University, UK
Background: Mitochondrial disease in
children is an immensely-diverse group of disorders caused by nuclear or mitochondrial
DNA defects. Prevalence of cardiac involvement in childhood mitochondrial disease is not
well-characterised, thus resulting in uncertainties about cardiac health
surveillance.
Aims: To determine the spectrum of cardiac
abnormalities in genetically-confirmed childhood mitochondrial diseases.
Methods: Children and adolescents under 18
years were identified from the UK mitochondrial Disease Patient Cohort (REC 13/NE0326)
and were followed up regularly in Newcastle, UK. Medical records, electro-cardiographies
(ECG), echocardiograms and genetic reports from 2009 to 2018 were reviewed.
Results: Fifty two children with
pathogenic mitochondrial-DNA (mt-DNA) and fifty three children with nuclear-DNA (n-DNA)
mutations were identified. The most common mt-DNA mutation was the m.3243A>G followed
by m.11778G>A, m.8993T>C, m.8993T>G and m.9176T>C. The most common n-DNA
defect was the POLG gene mutation followed by the Complex I subunit
assembly genes, the SUCLA2 and the SURF1 genes. Out of
the 96 clinically-affected children and adolescents, the three most prevalent phenotypes
were ataxia, weakness and developmental delay.
Overall, cardiac abnormalities were identified in 9
patients (9.4%): mt-DNA mutation(n=7) and n-DNA defect(n=2). In the mt-DNA group,
hypertrophic cardiomyopathy was identified in four patients (m.3243A>G, m.8993T>C,
m.8993T>G and m.4300A>G). One patient who harboured the m.3243A>G mutation had
coarctation with multiple ventricular septal defects while the other patient with the
same mutation had dysplastic aortic valve with stenosis and regurgitation. Asymptomatic
right bundle branch block was identified in a 17-year-old who harboured the
m.13513G>A mutation.
In the n-DNA group, only two patients had hypertrophic
cardiomyopathy (recessive mutations in AGK and
NDUFS1). The patient who harboured the AGK mutations
developed hypertrophic cardiomyopathy with biventricular systolic and diastolic
impairment. His two elder siblings (adults) had severe cardiomyopathy requiring heart
transplant at age 13 and 14 years, respectively. The cardiac manifestations were
significantly more frequent in mt-DNA defects (15.9% vs 3.8%, p=0.047).
Conclusion: Cardiac manifestations
featured more frequently in those with mt-DNA mutations than those with n-DNA defects.
In the n-DNA defects, cardiac disease with mitochondrial disease is genotype-specific.
Therefore, cardiac surveillance should be targeted to mitochondrial DNA mutations and
high-risk nuclear genotypes.
Email:
albert.lim@ncl.ac.uk
Truncating MT-ATP6 mutations: expanding the molecular and phenotypic spectrum of
mitochondrial ATP synthase disorders
Enrico Bugiardini*,1,2 Emanuela
Bottani*,3,4 Silvia Marchet,5 Olivia V. Poole,1,2
Cristiane Beninca,3 Alejandro Horga,1 Cathy Woodward,6
Amanda Lam,7 Iain Hargreaves,7 Annapurna Chalasani,7
Alessandra Valerio,4 Eleonora Lamantea,5 Kerrie
Venner,8 Janice L. Holton,8 Massimo Zeviani,3 Henry
Houlden,1,2 Rosaline Quinlivan,1,9 Costanza
Lamperti,5 Michael G. Hanna,1,2 Robert D.S.
Pitceathly1,2
*Joint first authorship
1MRC Centre for Neuromuscular Diseases, UCL Queen Square Institute of
Neurology and National Hospital for Neurology and Neurosurgery, Queen Square, London,
UK
2Department of Neuromuscular Diseases, UCL Queen Square Institute of
Neurology, Queen Square, London, UK
3Mitochondrial Medicine group, Medical Research Council Mitochondrial
Biology Unit, Hills Road, Cambridge, UK
4Department of Molecular and Translational Medicine, University of
Brescia, Brescia, Italy
5Medical Genetics and Neurogenetics Unit, Fondazione IRCCS Istituto
Neurologico, “C. Besta”, Milan, Italy
6Neurogenetics Unit, National Hospital for Neurology and
Neurosurgery, Queen Square, London, UK
7Neurometabolic Unit, National Hospital for Neurology and
Neurosurgery, Queen Square, London, UK
8Division of Neuropathology, UCL Queen Square Institute of Neurology,
Queen Square, London, UK
9Dubowitz Neuromuscular Centre, Great Ormond Street Hospital, London,
UK
e.bugiardini@ucl.ac.uk
Background: Mitochondrial DNA
(mtDNA)-related ATP synthase disorders cause maternally-inherited Leigh syndrome and
neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP). Most MT-ATP6/8
mutations are missense with relatively uniform levels across tissues, and high mutant
loads frequently correlate with severe clinical phenotypes.
Aims: To describe the clinical phenotypes
and functional consequences of truncating MT-ATP6 mutations.
Methods: We report three unrelated
probands with mitochondrial encephalomyopathy harbouring truncating
MT-ATP6 mutations. Structural and functional consequences of the
MT-ATP6 variants were investigated with Blue-Native Gel
Electrophoresis (BNGE), Microscale oxygraphy, reactive oxygen species (ROS) measurement
in patients’ tissues, and with transmitochondrial cybrid cell lines.
Results: Patient 1 presented with
adult-onset ataxia, chronic kidney disease and diabetes, while Patient 2 had myoclonic
epilepsy and ataxia; both harboured the novel m.8782G>A; p.(Gly86*) mutation. Patient
3 exhibited cognitive decline, with posterior white matter abnormalities on brain MRI,
and severely impaired renal function requiring transplantation. The m.8618dup;
p.(Thr33Hisfs*32) mutation, previously associated with NARP, was identified. All three
probands demonstrated a broad range of heteroplasmy across different tissue types.
Blue-native gel electrophoresis of cultured fibroblasts and skeletal muscle tissue
confirmed multiple bands, suggestive of impaired complex V assembly. Microscale
oxygraphy showed reduced basal respiration and ATP synthesis, and increased reactive
oxygen species generation. Transmitochondrial cybrid cell lines studies confirmed the
deleterious effects of the novel m.8782G>A; p.(Gly86*) mutation.
Conclusion: We expand the clinical and
molecular spectrum of MT-ATP6-related mitochondrial disorders to
include leukodystrophy, renal disease and myoclonic epilepsy with ataxia. Truncating
MT-ATP6 mutations may exhibit highly variable mutant levels across
different tissue types, an important consideration during genetic counselling.
Targeting metabolic-epigenetic axis to promote oxidative phosphorylation activity
in the presence of mitochondrial dysfunction
Monika J Madej*, Ben
O’Callaghan, Michael G Hanna & Henry Houlden
University College London, MRC Centre for Neuromuscular Diseases London,
UK
*Email- m.madej@ucl.ac.uk
Background: Mitochondrial impairment,
which results in a metabolic imbalance, determines changes in activities of
metabolites-dependent enzymes. Chromatin modifying enzymes reliant on metabolites link
metabolism with gene transcription and overall cell function. Therefore, we aim to shift
this program towards the oxidative respiration. The rationale of this therapeutic
strategy is to overcome the oxidative metabolism deficit threshold at which disease
manifests.
Aims: This project aims to delineate
compounds, which boost oxidative mitochondrial function. This strategy has a potential
to target a broad spectrum of diseases, where mitochondrial function is compromised
including neuromuscular and neurodegenerative disorders.
Methods: We take advantage of the
established in vitro human model system where human induced pluripotent stem cells
derived (iPSCs) derived-myogenic progenitors are induced towards the mature myotubes
characterized by expression of oxidative and glycolytic Myosin Heavy Chains (MYH)
isoforms. Those two distinct fibre types correlate with oxidative and glycolytic
metabolism, respectively. To this end, iPSCs-derived myogenic progenitors carrying
mitochondrial mutations vs. isogenic control lines are differentiated in the presence of
small compounds. Small compounds compatible with this assay are available commercially.
Treatments, which enhance or cause a shift from glycolytic to oxidative fibre types are
selected for further analysis. Fibres isoforms are detected based on immunofluorescent
staining and quantified as an output read-out.
Results: We have performed the proof of
concept experiment, which demonstrates an absence of MYH isoforms in the presence of the
mitochondrial mutation (m.3243A>G), indicating lack of fibres maturation. The same
mutant line subjected to the treatment with one of the tested compounds - inhibitor of
epigenetic reader targeted against bromodomain (I-BET762) induces Fast MYH. Therefore,
treatment with epigenetic modifiers can prove to be of therapeutic benefit applicable to
disorders where mitochondrial dysfunction plays a major role.
Neuromuscular disorders and PREFER project: understanding patient preferences and
their importance in the drug development process
Jimenez-Moreno A.C.1 and Gorman
G.1 on behalf of the PREFER project WP3.6 team members.
1Welcome Trust Mitochondrial Research Centre, Institute of
Neurosciences, Newcastle University, UK.
Cecilia.jimenez-moreno@ncl.ac.uk
Background: Patient preferences are
defined by the Food and Drug Administration (FDA) as “The relative desirability or
acceptability to patients of specified alternatives or choices among outcomes or other
attributes that differ among alternative health interventions”. Over the last decades
there has been an increasing recognition of the importance of patient preference
information in the decision making process along the drug development. Rare diseases in
particular have been highlighted as sensitive groups that could beneficiate from patient
preferences. However, there is not yet consensus on how and when better to incorporate
patient preferences into decision making to better inform concerning stakeholders. In
2016, the European public-private partnership PREFER (‘Patient Preferences in Benefit
and Risk Assessments during the Drug Life Cycle’) launched, a five year project funded
by the Innovative Medicines Initiative-2 Joint agreement. The PREFER project aims to
assess when and how patient preferences should be incorporated into the drug development
process. More specifically, provide a set of systematic recommendations to assess,
engage and include patient preferences during the development, approval, and
post-approval of new therapies.
Methods: As part of the PREFER project’s
work-package 3, a case study in neuromuscular disorders will be completed that will
inform the PREFER aim. This case study will assess patient and caregiver unmet health
priorities and risk tolerance for neuromuscular treatments by systematic qualitative and
quantitative approaches. An initial series of ten face-to-face interviews and four focus
groups will be completed to elicit a set of attributes that will later be included on a
large-scale survey expected to reach up to 700 participants (patients and caregivers)
with myotonic dystrophy type 1 and mitochondrial disorders.
Results: In parallel to this conference,
focus groups with patients and caregivers will be ongoing as part of the case study.
Conclusion: This study will set precedents
that not only will inform the PREFER project aims but also stakeholders interested in
these diseases about current unmet health needs that patients and caregivers established
as priority and the risks they are willing to undertake to treat them.
Other diseases and diagnostics
The development of an immunoblotting external quality assurance scheme
Yolande
Parkhurst1, Matt Henderson1, Julie
Marsh1, Richard Charlton1, Rita Barresi1,2.
1 Muscle Immunoanalysis Unit, Newcastle upon Tyne Hospitals NHS
Foundation Trust, Newcastle upon Tyne, UK; 2 John Walton Muscular Dystrophy
Research Centre, MRC Centre for Neuromuscular Diseases, Institute of Genetic Medicine,
Newcastle University, Newcastle upon Tyne, UK.
Background: The Muscle Immunoanalysis Unit
(MIU) is an ISO 15189 accredited laboratory which offers a highly specialised national
referral service for the diagnosis of Limb Girdle Muscular Dystrophy (LGMD) -
progressive muscle diseases that produce weakness in a limb-girdle distribution.
Analysis is undertaken on frozen muscle biopsies, utilizing immunohistochemistry and
immunoblotting techniques to study protein expression and focus genetic analysis. The
unique immunoblotting process used in MIU was developed by the department and is not
offered by any other UK laboratory. As a result, there is currently no external quality
assurance (EQA) scheme available for this technique.
Aims: To develop an international scheme
to assess the quality of immunoblots by suitably accredited laboratories.
Methods: The scheme was trialled with
Hospital Sant Pau (Barcelona) and MIU as participants. The referring centre emailed an
electronic image of the blot to the assessing centre, which reviewed the image and
completed a standardized scoresheet. The referring centre checked the responses against
the original report to determine consistency.
Results: Results from the initial trial
assessment highlighted advantages and limitations of the methodology. Further discussion
and modifications of the scoresheet are in progress to establish guidelines for an
unambiguous standardized assessment.
Conclusion: We propose to develop a format
and procedure for an external quality assessment scheme for immunoblotting. This is
supportive of requirements necessary for ISO 15189 accreditation, in which medical
laboratories seek confirmation of confidence in the results of diagnostic testing
through participation in suitable comparisons. Further data is required and the
expansion of the scheme to involve additional participants will be explored.
Diagnostic outcomes of the Highly Specialised Services for Rare Neuromuscular
Disorders -Congenital Muscular Dystrophies and Myopathies (HSS CMD/CMY)
Anna Sarkozy1, Rahul Phadke1, 3, Rachael
Mein4, Stephanie Robb1, Lucy Feng1,3, Caroline
Sewry1, 5 and Francesco Muntoni1, 6
1Dubowitz Neuromuscular Centre, Great Ormond Street Hospital for
Children NHS Foundation Trust; 2UCL Institute of Child Health & MRC
Centre for Neuromuscular Diseases, London, UK; 3Queen Square Institute of
Neurology, London, UK; 4Genetics Department, Molecular Genetics Laboratory
Viapath, Guy’s Hospital, London, United Kingdom; 5Wolfson Centre for
Inherited Neuromuscular Diseases, RJAH Orthopaedic Hospital, Oswestry, United Kingdom;
6NIHR Great Ormond Street Hospital Biomedical Research Centre, Great
Ormond Street Institute of Child Health, University College London, & Great Ormond
Street Hospital Trust, London, UK
Background: Congenital muscular
dystrophies (CMD) and congenital myopathies (CM) are rare neuromuscular conditions with
major clinical, pathological and genetic heterogeneity. Diagnostic gold standard is
identification of pathogenic variant/s in a disease causing gene. Diagnostic abilities
are hindered by the clinical and genetic complexity of CMD and CM, size and polymorphic
nature of many disease genes, such as RYR1, TTN and
NEB genes, and the high number of private mutations identified in
patients.
Aims: To describe diagnostic outcomes of
the HSS for CMD and CM in the UK.
Methods: Review of the clinical, pathology
and genetic diagnostic outcomes in HSS CMD/CMY in the 2016-2017 financial year. All
patients referred to the service underwent gatekeeping and specialised advisory service,
with multidisciplinary review of pathology and genetic results. Sequencing of full
coding and short flanking intronic sequences and duplication/deletion analysis of 82 CMD
and CM genes was performed by next generation sequencing.
Results: A total of 516 patients were
referred to the service. A definite genetic diagnosis was achieved in 160 patients
(31%). Ninety of these patients (17%) had parallel referrals to the
clinical/pathological/genetic services. Clinical assessment was offered to 201 patients
and majority of these had muscle biopsy and muscle MRI review as well, if available. A
definite genetic diagnosis was achieved in 117/201 patients seen in our clinic (58%).
Fifty patients had a diagnosis of CM (25%) and 13 of CMD (5%). A muscle/skin biopsy
review was completed in 128/516 patients and 95/128 (74%) obtained a definite
pathological diagnosis. A diagnosis of CMD or CM was excluded on clinical and/or
pathological grounds in 86/516 patients.
Conclusion: This review highlights the
importance of specialist validation of genetic analysis results by expert clinical and
pathological review. The higher diagnostic yield in patients with clinical assessments
at GOSH reflects the benefits of the combined muscle pathology and muscle MRI imaging
review, systematically applied to all patients seen in clinic. This outcome review also
highlights a high number of patients in whom a diagnosis of CMD/CM was ruled out,
helping to redirect diagnostic investigations.
Novel recessive MYO18 gene pathogenic variants in a patient with
congenital myopathy and mini core pathology
Anna
Sarkozy1, Irina Zaharieva1,
Ian Horrocks2, Rachel Mein3, William Stewart4, Rahul
Phadke1, Cheryl Longman5 and Francesco Muntoni1,6
1Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health & MRC Centre for Neuromuscular Diseases, London, UK
2Paediatric Neurosciences Research Group, Royal Hospital for
Children, Glasgow, UK
4Laboratory Medicine, Queen Elizabeth University Hospital and
Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United
Kingdom.
5West of Scotland Regional Genetic Service, Queen Elizabeth
University Hospital, Glasgow, United Kingdom.
6NIHR Great Ormond Street Hospital Biomedical Research Centre, Great
Ormond Street Institute of Child Health, University College London, & Great Ormond
Street Hospital Trust, London, UK
Background Recessive variants in
MYO18B, encoding an unconventional myosin protein, have been
described in three unrelated patients with congenital myopathy, with classic nemaline
rods or dense bodies reminiscent of nemaline rods in two patients. Cardiomyopathy, short
stature, Klipper-Feil anomaly and dysmorphisms were variably present.
Aims: To describe a novel patient with
congenital myopathy and MYO18B variants.
Patient: The patient, of Philippine
origin, presented with severe motor delay, achieving independent ambulation at age 5
years. Now aged 17 years he has facial weakness with ptosis, axial weakness with
progressive thoracic scoliosis and proximal more than distal limb weakness, affecting
lower more than upper limbs. He remains ambulant but cannot rise independently from the
floor. Generalised hypermobility with ankle contractures is present. Respiratory
function is normal, but he has mild feeding difficulties. CK was normal at age 12 years.
Lower limb muscle MRI at age 15 years showed asymmetric fatty infiltration and bulk
reduction of glutei, hip extensors, flexors and adductors, soleus and gastrocnemii, with
asymmetric involvement of semitendinosus and tibialis anterior muscles. Muscle biopsy
showed myopathic size variation, unevenness of oxidative staining with few distinct
mini-cores, slow fibre uniformity and a distinct population of very small fetal
myosin-positive fibres uniformly dispersed across fascicles. No nemaline rods were
observed on light and electron microscopy.
Results: Next generation sequencing of 55
genes responsible for congenital myopathies identified two novel heterozygous
MYO18B gene variants. The c.4131delC p.(Ala1378fs) results in a
premature termination of the MYO18B protein and is predicted to be pathogenic. The
second variant, c.39+5G>T, has not been previously reported and bioinformatics
splicing predictions were not informative. The variants were confirmed in
trans. To assess the significance of the c.39+5G>T variant, transcript
analysis was performed on muscle tissue with RT-PCR of mRNA and amplification of cDNA.
The assay showed an abnormal fragment length, indicating and effect of the variant on
mRNA splicing.
Conclusions: We have identified a novel
patient with recessive MYO18B gene variants. This report further
expands the current knowledge on this rare form of myopathy, suggesting that mini-cores
without nemaline rods can also be part of the associated pathological spectrum.
A novel patient with multiple pterygium syndrome and nemaline myopathy due to
recessive TPM2 gene variants
Anna
Sarkozy1, Schaida Schirwani2,
Anne-Marie Childs3, Rachel Mein4, Rahul Phadke1 Audrey
Smith5, Karen Pysden3, Emma Hobson2 and Francesco
Muntoni1-6
1Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health & MRC Centre for Neuromuscular Diseases, London, UK
5Manchester Centre for Genomic Medicine, St Mary’s Hospital,
Manchester University NHS Foundation Trust, Health Innovation Manchester, Manchester
M13 9WL, UK.
6NIHR Great Ormond Street Hospital Biomedical Research Centre, Great
Ormond Street Institute of Child Health, University College London, & Great Ormond
Street Hospital Trust, London, UK
Background: Pathogenic variants in
TPM2 have been associated with a variable clinical spectrum,
including congenital myopathies and distal arthrogryposis, all but one with dominant
inheritance. A homozygous pathogenic variant in TPM2 has been reported
in a single family with nemaline myopathy (NM) and Escobar variant multiple Pterygium
syndrome (EVMPS).
Aims: We report the second patient with
recessively inherited TPM2-related EVMPS and NM in a patient from a
consanguineous family.
Patient: Reduced fetal movement and
scoliosis were antenatally detected. He was hypotonic at birth, with kyphoscoliosis,
multiple upper and lower limb contractures, pterygia at shoulders and knees and mild
ptosis. He required nasogastric tube feeding and gastrostomy from 10 months of age.
Creatine kinase was normal. He sat at 18 months and was able to pull up on furniture and
do a few steps at age 3.5 years. Intelligence and speech development was normal. He had
non progressive thoracolumbar kyphoscoliosis with lower thoracic and lumbar spine fusion
(T4-S1). There was no respiratory compromise.
Results: Muscle biopsy from right vastus
lateralis muscle showed myopathic size variation and type I fibre predominance. Few
cores/mini cores and sparse, small nemaline rods were seen only at the ultrastructural
level. A novel homozygous intronic sequence variant, c.564-2A>C was found in the
TPM2 gene, predicted to abolish the consensus acceptor splice site
for exon 6b. Unaffected parents were heterozygous for the variant and expected to
produce 50% of TPM2.2 which appears to avoid muscle weakness.
Conclusions: We have identified a novel
homozygous intronic TPM2 gene variant affecting exon 6b splicing in a
patient with EVMPS and NM. Interestingly, the only other homozygous TPM2 variant was
also found in exon 6b and expressed only in two of four TPM2 isoforms (TPM2.2 and
TPM2.3). This report confirms role of TPM2 as cause of EVMPS and the importance of TPM2
expression in early period of prenatal life.
1Newcastle University, John Walton Muscular Dystrophy Research
Centre, Institute of Genetic Medicine, MRC Centre for Neuromuscular Disease, Newcastle
upon Tyne, UK
2Ultragenyx
3National Centre of Neurology and Psychiatry, NCNP Hospital, Tokyo,
Japan
4Hadassah Medical Centre, Jerusalem, Israel
5Neuromuscular Disease Foundation, USA
Background: The GNE Registry is an
international, patient reported, disease specific database which provides the
neuromuscular community with a valuable, flexible and sustainable resource of disease
specific information. The registry can be used to support planning and recruitment for
clinical trials, and to capture real-world patient data (including data that may
contribute to post marketing surveillance and standards of care). The GNE Registry is
managed by the John Walton Muscular Dystrophy Research Centre (JWMDRC, Newcastle
University, UK) and supervised by the Steering Committee. The GNE myopathy registry
helps to facilitate translational research. The registry is available in 7 languages,
collecting data since March 2014 with over 350 participants from over 30 countries
worldwide (www.
http://www.treat-nmd.eu/gne/patient-registries/international-registry).
Aims: To contribute to the disease
understanding by collecting reliable information; facilitating clinical trial readiness
and translational research by helping the recruitment of patients in the clinical
trials; helping to overcome the scarcity of resources and the geographic isolation of
patients; informing patients and the GNE myopathy community of latest developments in
scientific research and disease management, via newsletters.
Methods: Patient reported data gathered
through disease specific questionnaires (GNEM-FAS, quality of life (SF12) and other
non-validated questionnaires.
Results: The registry has allowed a
genotype-phenotype study to be conducted (Reference). Ten newsletters have been produced
in 7 languages and distributed to the registry participants and to the neuromuscular
community via TREAT-NMD network. The registry helped to facilitate recruitment for
patient advocacy meetings, clinical trials (NCT02736188, NCT02377921) and natural
history study (NCT01784679). The registry has also participated in EURORDIS and MDUK
research, namely The Voice of rare disease patients and GNE patient’s day.
Conclusions: The International HIBM
Patient Registry is a valuable tool for this ultra-rare disease community
lucy.imber@ncl.ac.uk
Self-management in neuromuscular diseases: a qualitative exploration of the patient
perspective
1Department of Neuromuscular Diseases, UCL Queen Square Institute of
Neurology
2Faculty of Health Social Care and Education, Kingston University and
St George’s University of London
Background: The concept of self-management
has seen a growth in interest from health policy-makers over the past two decades and is
frequently regarded as a way of reducing the burden of long-term illness and demand on
health services, whilst simultaneously improving outcomes for individual patients. It
has consequently become a key focus in many areas of healthcare but has had little
exploration in people living with neuromuscular diseases (NMDs). This study seeks to use
qualitative research methods to explore the concept of self-management from the
perspective of patients with NMDs, to inform future interventions and research in the
area.
Methods: This study will use
semi-structured interviews with patients at one regional specialist centre for
neuromuscular diseases. Recruitment will continue until data saturation, indicated by no
new themes arising from the data. Semi-structured interviews will explore 1) how people
manage living well with their condition; 2) successful strategies; challenges; 3) unmet
support needs; 4) areas of importance for living well with NMDs; 5) resources used and
role of staff in self-management support. Interviews will be audio recorded and
transcribed. Sampling will be purposive to ensure a breadth of people with a range of
diagnoses and ages. Three main groups will be targeted with 5-10 participants in each
group: 1) Early disease onset with rapid progression; 2) early disease onset with slow
progression over the lifespan; 3) late disease onset with slow progression. Thematic
analysis will be used to code data and identify the key domains and themes. An
exploratory comparison of themes will be undertaken between the three identified groups
and those with and without affected family members. Preliminary themes will be sent to
participants for respondent validation to gain feedback and refine interpretation of
data.
Conclusion: This study is the first to
explore self-management from the perspective of NMD patients using qualitative methods.
This study seeks to use qualitative research methods to explore the concept of
self-management from the perspective of patients with NMDs, to inform future
interventions and research in the area.
The clinical and genetic spectrum of a UK cohort of paediatric and adult patients
with MYH7 gene related skeletal myopathies
Shpresa Pula1, Rahul
Phadke1, Ros Quinlivan1,4, Rachael Mein2,
Adnan Manzur1, Stephanie Robb1, Pinki Munot1, Lucy
Feng1, Caroline Sewry1, Francesco Muntoni1,3 and Anna
Sarkozy1
1Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health & MRC Centre for Neuromuscular Diseases, London, UK Department of
Neurosciences, Great Ormond Street Hospital for Children, NHS Foundation
Trust
2Genetics Department, Molecular Genetics Laboratory Viapath, Guy’s
Hospital London, UK
3NIHR Great Ormond Street Hospital Biomedical Research Centre, Great
Ormond Street Hospital, London, UK
4MRC Centre for Neuromuscular Diseases, National Hospital for
Neurology and Neurosurgery, London, UK
Background: Pathogenic variants in the
MYH7 gene, encoding for the slow beta cardiac myosin heavy chain, are responsible for a
wide spectrum of mostly dominantly inherited myopathies affecting cardiac and skeletal
muscles, including hypertrophic or dilated cardiomyopathy, Laing type distal myopathy,
congenital fibre type disproportion, scapuloperoneal myopathy and myosin storage
myopathy MYH7 gene variants have so far been reported only in two UK families with
skeletal myopathy.
Aims: To describe clinical and genetic
spectrum of a UK cohort of patients with MYH7 gene related congenital
myopathy.
Methods: Patients were recruited at the
Dubowitz Neuromuscular Centre and National Neurology and Neurosurgery Hospital in
London. A retrospective review of medical notes of patients with genetically confirmed
MYH7 gene related myopathy was done, including demographics, family
pedigrees, clinical features, neurophysiology, skeletal muscle MRI, and the muscle
biopsies.
Results: We identified 8 novel patients
from 5 families with MYH7 gene pathogenic variants. Four were females
and four males, with age ranging between 2 and 44 years. Onset ranged between 1 and 7
years. Patients presented variable patterns and severity of muscle weakness and wasting
or muscle hypertrophy, with wide intra an inter-familial variability. Cardiomyopathy was
diagnosed in 2 patients and one commenced targeted cardiac treatment. One patient had
reduced respiratory function, with force vital capacity being 29% of the predicted value
at age 18 years. Muscle biopsies were available in 2/8 cases. Both showed myopathic
changes with minicores, and one case showed marked fast fibre predominance. There was no
evidence of myosin storage. Lower limb muscle MRI was performed in two patients and
showed hypertrophy of gastrocnemii and in one patient diffuse involvement of the muscles
with streaky fatty infiltration. We identified 5 MHY7 dominant variants
(one missense, and four in-frame single amino acid or single exon deletions).
Conclusions: This work further expands
current genotypic and phenotypic knowledge on MYH7 gene related
myopathies and aids diagnosis and clinical management of patients with this rare
disease.
High-Fidelity Disease Modelling of Skeletal Muscle Laminopathies Using Human Ips
Cells and Artifical Skeletal Muscles
Luca Pinton1,2,*, Heather
Steele-Stallard1,2,*, Shilpita Sarcar1, Daniel
Moore1, Tanel Ozdemir1,2, Sara M. Maffioletti1, Peter
S. Zammit2,** and Francesco Saverio Tedesco1,3,**
1Department of Cell and Developmental Biology, University College
London, WC1E 6DE London, UK
2Randall Centre for Cell and Molecular Biophysics, King’s College
London, SE1 1UL London, UK
3The Dubowitz Neuromuscular Centre, Great Ormond Street Institute of
Child Health, University College London
Background: Laminopathies are severe
heterogeneous genetic diseases caused by mutations in A-type lamins, which are encoded
by the LMNA gene. These proteins together with Lamin B1 and B2 form the
nuclear lamina: a mesh-like structure located underneath the nuclear membrane which
helps maintaining nuclear shape and regulating gene expression. Laminopathies affect
multiple cell types and can be tissue-specific or systemic, with some subtypes affecting
striated muscle, peripheral nerve and adipose tissue, while others cause multisystem
disease with accelerated aging. Although different mechanisms have been proposed, the
precise pathophysiology of laminopathies remains unknown; additionally, their rarity and
lack of easily accessible cell types for ex vivo studies negatively impact on therapy
development.
Aims & Methods: To bypass these
hurdles, here we used induced pluripotent stem (iPS) cells from patients with skeletal
muscle laminopathies such as LMNA-related congenital muscular dystrophy
and limb-girdle muscular dystrophy 1B, to model disease-associated phenotypes in
vitro. iPS cells from three skeletal muscle laminopathy patients were
differentiated into skeletal myogenic cells and myotubes.
Results: Characteristic cellular
phenotypes were observed in all LMNA-mutant iPS lines, including
nuclear shape abnormalities and mislocalisation of proteins of the nuclear lamina.
Notably, complex modelling in three-dimensional (3D) artificial muscle constructs
resulted in recapitulation of nuclear abnormalities with higher fidelity than standard
bi-dimensional (i.e. monolayer) cultures and identified nuclear length as a robust and
objective outcome measure. Finally, we will present and discuss current efforts and
future applications of this novel 3D organoid-like platform for therapy development and
drug screening for skeletal muscle laminopathies.
Conclusion: These results demonstrate that
patient-specific iPS cells can model phenotypic readouts of skeletal muscle
laminopathies with high fidelity upon 3D differentiation in vitro,
laying the foundation for future therapy screening platforms for skeletal muscle
laminopathies and other severe muscle disorders.
Natural history of sporadic inclusion body myositis: a longitudinal observational
study investigating outcome measures for clinical trials
Sangha G1, Skorupinska
I2, Germain L2, Bellin A2, Kozyra D2,
Parton M2, Hanna MG2, Miller J3, Hilton-Jones
D1, Freebody J2, Machado PM2
1Oxford University Hospitals NHS Foundation Trust, Oxford,
UK
2Department of Neuromuscular Diseases, Institute of Neurology,
University College London, London, UK
3Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle,
UK
gavinda.sangha@medsci.ox.ac.uk
Background: Sporadic inclusion body
myositis (IBM) is the most common age-related acquired myopathy. Distinct early clinical
features include selective weakness of quadriceps and forearm muscles. Pathological
features of the disease include both inflammatory and degenerative features, which,
together with the nature of progression and unresponsiveness to immunosuppressants
suggest a possible neurodegenerative mechanism.
Aims: Our aim was to longitudinally
evaluate clinical, quantitative (muscle strength) and functional characteristics of the
disease in order to develop reliable statistical models of progression over time that
can be used in clinical trials.
Methods: In a prospective cohort study, we
recruited 205 patients with a diagnosis of IBM made by a neuromuscular expert and
fulfilling any of the following research diagnostic criteria: 1) Griggs’ criteria, 2)
MRC (Hilton-Jones) criteria, or 3) ENMC 2011 criteria. Patients were enrolled across
three centres (London, Oxford and Newcastle). Patients underwent bilateral manual muscle
testing (MMT) on 23 muscle groups using the expanded (0-5) MRC scale, quantitative
muscle testing (QMT) using the HUMAC Norm CSMi dynamometer and grip testing using the
handheld dynamometer (where available) at each follow up visit. Disability scoring was
performed at each follow up visit using the IBM Functional Rating Scale (IBMFRS). The
time at which a patient first required a mobility aid was recorded. Linear multilevel
models were fitted to investigate the progression of MRC scores, IBMFRS scores and
quantitative myometry scores over time. Cox proportional hazards regression was used to
model time to use of a mobility aid in patients with IBM.
Results: In the preliminary analyses we
have been able to generate reliable multilevel statistical models of disease progression
using those muscle groups that are worst affected by the disease over time. We have also
demonstrated the progression of IBMFRS over time confirming its utility in monitoring
disease progression. We studied predictors of time to use of a mobility aid. Final
results will be presented at the meeting.
Conclusion: This study will prove to be
the largest prospective observational study in patients with IBM to date and will reveal
useful outcome measures that can be used to trial therapeutics in clinical trials.
Novel loss-of-function mutation in ACBD5 found in family with
ataxia
Roisin Sullivan1, Plamena
Angelova2, Emer O’Connor1, David Lynch1, Andrey
Abramov2, Mike Hanna3, Henry Houlden1
1Department of Movement Disorders, Queen’s Square Institute of
Neurology, UCL, London
2Department of Clinical and Movement Neurosciences, Queen’s Square
Institute of Neurology, UCL, London
3MRC Centre for Neuromuscular Diseases, Queen’s Square Institute of
Neurology, UCL, London
Hereditary autosomal recessive cerebellar ataxias are a
highly heterogeneous group of disorders that affect the cerebellum and connected regions
of the nervous system causing a range of symptoms including uncoordinated voluntary
movements, problems with eye movements and dysarthria. ACBD5 encodes
for the peroxisomal membrane protein Acyl-coA binding domain 5 (ACBD5) which is involved
with peroxisomal metabolism and selective autophagy. A homozygous deleterious indel
mutation has been previously reported in an ACBD5-deficient patient with ataxic
symptoms, with impaired β-oxidation of very-long-chain fatty acids (VLCFA)
(Ferdinandusse et al., 2017).
Aims: To investigate the autosomal
recessive mutation of ABCD5 found in a family with ataxia by exploring
its pathogenic mechanism and functional effects.
Methods: Blood and skin biopsies taken
from consented family members (two affected brothers, one unaffected sister and both
parents). DNA was extracted from blood and the skin biopsies were used to grow primary
fibroblast lines for all family members. Western blots using protein from primary
fibroblast lines, anti-ACBD5 antibody, cDNA sequencing and metabolic screening were used
to confirm type of mutation, pattern of inheritance and effect on protein ACBD5.
Results: Western blots, immunofluorescence
staining, and cDNA sequencing show a loss of function mutation in
ACBD5, with nonsense mediated decay (NMD). Metabolic screening revealed
an increase in very long chain fatty acids in the affected patients.
Conclusion: The molecular techniques used
show a loss of function mutation that shows NMD. Metabolic screening shows an increase
in very long chain fatty acids which hints at a possible peroxisomal mechanism. Future
work will centre around further exploration of a possible mechanism, focusing on
peroxisomal and mitochondrial metabolism and function, using an additional
ACBD5-deficient line and ABCD1-deficient cell lines that cause adrenoleukodystrophy
(ALD) that may have a similar mechanism.
References: ACBD5 deficiency causes a
defect in peroxisomal very long-chain fatty acid metabolism. Ferdinandusse S et al.,
2017. J Med Genet 54:330-337
The TREAT-NMD Advisory Committee for Therapeutics (TACT): Facilitating Drug
Development in Neuromuscular Rare Diseases
Cathy Turner*1, Volker
Straub1, Annamaria de Luca2, on behalf of TACT
catherine.turner@ncl.ac.uk
1John Walton Muscular Dystrophy Research Centre, Newcastle
University, UK
2Dipartimento Farmacobiologico, Università degli Studi di Bari,
Italy
Background: Established in 2009 as part of
TREAT-NMD, TACT is an expert multidisciplinary body that provides the neuromuscular
community with independent, confidential and objective guidance on advancing new
therapies (whether novel or repurposed) for rare neuromuscular diseases.
Aims: The goal of each TACT review is to
position the potential therapy along a realistic and well-informed pathway to or through
clinical trials, and eventual registration, by evaluating supporting preclinical data
and other critical drug development considerations necessary for objective
decision-making. The output is detailed advice leading to the improved design and
conduct of studies that generate more meaningful data and have the increased potential
to be funded longer term.
Methods: Applications are reviewed by a
bespoke panel of world-leading experts drawn from the TACT committee of around 65
members in response to the specific needs of a particular application. All reviews are
conducted under strict confidentiality agreements and conflict declarations. An online
and thorough review process is followed by a face-to-face meeting between the panel and
the applicant. A confidential and independent report detailing the panel’s advice is
provided to the applicant within 4-6 weeks of the meeting. TACT conducts two review
cycles each year, considering up to four applications each time.
Results: To date TACT has held 18 review
meetings, 10 in Europe and 8 in the North America, and has reviewed a total of 53
applications from both academic investigators and industry. TACT has worked closely with
existing infrastructures established by the National Institute of Health (NIH) and the
European Union, and major patient organisations in multiple regions around the world to
ensure the process assists the neuromuscular community as a whole.
Conclusion: Feedback shows that TACT has
generated recommendations that have greatly helped investigators, including industry, in
evaluating their potential compounds. It has encouraged applicants to consider their
development programs in a methodical fashion with clear go/no-go decisions and with
optimal use of funding and resources. Examples of the impact of TACT review advice are
given.
Frequency of Coronary Artery Disease in People with McArdle Disease
Aleksandra Pietrusz, Renata S. Scalco, Ros
Quinlivan
MRC Centre for Neuromuscular Diseases, UCL, London, UK
a.pietrusz@ucl.ac.uk
Background: McArdle disease (glycogen
storage disease type V; GSDV) is a rare metabolic disorder characterised by the
inability to use glycogen as an energy source in skeletal muscles. Consequently,
affected people experience pain during physical activity and exercise. Complications
include muscle contractures, muscle breakdown (rhabdomyolysis), compartment syndrome and
kidney failure. Exercise-related symptoms and anxiety about causing muscle damage can be
a major barrier to exercise and many patients lead sedentary lifestyle. Sedentariness
may increase the prevalence of coronary heart diseases (CAD) in this patient population.
Proving this theory may help improving patients’ care by supporting exercise training in
this patient population and early investigation for CAD.
Aims: Review the medical notes of patients
with McArdle disease to analyse the occurrence of CAD, risk factors and treatments
provided in this population.
Methods: A retrospective, single-site
study was performed in the UK. Medical notes from patients seen between April 2017 and
April 2018 (period of one year) were reviewed. Collected data included genetic
diagnosis, age at McArdle disease diagnosis, current age, gender, presence/absence of
CAD, age at CAD diagnosis, symptoms of cardiac disease, cardiac disease treatment type,
level of physical activity / sedentariness, other risk factors for cardiac disease
(smoking, hypercholesterolemia, hypertension, family history of cardiac disease, etc.),
body mass index and past medical history.
Results: Preliminary data from 73 patients
will be presented.
Conclusion: Preliminary data on CAD
frequency in a sample of patients with McArdle disease will be discussed.
Comorbidity and mortality in patients with sporadic Inclusion Body Myositis
(sIBM)
I. Skorupinska, D. Kozyra, L. Germain, A.
Bellin, M.J. Parton, M.G. Hanna, P.M. Machado
MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology, London
UK
Iwona.skorupinska@nhs.net
Background: Sporadic Inclusion Body
Myositis (sIBM) is an age-related acquired myopathy with male predominance. The disease
results in progressive limb weakness and loss of function leading to severe disability
and having a major impact on quality of life. In a recent meta-analysis, high
variability in sIBM prevalence estimates and quality of the studies was found. The
meta-prevalence using the four articles of highest quality was 45.6/million.The natural
history of sIBM remains largely unknown and available data on mortality and comorbidity
in these patients is extremely limited. Survival rates in patients with sIBM seem to be
comparable to the general population.
Aims: To evaluate the cause of death and
contributing factors in sIBM patients seen at the UCL Queen Square Centre for
Neuromuscular Diseases.
Methods: Data from patients with sIBM
fulfilling 2011 ENMC criteria were reviewed. Causes of death were obtained from the
death certificates, which were retrieved from the General Register Office. Collection
and review of clinical data of the living and deceased patients was done. Underlying
comorbidities were extracted.
Results: Seventeen deaths were recorded.
Mean age of death was 75.6±7.7 years. Mean age of death in the UK is 79 years for males
and 83 years for females (Office for National Statistics). The most frequent primary
cause of death was respiratory (60%). The most common associated comorbidities were
cardiovascular (21%) and rheumatological (19%).
Conclusions: These preliminary data
suggest that there might be a possible impact of sIBM on lifespan. Aspiration pneumonia
was the leading cause of death in our cohort. Regular monitoring including SALT
evaluation and advice should be undertaken.
A versatile, modular digital script for automated high-throughput multiparametric
myofibre analysis in brightfield and epifluorescent paradigms
Matthew Ellis1, Dominic
Scaglioni2, Francesco Catapano2, Silvia
Torelli2, Darren Chambers3, Lucy Feng3, Caroline
Sewry3, Mervyn Singer4, Jennifer Morgan1, Francesco
Muntoni1,5, Rahul Phadke3,6
1Department of Neurodegenerative Diseases, UCL Queen Square Institute
of Neurology, London, UK
2Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of
Child Health, London, UK
3Dubowitz Neuromuscular Centre, UCL Queen Square Institute of
Neurology & Great Ormond Street Hospital for Children NHS Foundation Trust,
London, UK
4Bloomsbury Institute for Intensive Care Medicine, University College
London, London, UK
5NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL
Great Ormond Street Institute of Child Health & Great Ormond Street Hospital for
Children NHS Foundation Trust, London, UK
6Division of Neuropathology, National Hospital for Neurology and
Neurosurgery, UCLH NHS Foundation Trust, London, UK
Email: d.scaglioni@ucl.ac.uk
Background: Digital scripts are vital for
unbiased, high-throughput multiparametric analysis of muscle landscapes in frozen/fixed
histology sections with useful diagnostic and research applications. By extrapolating
far greater quantities and parameters of pathological data compared to traditional
manual assessment digital scripts open up novel opportunities for deep subset analyses
and pathway mapping in tissue sections.
Aims: To develop a versatile, modular
digital script that can be tailored to desired study specifications applicable to a
variety of neuromuscular conditions.
Methods: Entire sections of skeletal
muscle stained either fluorescently or chromogenically were scanned using a digital
slide scanner. The images were exported to the Definiens Tissue Studio and Developer
software platform that was used to develop subsequent image analysis modules.
Results: Initially the script was
developed to map oxidative changes in the mixed fibre-type gastrocnemius muscle, with
COX-SDH staining, in a long-term rodent model of critical illness and recovery. Staining
intensity translated to digital heatmaps as surrogate indicators of mitochondrial
biogenesis. Reliable fibre separation was then achieved with subsequent introduction of
a ubiquitously expressed membrane marker stain (spectrin/laminin) to create a ‘mask’ for
defining the sarcolemma of each myofibre. Brightfield analysis of muscle fibre diameter
in ‘histologically normal’ paediatric muscle biopsies provided good correlation between
whole section counts and manually selected regions of interest, generating
age-stratified data on muscle fibre size. Current modules measure numerous indices
related to the level of expression of markers, and the coverage of markers above a given
threshold in myofibre subpopulations using multiplexed immunofluorescent staining. We
have developed an optimised module for multiparametric quantification of dystrophin and
associated proteins in biopsies from Duchenne and Becker muscular dystrophy patients.
Separate modules are under development for quantitative immunoanalysis of complex I/IV
defects in patients with primary mitochondrial disease and dot-quantitation for RNA
in-situ hybridisation assays.
Conclusion: Our unique modular approach
allows for continuous machine learning, increasing the script’s capacity to generate a
variety of high-throughput qualitative and quantitative datasets tailored to a wide
range of neuromuscular applications.
Vacuolar myopathy with valosin containing protein (VCP)-positive intranuclear and
cytoplasmic inclusions: report of two cases with early and late childhood-onset
disease
Rahul
Phadke1, Andrew
Dean2, Matthew Evans3, Alasdair Parker4, Damian
Maxwell5, Caroline Sewry6, Anna Sarkozy6, Francesco
Muntoni6
1Dubowitz Neuromuscular Centre, Great Ormond Street Hospital for
Children & UCL Queen Square Institute of Neurology, London, United
Kingdom
2Department of Neuropathology, Cambridge University Hospitals NHS
Foundation Trust, Cambridge, United Kingdom
3Department of Neurology, Guy’s and St Thomas’ NHS Foundation Trust,
London, United Kingdom
4Department of Paediatric Neurology, Cambridge University Hospitals
NHS Foundation Trust, Cambridge, United Kingdom
5Department of Neurology, Cambridge University Hospitals NHS
Foundation Trust, Cambridge, United Kingdom
6Dubowitz Neuromuscular Centre, Great Ormond Street Hospital for
Children & UCL Great Ormond Street Institute of Child Health, London, United
Kingdom
Email: r.phadke@ucl.ac.uk
Background: Paediatric-onset protein
aggregation and vacuolar myopathies (PAVM) are rare familial or sporadic disorders with
marked clinical, pathological and genetic heterogeneity. Extensive pathological work-up
in muscle biopsies may be required to narrow the hunt for the putative disease-causing
gene/protein. A multisystem proteinopathy (MSP) causing several phenotypes in adults
including rimmed vacuolar myopathy, motor neurone disease, frontotemporal dementia and
Paget disease of bone, unified by pathological aggregation of ubiquitin and TDP-43 has
been linked to mutations in VCP, HNRNPA2B1,
HNRNPA1 and SQSTM1. Intranuclear and cytoplasmic
VCP+ inclusions are present in most cases of VCP-MSP. VCP+ aggregates
are hitherto not reported in other PAVM except sporadic inclusion body myositis and some
extra-skeletal neurodegenerative disorders
Aims: Here we describe two clinically
diverse early and late-childhood onset cases with unifying muscle pathology of a
vacuolar myopathy with VCP-positive intranuclear and cytoplasmic inclusions resembling
the classic VCP-MSP.
Patients: PI, a 21-year-old male presented
at age 16 with aching pain in his left forearm and progressive difficulty straightening
the left hand fingers. Remaining neurological assessment was normal. PII, a 22-year old
female presented at age 9 with toe walking and delayed motor milestones. She developed
rapidly progressive weakness and lost ambulation at age 14. She is currently fully
wheelchair dependent, and uses long-term non-invasive ventilation. There is no family
history of neuromuscular disease in both patients.
Results: CK was normal, EMG myopathic in
left forearm finger flexors and intrinsic hand muscles, and muscle MRI showed oedema and
fatty infiltration affecting left forearm and finger flexors in PI. Muscle biopsies from
both patients following extensive histochemical, immunohistochemical and ultrastructural
work-up showed identical pathology resembling that of classic
VCP-inclusion body myopathy with chronic myopathic/dystrophic changes,
rimmed vacuoles, sarcoplasmic and intranuclear protein aggregates/inclusions
(VCP/ubiquitin/TDP43+) and tubulofilamentous inclusions. Extensive molecular genetic
studies to date are negative including whole exome sequencing in PII.
Conclusion: Further molecular genetic
diversity may underpin the unifying VCP+ inclusion body pathology in these atypical
paediatric presentations. Recruitment to other next-generation-sequencing platforms is
under consideration.
Clinical research activity in the John Walton Muscular Dystrophy Research
Centre
Anna Peel1, Agata Robertson1, Samantha
Fitzsimmons1, Becky Crow1, Katherine Frith1, Sabine
Specht1, Anna Mayhew1, Robert Muni Lofra1, Dionne
Moat1, Jassi Sodhi1, Meredith James1, Michelle
McCallum1, Ruth Wake1, Katie Waller1, Jayne
Banks1, Lian Meyers1, Lilian Hayward1, Volker
Straub1, Michela Guglieri1
1The John Walton Muscular Dystrophy Research Centre, Institute of
Genetic Medicine, University of Newcastle upon Tyne and Newcastle Upon Tyne Hospitals
NHS Foundation Trust, UK.
anna.peel1@newcastle.ac.uk
The John Walton Muscular Dystrophy Research Centre
(JWMDRC) team encompasses a number of specialists performing world-class translational
research to bring diagnosis, care and therapy to people with neuromuscular diseases.
These diseases range in scope from Duchenne Muscular Dystrophy and Spinal Muscular
Atrophy, to Limb Girdle Muscular Dystrophies and Myotubular and Centronuclear Myopathy.
The team aims to use the knowledge obtained from translational research to offer
patients with genetic and acquired neuromuscular diseases the opportunity to take part
in studies and clinical trials, which may lead to new treatments and improve the health
outcomes and quality of life for all patients and their families. Multidisciplinary in
nature, the JWMDRC team works together to collaborate on many different clinical trials
and studies in both adult and paediatric populations. Current and upcoming projects
include interventional clinical trials, translational research, natural history studies,
patient registries and BioBanks, plus exciting advancements in gene therapy. The team is
active in the conception and design of local, national and international commercial and
academic studies. The JWMDRC coordination team is responsible for obtaining MHRA
approval, Health Research Authority (HRA) and Research Ethics Committee approval, local
confirmation of capacity and capability from Research and Development, National
Institute for Health Research support and adoption and study management throughout the
whole process. Every member of the team is instrumental in conducting research in line
with Good Clinical Practice (GCP) that is facilitated by the coordination team to
produce the highest professional level of neuromuscular clinical research in Newcastle
and the North East.
Immune-array genotyping association analysis in a large cohort of sporadic
inclusion body myositis and controls
Alaa M. Khan1, Qiang Chang1, Pedro
Machado2, Michael Hanna3, Henry Houlden4
1Department of Molecular Neuroscience, Institute of Neurology,
University College London, Queen Square, London, UK; MRC Centre for Neuromuscular
Diseases, Institute of Neurology, University College London, Queen Square, London,
UK.
2Department of Molecular Neuroscience, Institute of Neurology,
University College London, Queen Square, London, UK; MRC Centre for Neuromuscular
Diseases, Institute of Neurology, University College London, Queen Square, London, UK;
Centre for Rheumatology, Division of Medicine, University College
3Department of Molecular Neuroscience, Institute of Neurology,
University College London, Queen Square, London, UK; MRC Centre for Neuromuscular
Diseases, Institute of Neurology, University College London, Queen Square, London, UK.
Electronic address: m.hanna@ucl.ac.uk.
4Department of Molecular Neuroscience, Institute of Neurology,
University College London, Queen Square, London, UK; MRC Centre for Neuromuscular
Diseases, Institute of Neurology, University College London, Queen Square, London, UK;
Neurogenetics Laboratory, Institute of Neurology, University College London, Queen
Square, London, UK. Electronic address: h.houlden@ucl.ac.uk.
Background: Sporadic inclusion body
myositis (sIBM) is characterized by a combination of inflammatory and degenerative
changes affecting muscles. While the primary cause is unknown. Genetic factors
contributing to the aetiology of sIBM. Our last whole exome sequencing association
analysis in sIBM and controls identified 15 common SNPs within Major histocompatibility
complex (MHC) region. This finding is inconsistent with the previous reports that MHC
associated with the risk of sIBM. However, because this region is in high degree of
linkage disequilibrium (LD) existing among alleles, it is very difficult to know the
causal allele among these in LD.
Aim: My aim from this work is to explore
the sIBM cases-controls genotyping array association by investigating the potential
association with HLA-related genes.
Methods: A total of 434 Caucasian patients
with IBM were recruited from many countries through International IBM Consortium Genetic
Study (IIBMCGS). These cohort will be genotyped by immunochip array a custom-designed,
high-density genotyping chip that covers genes known to be associated with a variety of
autoimmune diseases.
Expected result: We expected to identify
the most significant risk allele within HLA genes. This include the alleles identified
in the recent association study that showed a strong association with HLA-DRB1*03:01,
HLA-DRB1*01:01 and HLA-DRB1*13:01. Promising association might be identified with large
sample size. We are aiming to reach 550 sIBM cases.
Overnight Pulsoximetry for respiratory progression screening in a Neuromuscular
Service
Michelle McCallum, Anna Mayhew, Dionne Moat, Jas Sodhi, Meredith
James, Sabine Specht, Michela Guglieri, Volker Straub, Chiara Marini-Bettolo,
Robert Muni-Lofra
Robert.muni-lofra@ncl.ac.uk
The John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle University and Newcastle upon Tyne Hospitals NHS Foundation Trust,
Newcastle upon Tyne, UK#
Background: Progressive respiratory
involvement it’s been described in some patients with neuromuscular disorders (NMD)
being the result of progression of weakness in the inspiratory muscles in combination to
alteration of the mechanical properties of the lungs and chest wall (1). In patients
with NMD, clinical assessment of respiratory function and performance of pulmonary
function tests are of great value to screen for nocturnal respiratory disorders,
weakness of the diaphragm, hypercapnic respiratory insufficiency and potentially cough
deficiency(2). The screen the rate of progression and the need of potential referral to
specialized respiratory services for additional interventions becomes critical in over
time.
Aims: To explore the use of Overnight
Pulsoximetry (OP) as an initial screen to evaluate respiratory progression in
neuromuscular patients.
Methods or Patients or Materials: A
retrospective audit of OP tests was performed from January 2017 to July 2018. Reason for
referral and outcome of the OP were reviewed alongside diagnosis, age, FVC and FVC% and
presence of signs of nocturnal hypoventilation.
Results: A total of OP 83 tests were
audited (23% from pediatric patients). There was a range of diagnosis. Duchenne Muscular
Dystrophy (14%) and Fascioscapulohumeral Muscular Dystrophy (13%) were the most common
ones.
Main reason (75%) for referral was the presence of
hypoventilation signs. Low values of FVC%, drop in FVC from sitting to lying or
incapacity to perform FVC test in clinic were the other ones. The OP outcome for the
majority of patients (42%) indicated absence of objective changes in overnight traces.
Only 29% were referred to respiratory specialist services (13% to Sleep Services under
the suspicion of Obstructive Sleep Apnea). The remaining demonstrated either a
combination of normal SPO2 mean with an altered Sleep Apnea Index (SAI), moderately
altered SPO2 mean but stable in comparison to previous studies or moderately altered
SPO2 mean but with no symptoms. The presence of artefact traces resulting in the need of
repeating the study was found in 16%.
Conclusion: OP appears to be a valuable
screening assessment for those patients who report signs of hypoventilation with an
unclear respiratory involvement signs. Further exploration of confounding factors such
as fatigue, musculoskeletal pain, is strongly encouraged to discriminate from
respiratory progression.
References:
1. Chatwin M, Ross E, Hart N, Nickol A, Polkey M,
Simonds A. Cough augmentation with mechanical insufflation/exsufflation in patients with
neuromuscular weakness. Eur Respir J. 2003;21:502–8.
2. Priou P, Trzepizur W, Meslier N, Gagnadoux F.
Update on the respiratory management of patients with chronic neuromuscular disease
[Internet]. Vol. 73, Revue de Pneumologie Clinique. 2017 [cited 2019 Jan 8]. p. 316–22.
Available from: http://www.ncbi.nlm.nih.gov/pubmed/29174288
Exercise training outcome measures in Neuromuscular Diseases: a systematic
review
Renae J. Stefanetti, PhD1*,
Alasdair Blain1, PhD1 Cecilia Jimenez-Moreno PhD1,
Linda Errington, MSc2, PhD1, Robert McFarland, MD1,
Doug Turnbull, MD1, Jane Newman PhD1,3, Gráinne S. Gorman
MD1
1Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, UK
2Faculty of Medical Sciences, Newcastle University, UK
3 NIHR Newcastle Biomedical Research Centre. Campus for Aging and
Vitality. Newcastle University
Background: Efficacy of exercise
interventions in patients with myopathy has long been a contentious topic, heightened by
the variability of outcome measures across studies. We systematically reviewed outcome
measures utilised to assess exercise training in patients with muscle diseases and
performed a meta-analysis for outcome measures that were most frequently employed.
Aim: Evaluate the relevance and usefulness
of the most frequently utilised outcomes to evaluate the benefit of exercise training in
patients with muscle diseases.
Methods: A systematic review and
meta-analysis were performed according to PRISMA. We searched MEDLINE, Embase, Scopus,
WoS, SPORTDiscus, CDSR, CENTRAL, and DARE from inception to April 2018. Articles were
eligible according to pre-defined criteria. The most frequently utilised outcome
measures were evaluated by a random-effects meta-analyses pre, post and training,
non-training.
Results: Our search identified 33,727
records; 130 articles fulfilled inclusion ctiteria, comprising 1,808 patients with
muscle diseases. We observed a significant increase in maximal aerobic capacity and peak
power pre versus post exercise training (P<0.0001) and exercise
training versus control (P<0.05). Compared to post-training, 6
Minute Walk Test (P<0.05), timed sit to stand tests (P<0.05), rise from supine
(P<0.005), SF-36 (P<0005), fatigue severity scale (P<0·0001), central nuclei
(P<0.05), type I/type II muscle fibre area (P<0.005), and citrate synthase
(P<0·0001) were effected. We noted no effect on the Timed up and Go, Quality Of Life
domains, capillary density, fibre type composition, strength and creatine kinase
compared with non-exercise controls and/or post-exercise.
Conclusion: An array of outcome measures
currently exist to assess exercise efficacy and safety in patients with myopathy.
Greater consensus of the most appropriate outcome measures to be used in clinical
research and clinical practice is required. A comprehensive review of outcome measures
utilised will facilitate academics, clinicians, and pharmaceutical companies in trial
design. These findings have major implications in the context of clinical trial
readiness.
Physical activity interventions and therapeutic exercise in rare neurological
disorders: a protocol for a scoping review
Valentina Buscemi1, Jonathan
Marsden2, Helen Dawes3, Fiona Jones4, Lynne
Rochester5, Lorna Paul6, Thomas Jaki7, Rebecca
Playle8, Mike Robling8, Rachel Breen8, Annette
Boaz9, Monica Busse8, Gita Ramdharry1
1 UCLH Queen Square Institute of Neurology, London; 2
Plymouth University; 3 Oxford Brookes University; 4 Kingston
University; 5 Newcastle University; 6 Glasgow Caledonian
University; 7 University of Lancaster; 8 Cardiff University;
9 St. George’s, University of London
Email: valentina.buscemi@nhs.net
Background: Rare neurological conditions
have a significant cost burden on health and social care services. Neurological
impairments result in increased dependence and sedentary lifestyle. Although enhancing
physical activity is considered imperative in these conditions, the effectiveness of
interventions on physical activity and therapeutic exercise across rare neurological
disorders remains unclear. The aim of this protocol is: to synthesise the body of
evidence on the effectiveness of physical activity interventions and therapeutic
exercise for people with rare neurological diseases.
Methods: a scoping review will be
undertaken across a broad range of rare conditions, such as the ataxias (e.g.
Friederich’s ataxia), hereditary spastic paraparesis, Huntington’s disease,
neuromuscular diseases (e.g. polyneuropathies, myasthenia and muscular dystrophies),
motor neurone disease, atypical parkinsonisms. A two-phase search strategy will be
conducted: a preliminary search will be conducted using keywords such as physical
therapy, physiotherapy or exercise. Relevant keywords of systematic reviews will be
extracted to form a second list of terms that will be used to run the final searches
through the following databases: Pubmed, Embase and CINAHL. Only full-text systematic
reviews in English will we included with at least one outcome measure on physical
activity (e.g. walking, endurance or balance). Only studies concerning adults over the
age of 18 will be considered. Pharmacological studies will be excluded. Data extraction:
titles and abstracts will be screened for eligibility by one reviewer against
inclusion/exclusion criteria. The full-text of papers that are potentially suitable will
be retrieved and checked by a second reviewer, who will also independently review a
random sample of 10% of the total papers for inclusion or exclusion. Any discrepancies
will be discussed and resolved by a third party. Data will be extracted from each
eligible review including, where possible: research designs, population sample sizes,
details of interventions and any control, outcome measures, study results and effect
sizes. A narrative synthesis of findings will be undertaken.
Conclusion: this synthesis should help to
identify the available evidence on physical activity interventions and therapeutic
exercise and areas of unmet needs in individuals with rare neurological disorders.
Clinical presentation of patients with TANGO2 mutations
1Wellcome Centre for Mitochondrial Research, Institute of Genetic
Medicine, Newcastle University,
Newcastle upon Tyne, UK;
2Department of Neuropediatrics and Muscle Disorders, Medical Center –
University of Freiburg,
Faculty of Medicine, Germany;
3Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V,
Biomedical Research Department, Otto-Hahn-Str. 6b, 44227 Dortmund, Germany;
4Institute of Clinical Genetics and Tumor Genetics, Bonn,
Germany;
5Kid’s Neuroscience Centre, Children’s Hospital at Westmead, Sydney,
NSW2145 Australia;
6Wellcome Centre for Mitochondrial Research, Institute of
Neuroscience, Newcastle University, Newcastle upon Tyne, UK;
7 Discipline of Child and Adolescent Health, The University of
Sydney, NSW2006, Australia;
8John Walton Muscular Dystrophy Research Centre, Institute of Genetic
Medicine, Newcastle
University, Newcastle upon Tyne, UK;
9Departments of Clinical Biochemistry, Genetics, Pediatric Neurology
and Cardiology. Institut de Recerca Sant Joan de Déu and CIBERER, Instituto de Salud
Carlos III Barcelona, Spain;
10Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham,
UK;
11Department of Inherited Disease, St Thomas Hospital, London,
UK;
12South West Regional Metabolic Department, Bristol Royal Hospital
for Children, Bristol, UK;
13Centro Andaluz de Biología del Desarrollo, Uníversidad Pablo de
Olavide-CSIC-JA and CIBERER, Instituto de Salud Carlos III, 41013 Sevílla,
Spain;
14Secció d’Errors Congènits del Metabolisme –IBC, Servei de
Bioquímica I Genètìca Molecular, Hospital Clínìc, IDIBAPS.CIBERER. Barcelona,
Spain;
15Neurometabolic Diseases Laboratory, Institut d’Investìgacío
Biomedíca de Bellvitge (IDIBELL), and Centre for Biomedical Research on Rare Diseases
(CIBERER), Instituto de Salud Carlos III, Spain
16Catalan Institution of Research and Advanced Studies (ICREA),
Catalonia Barcelona, Spain;
17 Children’s Hospital of Eastern Ontario Research Institute,
University of Ottawa, Ottawa, Canada and Division of Neurology, Department of
Medicine, The Ottawa Hospital, Ottawa, Canada
18Department of General Pediatrics, Adolescent Medicine and
Neonatology, Medical Center – University of Freiburg, Faculty of Medicine,
Germany
19Pediatric Neurology, University Childrens Hospital, University of
Duisburg-Essen, Faculty of Medicine, Essen, Germany
20Department of Clinical Neurosciences, University of Cambridge,
Cambridge, UK
Background: Autosomal recessive mutations
of the Transport And Golgi Organization protein 2 (TANGO2) have recently been identified
as the cause of a rare metabolic disorder with a distinct clinical and biochemical
phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis,
arrhythmias and encephalopathy with cognitive decline.
Aims: To further elucidate the clinical
presentation of patients with TANGO2 mutations.
Patient and methods: We report nine
patients from seven independent families carrying TANGO2 mutations with
a complex presentation including developmental delay, recurrent metabolic crises, muscle
weakness, rhabdomyolysis, cardiac arrhythmias, encephalopathy and refractory seizures
with onset in early childhood. We studied muscle histology, biochemical measurement of
respiratory chain enzyme activities in skeletal muscle and immunoblot analysis of
fibroblasts and muscle.
Results: All nine patients carried
autosomal recessive pathogenic TANGO2 mutations. One patient was
homozygous for the reported deletion of exons 3-9, another carried the same deletion at
hemizygosity with a 22q11.21 microdeletion inherited in trans. The
other patients carried four novel homozygous mutations (c.262C>T/p.Arg88*;
c.220A>C/p.Thr74Pro; c.380+1G>A exon 5 essential splice-site variant), and a
further novel heterozygous mutation (c.11-13delTCT/p.Phe5del) which was hemizygous in
cDNA, indicating the loss of the other allele. These variants are very rare and
predicted to be pathogenic. Immunoblot analysis in fibroblasts (3 patients) and muscle
(1 patient) detected a decrease of TANGO2 protein levels.
Conclusions: These nine new cases expand
the clinical presentation, and further elucidate the molecular mechanism contributing to
the phenotype, of patients with TANGO2 mutations.
MDUK Oxford Neuromuscular Centre - Changing the landscape for clinical trials by
developing a major centre in the UK
Jaclyn Nicole Le Grand1, Kay
Davies2, Kevin Talbot3, Matthew Wood1
nicole.legrand@paediatrics.ox.ac.uk
1MDUK Oxford Neuromuscular Centre, University of Oxford, Department
of Paediatrics; 2Department of Physiology, Anatomy and Genetics;
3Nuffield Department of Clinical Neurosciences
Neuromuscular diseases (NMD) are an important group of
inherited and acquired disabling conditions caused by impairment of peripheral nerve and
/ or skeletal muscle function, and leading to premature death or major chronic
disability, often exacerbated by cardio-respiratory involvement. Over the last decade
there has been dramatic progress in the development and testing of experimental
therapies for NMD, exemplified by the unprecedented number of possible therapies for
Duchenne muscular dystrophy (DMD) coming to trial, the extensive patient registries and
work on outcome measures and regulatory interactions led by TREAT-NMD and other
groups.
However, to date, involvement of clinical trial sites
in the UK outside the main centres at Newcastle and London has been limited, especially
in industry funded studies. There are thus two current strategic imperatives: first, to
invest in and accelerate the pre-clinical discovery, development and translation of
improved, second generation experimental therapies for DMD and other NMDs; and second,
to dramatically increase UK clinical trial capacity.
As such, the MDUK Oxford Neuromuscular Centre, directed
by Professor Matthew Wood, was founded in 2019 by Muscular Dystrophy UK and the
University of Oxford to focus on the urgent mission of accelerating the discovery,
development and deployment of new medicines to combat devastating neuromuscular
diseases. The Centre builds on the already excellent research, training and patient care
in Oxford to drive the development of novel experimental therapies more rapidly and
increase national clinical trial capacity in neuromuscular diseases.
The core strategic and scientific objects of the Centre
are to:
Develop enhanced clinical infrastructure
and clinical trial capacity in adult and paediatric neuromuscular
disease;
Deliver new experimental medicines to impact
neuromuscular disease;
Advance neuromuscular gene
discovery to identify new therapeutic targets and
biomarkers;
Develop new and improved stem cell and
animal models for neuromuscular disease;
Build
capacity through recruitment and a PhD training
programme;
Build partnerships with industry to
advance the core translational objectives.
