Understanding and Ameliorating perturbed signalling and pathogenesis in FSHD

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy

Abstract

Diseases can be treated more effectively if the causes/mechanisms underlying the symptoms are known. For muscular dystrophy, the causative defect is changes in genes or DNA that in turn, produce changes to proteins. However, it is generally poorly understood how such defects result in the progressive skeletal muscle weakness and wasting typical of muscular dystrophies.

Every cell in the body contains the same DNA, which is divided into genes, each of which carries the instructions to make a protein(s): the molecules that build, maintain and operate the body. The selection of genes that are expressed (active) in a particular cell type, such as in a muscle fibre, dictate which proteins are made. This gives the cell its special characteristics and functions. For example, muscle genes are not expressed in skin cells, and skin genes are not expressed in muscle cells.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by a change in a particular region of DNA that leads to production of a protein called DUX4, that is not normally present in muscle. DUX4 is a 'transcription factor' meaning that it can control the expression of other genes by binding to their regulatory regions, and so can alter the type of proteins that are made by a cell. Thus the carefully coordinated pattern of gene expression and protein production that enables skeletal muscle to function effectively, is perturbed by the presence of DUX4. This ultimately leads to muscle weakness and wasting.

We have examined gene expression changes during human muscle formation in FSHD cells and used mathematical tools to understand which signalling process are perturbed. We found that pathways that control generation of mitochondria and processes associated with mitochondrial function such as dealing with by-products of metabolism/respiration called reactive oxygen species (oxidative stress) are suppressed. It is well known that FSHD cells are more sensitive to oxidative stress, and a recent clinical trial (clinicaltrials.gov number: NCT01596803) reported that administration of anti-oxidants, improved aspects of muscle function in FSHD patients.

We have found that inhibiting pathways that control generation of mitochondria in healthy human muscle cells makes them appear like FSHD muscle fibres. Conversely augmenting the action of these pathways controlling generation of mitochondria in FSHD muscle cells makes them more like healthy human muscle cells.

This project will investigate how regulation of this pathway controlling generation of mitochondria is perturbed in FSHD and test potential therapeutic strategies to improve its function.

Thus in summary, we will generate tools and models of FSHD that will both further reveal disease mechanisms and also provide a platform for testing potential therapies for FSHD, to underpin possible clinical trials. Better understanding the disease mechanism in FSHD may also highlight other potential therapeutic interventions.

Technical Summary

Facioscapulohumeral muscular dystrophy (FSHD) is the third commonest inherited myopathy, characterised by a descending, often asymmetric, dystrophy with initial involvement of facial and proximal upper limb musculature, and later progression to certain lower limb muscles.

We have examined gene expression changes during muscle formation in human FSHD cells and used mathematical tools to understand which signalling process are perturbed. We found that pathways that control generation of mitochondria and processes associated with mitochondrial function such as dealing with reactive oxygen species are suppressed. It is well known that FSHD cells are more sensitive to oxidative stress, and a recent clinical trial (clinicaltrials.gov number: NCT01596803) reported that administration of anti-oxidants improved aspects of muscle function in FSHD patients.

We have found that inhibiting pathways that control generation of mitochondria in healthy human muscle cells generates myofibres with an atrophic phenotype: characteristic of FSHD muscle fibres. Conversely augmenting the action of these pathways controlling generation of mitochondria in FSHD muscle cells makes them generate myofibres that are larger and indistinguishable from healthy human muscle.

This project will investigate how regulation of this pathway controlling generation of mitochondria is perturbed in FSHD and test potential therapeutic strategies to improve its function.

Main objectives
1. Determine if drugs that increase mitochondrial biogenesis improve the FSHD phenotype in vivo.
2. Validate the mechanism of perturbed mitochondrial biogenesis in FSHD myoblasts.
3. Decipher the action of drugs that improve mitochondrial biogenesis in ameliorating FSHD.
4. Understand the regulation of mitochondrial biogenesis pathways in FSHD.
5: Determine if mitochondrial biogenesis improves DUX4 induced myopathy in vivo.

Planned Impact

This research is directed at understanding pathology of the FSHD, which will inform development/optimisation of treatments. FSHD is autosomal dominant with poorly understood pathophysiology and there is no cure. FSHD presents as an asymmetric, progressive descending skeletal muscle weakness and wasting affecting many muscles that severely affects quality of life: around 30% use a wheelchair. For example, patients can undergo surgery to fix the scapular to the ribcage to stabilize the shoulder, which significantly incapacitates patients for months. FSHD is also associated with sensorineural hearing loss, which can progress to deafness, and a retinal vascular abnormality, which can compromise vision. Lifespan is not attenuated, but means that economic burden lasts many years.

FSHD is the third most common inherited myopathy (prevalence ~1/20,000 http://www.orpha.net). Recent figures indicate that 52 people/year are newly diagnosed with FSHD in the Netherlands, a prevalence of 12/100,000 (1/8333).

Financial Costs: An estimated 70,000 people in the UK in 2010 had a neuromuscular condition, (http://www.muscular-dystrophy.org/assets/0002/0463/Cost_of_Living_with_Muscle_Disease.pdf), creating a considerable financial burden. The Access Economics research published in 2007 put costs of muscular dystrophy for the 3500 Australian patients in 2005 at £258 million a year (http://www.mda.org.au/media/accesslaunch/Gullotta.asp). More recently, calculated annual per-patient costs for neuromuscular disease in the US are $63,693 for ALS, $50,952 for DMD, and $32,236 for DM. Population-wide national costs were $1,023 million (ALS), $787 million (DMD) and $448 million (DM) (Larkindale et al. Muscle Nerve (2014) 49: 431-8). For FSHD, cost per patient year in Germany is 26,240 Euros (Schepelmann et al. J. Neurol. (2010) 257: 15). In addition, > 50% of adult members of the FSHD registry reported that symptoms adversely affected their employment by forced disability, early retiremen, or job loss, or that their job had been modified to accommodate physical limitations (http://www.ncbi.nlm.nih.gov/pubmed/22155025).

Quality of Life: 29.6% of FSHD registry members report use of wheelchairs for short or long distances, with 8.3% reporting as "always" using wheelchairs for both. 11.3% of FSHD registry members report use of leg braces at enrolment. The EQ-VAS quality of life score is 0.75 +/- 0.21 (0.81) for FSHD and such a low score associated with increased risk of depression (Winter et al. J. Neurol. (2010) 257(9):1473-81). FSHD registry paper: Moreover, psychological problems occurred in 28.1% of FSHD registry members, with more than one-third reporting having received psychological counselling (Hilbert et al. Contemp. Clin. Trials (2012) 33:302-11).

Direct beneficiaries with immediate impact from the work will include academics, clinicians, and the pharmaceutical industry. Researchers/clinicians will benefit as better understanding pathomechanisms will inform treatment and development of therapies, particularly relevant to progressing from the recent clinical trial involving antioxidants for FSHD that showed improvement in certain parameters (clinicaltrials.gov number: NCT01596803). Pharmaceutical companies and translational researchers will benefit as this will inform planning pre-clinical/clinical trials.

Longer term, patients and carers will benefit from relief of disease burden from better understanding disease pathology and improved therapy. Thus society and the economy benefits from improved treatments, due to reduction in NHS and care costs and both reduced loss of earnings and benefit payments for patients and carers. Biochanin A is available in food supplements and clinically tested, so could be rapidly adopted for FSHD, also encompassing MRC Priorities for Nutrition Research
https://www.mrc.ac.uk/funding/science-areas/population-systems-medicine/mrc-priorities-for-nutrition-research/

Publications

10 25 50
 
Description Cellular and sub-cellular sampling using laser capture microdissection to understand disease mechanisms
Amount £198,139 (GBP)
Funding ID 212917/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 09/2023
 
Description DUX4 and PAX7 interaction in FSHD pathology
Amount £224,210 (GBP)
Funding ID 19GRO-PG36-0330 
Organisation Muscular Dystrophy UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2020 
End 12/2022
 
Description Ameliorating oxidative stress with drugs 
Organisation Pablo de Olavide University
Country Spain 
Sector Academic/University 
PI Contribution Helping a team at Universidad Pablo de Olavide (Sevilla, Spain) to analyse muscle function in a mouse model of mitochondrial disease.
Collaborator Contribution Team at Universidad Pablo de Olavide (Sevilla, Spain) are collaborating on dosing regimes and techniques for delivering drugs to counter mitochondrial disfunction.
Impact This collaboration will help progress projects in both labs and lead to joint ventures.
Start Year 2018
 
Description HIF1A in Muscular dystrophy 
Organisation University of Mons
Country Belgium 
Sector Academic/University 
PI Contribution Exploring the role of HIF1alpha in oxidative stress response in muscular dystrophy using engineered human myoblasts.
Collaborator Contribution Supplying engineered human myoblasts and expertise on analysing HIF1alpha for a complementary project at University of Mons in Belgium. Supplying RNA-Sequencing data.
Impact Cross-fertilisation with a group experienced in analysing HIF1alpha in other systems.
Start Year 2018
 
Description Measuring oxidative stress 
Organisation Ludwig Boltzmann Society
Department Ludwig Boltzmann Institute for Experimental and Clinical Traumatology
Country Austria 
Sector Academic/University 
PI Contribution We have generated human cell samples to send to Ludwig Boltzmann Institute for Experimental and Clinical Traumatology for analysis of free radicals
Collaborator Contribution Ludwig Boltzmann Institute for Experimental and Clinical Traumatology analyse our samples of human cells for free radical content.
Impact A better understanding of free radical generation in muscular dystrophy will be the outcome, with joint publications planned.
Start Year 2018
 
Description Performed RNASeq in primary cells from FSHD patients 
Organisation Radboud University Nijmegen Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution Perform and analyse RNASeq in primary cells from FSHD patients supplied by Radboud University Nijmegen Medical Center, and shared the data.
Collaborator Contribution Supplied cells from FSHD patients supplied by Radboud University Nijmegen Medical Center.
Impact Cells being collected and RNA prepared.
Start Year 2019
 
Description RNASeq of primary FSHD cells 
Organisation University of Montpellier
Country France 
Sector Academic/University 
PI Contribution Performed RNASeq in primary muscle cells from FSHD patients supplied by University of Montpellier, and shared the data.
Collaborator Contribution Performed and analysed RNASeq in primary muscle cells from FSHD patients and healthy controls supplied by University of Montpellier, and shared the data.
Impact Data has contributed to a paper under review for Human Molecular Genetics.
Start Year 2019
 
Description Amis FSH Site Visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact Board and members of charity AMIS FSH performed a site visit to King's College London on 20/2/2020. This involved visits to the lab and then talks/discussions with Zammit group members explaining facioscapulohumeral muscular dystrophy and our research activity in lay terms. This better informed the participants about the disorder and research approaches, that will also be fed back to other charity members. Outcomes are dissemination of information and potential for research funding.
Year(s) Of Engagement Activity 2020
URL http://www.amisfsh.fr
 
Description Patient Group Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact FSHD Information Day - Muscular Dystrophy UK
Talk: - title - What causes FSHD and how can we treat it?
York Racecourse, Knavesmire Road, York YO23 1EX
23/11/19
Talk explaining FSHD and current research to a lay and specialist audience. Many comments about this being the first time that they had actually understood the causes of their disease. Pdf of the talk was requested for dissemination to patents/carers/supporters.
Year(s) Of Engagement Activity 2019
URL https://www.musculardystrophyuk.org/wp-content/uploads/2019/11/Information-Day-November-2019-full-pr...
 
Description School Visit to King's College London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Approximately 80 male and female year 7 pupils, with 6/7 school staff undertake histological staining and examining tissue sections under light microscopes. They were highly engaged with the topic, generating an active discussion and expressions of interest in STEM subjects. Useful experience for Zammit lab members who reported it an enlightening and rewarding activity.
Year(s) Of Engagement Activity 2018,2019,2020
 
Description Site visit by executives of Charity FSHD Soc 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact FSHD Soc charity site visit by Chief Science Officer and Chief Executive Officer of one of the major funders in FSHD research, to discuss our research activity, funding opportunities and further engagement by the Zammit group in charity events.
Year(s) Of Engagement Activity 2020
URL https://www.fshdsociety.org