Understanding and Ameliorating Pathogenesis in FSHD

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


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

Facioscapulohumeral muscular dystrophy (FSHD) is caused by production of a protein (DUX4) that is not normally present in muscle. DUX4 is a 'transcription factor' that controls other genes, thus upsetting the carefully coordinated pattern of gene expression in muscle, leading to weakness and wasting.

One the many proteins that DUX4 controls is called HIF1alpha. This protein is very important in the way that a cell responds to low oxygen levels. However, abnormal sustained HIF1alpha levels can lead to cell death. FSHD cells are also less efficient at dealing with by-products of metabolism/respiration called reactive oxygen species (oxidative stress), which are normally dealt with by compounds called anti-oxidants, such as Vitamin C. Importantly, a recent clinical trial (clinicaltrials.gov number: NCT01596803) reported that administration of Vitamin C, as part of a panel of anti-oxidants, improved muscle function in FSHD patients.

This project will help better understand the mechanism behind this encouraging observation in exploring the effects of manipulating HIF1aplha levels and associated signalling factors and how Vitamin C exerts its effects in improving muscle
function in FSHD.

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. Better understanding the disease mechanism in FSHD may also highlight other potential therapeutic interventions.

Technical Summary

Hypothesis: DUX4 leads to down-regulation of PAX7 target genes, resulting in sustained HIF1alpha activity that both facilitates apoptosis and suppresses canonical Wnt/beta-catenin signalling to affect muscle differentiation and function in FSHD.

Research Objectives
1: Understand DUX4-Pax7-HIF1alpha axis. Express a panel of DUX4 or PAX7 constructs via viral-mediated delivery (including constitutively-active and dominant-negative versions) in FSHD/control myoblasts to assay effects on HIF1alpha and its downstream target genes (RT-qPCR, Western and immunostaining).
2: Determine if Pax7 interferes with HIF1alpha levels. Investigate whether constitutive Pax7 expression will suppress HIF1alpha activity in FSHD myoblasts. Conversely, inhibit Pax7-target genes using dominant-negative PAX7 or DUX4 constructs to examine effects on HIF1alpha activity.
3: Determine if elevated HIF1alpha contributes to apoptosis in FSHD. Inhibit HIF1alpha with specific inhibitors, and siRNA and/or CRISPR\Cas9 in FSHD myoblasts to determine if cell survival is improved. Examine interactions between HIF1alpha and P53.
4: Inhibit/overexpress/stabilise Hif1alpha in myoblasts and examine if canonical Wnt signalling is altered using TopFLASH/FopFLASH luciferase activity assays. Determine whether increased canonical Wnt signalling/beta-catenin activity affects HIF1alpha and rescues myogenesis/survival under oxidative stress.
6: Determine if antioxidants including vitamin C, vitamin E, CoQ10 reduce HIF1 activity while improving myogenic differentiation in FSHD myoblasts. Is vitamin C as effective when HIF1alpha activity is inhibited/enhanced/stabilised?
7: Perform RNASeq on healthy and FSHD myoblasts exposed to antioxidants to further decipher the signalling pathways that are altered to improve myogenic differentiation/survival under oxidative stress.

This research will both further reveal disease mechanisms and also allow better understanding of potential therapies for FSHD.

Planned Impact

This research is directed at understanding pathology of the muscular dystrophy facioscapulohumeral muscular dystrophy (FSHD), which will inform development/optimisation of potential treatments. FSHD is an autosomal dominant condition yet pathophysiology is poorly understood and there is currently no cure. Clinically, FSHD presents as an asymmetric, progressive descending skeletal muscle weakness and wasting affecting a variety of muscles that severely effects quality of life. For example, some FSHD patients undergo scapular fusion surgery, involving fixation of the scapular to the ribcage to stabilize the shoulder, which renders patients significantly incapacitated for several months. Approximately 30% of patients use a wheelchair. Moreover, FSHD is associated with sensorineural hearing loss, which can progress to deafness, and a retinal vascular abnormality, which can compromise vision. Fortunately 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 are newly diagnosed with FSHD every year in the Netherlands, giving 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. For example, 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, in Germany cost perpatient year 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 retirement, 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). Moreover, psychological problems occurred in 28.1% of FSHD registry members, with more than one-third reporting having received psychological counselling (http://www.ncbi.nlm.nih.gov/pubmed/22155025).

Direct beneficiaries with immediate impact from the work will include academics, clinicians, and the pharmaceutical industry. Researchers/clinicians will benefit as better understanding the pathomechanims will inform treatment and development of new 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 those involved in translational research will benefit as this work will inform planning pre-clinical and clinical trials.

In the longer term, patients and carers will benefit from a relief of disease burden from a better understanding of disease pathology and improved therapy. Thus society and the wider 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 their non-professional carers.
Description Advisory board of French Charity
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
Impact Advise on how funds should be awarded by a French Charity that supports research and improvements to quality of life in patients suffering from muscular dystrophy.
Description Research policy of a major charity
Geographic Reach North America 
Policy Influence Type Membership of a guideline committee
URL https://www.fshsociety.org/wp-content/uploads/2018/09/2018_FSHD_IRC_-Priorities.pdf
Title RNA-Sequencing data 
Description RNA-Sequencing data of muscle cells from muscular dystrophy patients and family controls. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Freely available through GSE for other researchers to re-analyse and use. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE123468
Description Measuring oxidative stress 
Organisation Ludwig Boltzmann Society
Department Ludwig Boltzmann Institute for Experimental and Clinical Traumatology
Country Austria 
Sector Charity/Non Profit 
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
Title FSHD biomarker 
Description A gene set biomarker to distinguish healthy muscle biopsy/cells samples from those obtained from muscular dystrophy patients. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Initial development
Year Development Stage Completed 2018
Development Status Under active development/distribution
Impact Improved biomarker for identifying samples from muscular dystrophy patients, and could be used for monitoring effectiveness of clinical interventions. 
Title Biomarker analysis software 
Description Software that gives a biomarker score for a muscular dystrophy using inputted RNA-Sequencing data. To facilitate consideration of PAX7 target gene repression in the analysis of FSHD transcriptomic data, we provide a simple pipeline for extraction of PAX7 target gene repression, as well as three validated DUX4 target gene expression biomarkers, from normalized gene level expression data. Available as supplementary data from a publication - doi: 10.1093/hmg/ddz043 
Type Of Technology Software 
Year Produced 2019 
Impact A simple pipeline for extraction of PAX7 target gene repression, as well as three validated DUX4 target gene expression biomarkers, from normalized gene level expression data 
Description House of Lords - MDUK Vice-President's lunch 
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 MDUK Vice-President's lunch at the House of Lords that is attended by patients suffering from muscular conditions/carers/sponsors/donors/scientists/clinicians. Important occasion to explain the causes of muscular conditions and the research that is being conducted to understand and treat such disorders.
Year(s) Of Engagement Activity 2017,2018
Description Patient Group Workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Supporters
Results and Impact Lay talk at 2018 FSHD Connect: The FSH Society's International Network Meeting for FSHD Families, Clinicians, and Researchers, Las Vegas, June 2018 (https://www.fshsociety.org/2018/06/2018-fshd-connect-conference/).
Year(s) Of Engagement Activity 2018
URL https://www.fshsociety.org/2018/06/2018-fshd-connect-conference/