THE WOLFSON CENTRE FOR INHERITED NEUROMUSCULAR DISEASE (CIND).
The Clinical Research Team: Dr Tracey Willis, Mr Nigel Kiely, Dr Richa Kulshrestha, Dr Yvette Easthope-Mowatt, Mr Nick Emery, Mrs Kathryn Titchen, Dr Deborah Short.
The Laboratory Research Team: Prof. Caroline Sewry, Prof. Glenn Morris, Dr. Heidi Fuller, Dr Ian Holt, Dr Le Thanh Lam, Dr Emma Wilson, Dr. Nguyen Thi Man,
This year has seen the completion of major research projects funded by three medical charities in the USA and by the British Heart Foundation in the UK. These projects were aimed at finding treatments for several different forms of muscular dystrophy. Many muscular dystrophy patients also have heart problems which become more frequent as patients survive longer with improved care. In addition, the Institute of Orthopaedics is supporting new pump-priming projects on muscular dystrophy and nemaline myopathy, while the Trust Charitable Funds at RJAH are supporting a project on bone loss caused by anti-epileptic drugs (with Dr Mark Garton). We welcome back Dr Emma Humphrey (now Emma Wilson) to work on this project.
The MDA Monoclonal Antibody Resource.
For more than 10 years, funding from the Muscular Dystrophy Association, a US Charity, has enabled Dr Le Thanh Lam of our Wolfson Centre to distribute monoclonal antibodies all over the world to researchers and clinicians involved in finding treatments, or improving diagnosis, for a wide variety of neuromuscular disorders. The antibodies were all produced by members of Prof. Morris’ laboratories over the past 35 years and are made freely available internationally. The neuromuscular disorders to which the antibodies relate are Duchenne and Becker muscular dystrophies, myotonic dystrophy, congenital, limb-girdle and Emery-Dreifuss muscular dystrophies, spinal muscular atrophy and nemaline myopathy. Between October 2007 and June 2014, the Resource distributed 3,288 units of 377 different antibodies to 238 laboratories (35% in North America, 33% EU, 25% UK and 8% Rest of the World). In addition, the Iowa Hybridoma Bank has distributed over 1,000 units on our behalf (mainly within the USA).
One of our antibodies against dystrophin, MANDYS106, has been used almost exclusively in the search for novel treatments for Duchenne muscular dystrophy using exon skipping. Different drugs (Drisapersen and Eteplirsen) were developed by GlaxoSmithKilne and Sarepta Therapeutics respectively, both having used MANDYS106 to test for successful up-regulation of dystrophin protein in patients treated with the new drugs. The FDA (Federal Drugs Administration) in the USA have not yet given “fast-track approval” for these drugs and Prof. Glenn Morris gave an invited talk about MANDYS106 at a FDA-sponsored conference on “Measuring Dystrophin in Dystrophinopathy Patients and Interpreting the Data” in Washington DC on March 20th 2015.
Spinal Muscular Atrophy
Heidi Fuller continues to work on her SMA Trust-funded Fellowship project aimed at understanding the consequences of reduced SMN in induced-pluripotent stem cell (iPSC)-derived motor neurons from SMA patients. In collaboration with a world-leading stem cell expert in Los Angeles, she is applying cutting-edge proteomics and bioinformatics tools to understand more about why motor neurons are so vulnerable in SMA.
Several other SMA-related collaborations have been fruitful this year. Together with long-standing collaborators in Edinburgh, Heidi was involved in a study to determine the stability of the SMN protein. Published in Neuromuscular Disorders, the study highlighted the fragility of the SMN protein, and has important implications for clinical studies where SMN levels are being measured.
In collaboration with Dr Mark Garton and Prof Glenn Morris, Heidi was awarded £25,000 from the RJAH Charitable Funds to investigate the link between anti-epileptic drugs and bone loss. The grant is supporting Dr Emma Wilson (nee Humphrey), who re-joined the research group in January this year, and builds upon our previous work showing that an anti-epileptic drug suggested for SMA therapy causes reduction of key bone proteins in cultured cells.
Heidi Fuller (with Dr Monte Gates, Keele) was awarded £16,500 continuation funding from the Henry Smith Charity to characterise proteins and cellular pathways that are associated with the growth and development of spinal motor circuitry. Such analyses have the potential to identify proteins that may be exploited to facilitate regeneration of spinal-motor connections after spinal cord injury or motor neuron disease, such as SMA. Visiting EPSRC and MRC Centre for Doctoral training student, Lia Blokpoel-Ferreras, continued to build upon this work during her two-month mini-project placement at RJAH during the spring of this year.
Heidi continues to collaborate with RJAH and Keele researchers on a range of projects involving proteomics and bioinformatics analysis. During the last year, these additional collaborations have resulted in two publications and an Arthritis Research UK grant award totalling £189,195, led by Dr Karina Wright, Spinal Studies.
Limb-girdle muscular dystrophy and dystroglycanopathies.
Dystroglycanopathies, including the limb-girdle muscular dystrophy, LGMD2I, are caused by failure to carry out vital modifications of a dystrophin-associated muscle membrane protein called dystroglycan. Failure to modify dystroglycan correctly can have severe consequences, not only in skeletal muscle, but also in heart and brain. Our laboratory research, conducted by Drs Emma Wilson (Humphrey), Heidi Fuller, Ian Holt and Le Thanh Lam is aimed at producing antibody reagents that will identify the dystroglycan modifications that have failed in the muscle of individual patients. The antibodies will be used for diagnosis of the different types of dystroglycanopathies, and also to help understand disease progression and therefore potential treatments. Our initial work on the production of the monoclonal antibody, DAG-6F4, was published in Neuromuscular Disorders in 2014. Since then, two further antibodies, DAG-1F7 and DAG-1G4, have been produced and show improved performance in diagnostic tests on muscle biopsies. This work was funded by two USA charities, CureCMD and LGMD2I.
This disorder is caused by genetic mutations in the muscle fibre proteins, actin, tropomyosin, troponin or nebulin. Nebulin is the largest of these proteins. Dr Le Thanh Lam has successfully produced new antibodies against specific exons 143 and 144 of nebulin. These regions are of interest because all nebulin molecules contain either 143 or 144, but never both. It is hoped that the new antibodies will help us to understand how the two isoforms of nebulin differ in their functions. This project is collaboration with world Nemaline expert Dr. Carina Wallgren-Pettersson (University of Helsinki). Prof Caroline Sewry (CIND) has organized a workshop on this disorder, bringing together participants from all around the world in London on Sep 28th, 2015, to decide the best way to fund research to help nemaline patients.
Legend: The new anti-nebulin antibodies produced by Dr. Lam stain the functional myofibrils within the muscle fibre of this human muscle biopsy.
Nesprins and cardiomyopathy in Emery-Dreifuss dystrophy.
Nesprins are large proteins, similar to dystrophin in structure, but over twice the size (ca, 1000kDa) and they are anchored in the outer nuclear membrane by SUN proteins, which in turn interact with emerin and lamins at the inner nuclear membranes. Nesprins anchor nuclei to the actin cytoskeleton in the cell cytoplasm and are thought to be important in maintaining structural integrity, especially in cells subject to mechanical forces, like the beating heart. This whole assembly of interacting proteins has been called the LINC complex. Mutations in emerin, lamin A, nesprins and, most recently, SUN proteins all cause Emery-Dreifuss MD, a neuromuscular disease with cardiac problems as its characteristic feature.
Legend: Our new antibody specific for the short isoform of nesprin-1 (the alpha2 isoform) shows that this isoform is absent from dividing myoblasts but present after they develop into multi-nucleated muscle fibres in cell culture.
Mirjana Babic, a PhD student from Professor Goran Simic’s group at the Croatian Institute for Brain Research in Zagreb, visited the Centre for 5 weeks in May/June 2014 to work with Dr Ian Holt on the nesprin project. The aim of her visit was to determine the levels of mRNAs for all nesprin isoforms in different regions of the human brain, a study which is still on-going.
Legend: Left to right: Glenn Morris, Mirjana Babic, Heidi Fuller, Ian Holt.
The Wolfson Centre will host the 7th UK Conference on Nuclear Envelope Diseases in June 2015, just 9 years after it hosted the very first such Conference in March, 2006. Over 50 researchers from around the UK, with a few international participants, are expected to attend.
ORLAU (ORTHOTIC RESEARCH AND LOCOMOTOR ASSESSMENT UNIT)
This year has seen a number of projects coming to completion. Justine Bee has carried out two research projects whilst on secondment to ORLAU. The first project Functional Electrical Stimulation (FES) in patients prior to knee replacement provided the material for her MPhil thesis, which she successfully completed this year. Justine has subsequently worked on a grant looking at gait patterns in patients following anterior cruciate ligament (ACL) surgery. She has recruited a number of patients and control volunteers during the year. Previously we measured high joint loads and muscle activity in patients with advanced knee arthritis. Justine was keen to see whether patients after ACL repair were also overloading their joints. Because these patients tend to be very active she extended her analysis beyond walking to including running, jumping, squatting and hopping. Recruitment has now finished and Justine has returned to her full time clinical role in the Therapies Department, however we plan to analyse and publish her results over the coming months.
We have also completed our work on the Motorised Os-Stretch device, a project funded by the League of Friends and led by Sarah Jarvis. The motorised Os-Stretch was designed and built in ORLAU and compromises a tilting platform for patients to stand on. As the platform tilts their calf muscles are slowly stretched. Sarah has recruited 18 volunteers (10 children with no known pathology, 4 with cerebral palsy and 4 idiopathic toe walkers). Attention has now turned to the analysis of the very large dataset which this project has produced. Preliminary results were presented at the ESMAC (European Society of Movement Analysis in Adults and Children) meeting in Rome in September 2014. A number of interesting insights are emerging and we hope to use the results to improve our treatment of children with tight calf muscles.
Victoria Kidgell, our clinical scientist trainee, has led the data collection and analysis for the FAST project, which has also reached its conclusion in recent weeks. This study has examined the walking patterns of patients who have had their ankle joint surgically fused. The study has investigated whether patients can be helped by wearing shoes with a rocker sole. Orthopaedic footwear, incorporating a rocker, can be provided but this is an expensive option. Victoria has been comparing specialist orthopaedic footwear with commercially available rocker soled shoes. Differences were found between different types of footwear and subjects varied in the shoes they preferred. For patients to feel comfortable, rockers had to be provided on both shoes (not just on the affected side). Commercially available rockers proved popular for some, though a rocker running the full length of the foot did lead others, and particularly the older subjects, to feel unstable.
Victoria has also been working with Keith Miller and the rehabilitation engineering team to develop two Hip Spica Perches (infant and junior sizes) in conjunction with Alice Ward staff and Mr Kiely. The new perches are designed to replace the old bespoke ‘wooden’ pattern and are manufactured using aluminium alloy and polymer materials. They have been developed to be adjustable to fit a wide range of patients and are reusable to reduce the costs to the Trust. The first patient has just completed a trial of the new device with a successful outcome. More trials are planned.
We were disappointed, early in the year, to learn that we had not been selected as a specialist centre to provide selective dorsal rhizotomy (SDR) operations for children with cerebral palsy. We were the first centre in the UK to offer this surgery 20 years ago and so have a substantial database of patient experience. This database has allowed us to collate our long term results into a paper which is currently going through the review process. This was done with the assistance of Cardiff University medical student John McFall. Andrew Roberts was also invited to write a review paper for the Gait & Posture journal, documenting the role of gait analysis in the treatment of children by means of SDR. These two papers have provided us with a useful means of putting our experience into the public domain.
As a number of projects draw to a conclusion, two new grants have been awarded. ORLAU staff have joined a partnership, led from Newcastle, which applied to the NIHR HTA scheme for funds to investigate the potential of carrying out a clinical trial of standing frames. In ORLAU we have developed our own design of standing frame so we are interested to know how our patients’ experience compares with those using other commercial systems. Funding has been granted and work will commence this Summer.
Another successful application was made to the Health Technology Co-operative (HTC) scheme which funds research to benefit patients with brain injury. The funds awarded will be used to develop the ORLAU Buzz, a new device which combines some of the supportive features of a standing frame with a vibrating plate for the patient to stand on. The rehabilitation engineering team have been hard at work developing a design and prototype. Once ethical approval has been secured 3 brain injury patients will try out the new device to see if it has a beneficial impact on their troublesome muscle spasms.
Through the year ORLAU has continued to work on building academic partnerships and further grants have been submitted. Despite continuing pressures from our busy clinical services we are keen to continue investigating human walking and developing new devices so we can treat our patients better in the future.
Junior Hip Spica Perch