Genes, mechanisms, models and treatments for hereditary myasthenia

Lead Research Organisation: University of Oxford
Department Name: Clinical Neurosciences

Abstract

Signalling between nerves is achieved largely by the opening and closing of ion channels, in their cell surface, at specialised sites called synapses. We believe an understanding of how synapses malfunction will help in finding the causes of many neurological disorders and in devising new therapies. We study inherited diseases, called congenital myasthenic syndromes, that cause severe disability in children and adults and affect ion channels, called acetylcholine receptors (AChR), located at the connection between nerves and muscles. These inherited mutations cause disease by affecting AChR in different ways. Understanding how the mutations cause disease enables us to give patients correct treatments and genetic counselling, and provides the knowledge base required for exploring new therapies. Several different genes have been shown to cause these disorders but in around 40% of cases the underlying defect is not known. Recently we identified mutations in a new gene, called DOK7. Standard treatments used in congenital myasthenia often make patients with DOK7 mutations worse. It is thought the faulty gene underlies a defect in the process that places the receptor in the correct position opposite the nerve ends. Here, in studies of this process we will learn why the signal from the nerve is not received by the muscle. Knowledge gained will help identify new genes involved in our disease, and help us understand why these patients do not respond to standard treatments. At present many patients with these mutations go undiagnosed or are misdiagnosed. Success in this project should prevent misdiagnosis, and enable us to provide better treatment for our patients.

Technical Summary

Congenital myasthenic syndromes (CMS) stem from genetic defects that affect transmission of information from nerves to muscles at the neuromuscular junction (NMJ) and result in fatiguable muscle weakness. They comprise a disabling and sometimes potentially lethal set of disorders with subtly different clinical features that arise from different underlying molecular mechanisms. Understanding the different disease mechanisms enables appropriate treatments to be given to patients, facilitates investigations of more common CNS disorders, and provides prototypic examples of synaptic dysfunction that may be used to evaluate new treatment strategies.
Acetylcholine receptors (AChR) are the ion channels that receive the signal for muscle contraction. Many mutations in CMS patients are in the AChR, but it is now apparent that an equal number will be identified in proteins that are responsible for the localisation and clustering of the AChR and also in maintaining the synaptic structure. We have detected a large number of mutations both in the AChR-clustering protein rapsyn and the newly identified muscle protein, Dok-7. Dok-7 has been identified as an essential component of the AChR clustering pathway.

The objective of this proposal is threefold: i) to investigate the molecular mechanisms of pathogenic mutations that impair AChR clustering, ii) to identify novel CMS-associated genes that are expressed on the postsynaptic side of the neuromuscular junction, and iii) to study current and novel therapies for congenital myasthenic syndromes in cellular and mouse models of disease.

In studies of rapsyn and Dok-7 mutations we have developed and are acquiring an exceptional set of experimental tools, including EGFP-tagged functional AChR, a series of pathogenic mutants, ?knock out? muscle cell lines and transgenic disease models, including a model of DOK7 CMS. We shall use these in a combination of microscopic, biochemical and genetic techniques to investigate the defective signalling from MuSK to the AChR that results in reduced and less stable rapsyn-associated clustering. We will use a multifaceted approach, including linkage analysis, the study of fetal akinesis, and knowledge of the MuSK signalling pathway to identify new candidate CMS genes. Therapies for CMS will be given to our transgenic mouse disease models and their response will be analysed in vivo by electromyography and ex vivo by electrophysiology, microscopy, and biochemical analysis

Publications


10 25 50
Burke G (2009) A treatable muscle disease. in Practical neurology
Cossins J (2012) The search for new antigenic targets in myasthenia gravis. in Annals of the New York Academy of Sciences
Cossins J (2013) Congenital myasthenic syndromes due to mutations in ALG2 and ALG14. in Brain : a journal of neurology
 
Description Defined treatment for patient subsets
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact Defined that ephedrine or other b2-adrenergic receptor agonists have a dramatic beeneficial effect for patients with DOK7 CMS. Thus this is now standard treatment for this genetic condition. many patients regain ambulation from previous requirement of a wheel chair. We have now defined other patient subsets for which appropriate treatment combinations leads to life-transforming improvement in quality of life. May patients regain ambulation from previously being wheel chair-bound.
 
Description Charitable Trust
Amount £90,000 (GBP)
Funding ID N/A 
Organisation J P Moulton Charitable Foundation 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2013 
End 09/2014
 
Description Fellowship within Wellcome Trust Ion channels in Disease strategic award
Amount £5,000,000 (GBP)
Funding ID Wellcome Trust WT084655MA 
Organisation The Wellcome Trust Ltd 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2008 
End 09/2017
 
Description MDC Prize studentship
Amount £90,000 (GBP)
Funding ID RA3/792 
Organisation Muscular Dystrophy UK 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2009 
End 09/2013
 
Description MDC Prize studentship
Amount £90,000 (GBP)
Funding ID RA3/839 
Organisation Muscular Dystrophy UK 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2010 
End 09/2014
 
Description MRC Medical Research Foundation
Amount £18,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2014 
End 01/2015
 
Title Expression plasmids 
Description cDNA clones expressing CHRNA, CHRNB, CHRNG, CHRND, CHRNE, RAPSN, AGRN, DOK7, 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Provided expression constructs to numerous laboratories throughout the world for studies of the neuromuscular junction, both pre and post 2006. 
 
Title Model of AChR deficiency syndrome 
Description Mouse model of AChR deficiency syndrome 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Cossins et al., 2004. Used to establish optimum therapeutic strategies for difficult to treat patients. 
 
Title Model of slow channel syndrome 
Description Transgenic mouse model of disease 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Used to study novel treatments, and optimise combinations of presently used treatments 
 
Title cell line 
Description Cell lines from patients with congenital myasthenic syndromes 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? Yes  
Impact Used in determining underlying disease mechanism and in determining if DNA variants are pathogenic 
 
Description GOSH collaboration on CMS 
Organisation University College London (UCL)
Department UCL Institute of Child Health
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Enables enough data on patients to be gathered so that general patterns of genotype-phenotype correlations can be ascertained
Collaborator Contribution Pooling of Patients and patient data in orderto gain further knowledge about rare muscle conditions
Impact To date 3 papers on phenotypic features on patients with congenital myasthenic syndromes
Start Year 2006
 
Description MRC neuromuscular centre 
Organisation Medical Research Council (MRC)
Department MRC Centre for Neuromuscular Diseases
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Supplied patient data, mutation data, functional laboratory studies
Collaborator Contribution Provision of patients and scientific data
Impact Publications identifying a new gene in which mutations can underlie congenital myasthenic syndromes, and characterisation of the syndrome phenotype
Start Year 2010
 
Description OXION collaboration 
Organisation Centre for Life
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Collaborate over access to patient data, genetic screening.
Collaborator Contribution Uncovering the pathogen mechanism of DEND syndrome (juvenile hereditary diabetes). We collaborated to find out is deficit was of due to dysfunction in the CNS or at the NMJ.
Impact A series of papers have been generated in which phenotypic characteristics of the different types of congenital myasthenic syndromes are described
 
Description OXION collaboration 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Collaborate over access to patient data, genetic screening.
Collaborator Contribution Uncovering the pathogen mechanism of DEND syndrome (juvenile hereditary diabetes). We collaborated to find out is deficit was of due to dysfunction in the CNS or at the NMJ.
Impact A series of papers have been generated in which phenotypic characteristics of the different types of congenital myasthenic syndromes are described
 
Description WGS500 consortium 
Organisation Wellcome Trust Centre for Human Genetics
Department DNA Sequencing Hub (Oxford)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Providing patient DNA and analysis of identified variants for identification rare Mendelian disorders
Collaborator Contribution Identification of appropriate DNA samples Functional analysis of variants to determine if they are pathogenic
Impact Pathogenic mutations in new genes identified Cossins et al. (submitted)
Start Year 2011
 
Description WGS500 consortium 
Organisation Wellcome Trust Centre for Human Genetics
Department Wellcome Trust Case Control Consortium 2 (WTCCC)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
PI Contribution Providing patient DNA and analysis of identified variants for identification rare Mendelian disorders
Collaborator Contribution Identification of appropriate DNA samples Functional analysis of variants to determine if they are pathogenic
Impact Pathogenic mutations in new genes identified Cossins et al. (submitted)
Start Year 2011
 
Title ephedrine 
Description We demonstrated this commonly available drug has a remarkable beneficial effect for patients with DOK7 CMS. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Refinement. Clinical
Year Development Stage Completed 2006
Development Status Under active development/distribution
Impact Use of ephedrine in DOK7 CMS patients results in a dramatic improvement in disability scores. For example patients who were in wheel chairs are now often able to run. Ephedrine and salbutamol (which acts in a similar way) are now therapies of choice for DOK7 CMS 
 
Title salbutamol 
Description We have been able to establish salbutamol and ephedrine as treatment of choice for the DOK7 CMS patient subgroups (and other forms of CMS). 
Type Therapeutic Intervention - Drug
Current Stage Of Development Wide-scale adoption
Year Development Stage Completed 2012
Development Status Under active development/distribution
Impact Salbutamol and ephedrine are already available cheap drugs that have a dramatic beneficial effect of a subgroup of patients that we have defined. Further work is ongoing to establish additional uses for these drugs. 
 
Description Articles in the MGA news letters 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact We provide updates on latest research in the field

Do not usually get feedback from this but believe the patients groups are keen to keep up with what is going on.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Congenital myasthenic syndrome open day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Patient day. Patients are given a series of lectures on scientific research updates, and then in the afternoon there are a series of workshops for particular patient groups where needs that have been identified or areas of special interest are addressed. Typically this might involve treatments, exercise, benefits, future therapy updates.

Patients and researchers are both enthused about the research effort
Year(s) Of Engagement Activity 2006,2008,2010,2013,2014
URL http://www.ouh.nhs.uk/services/referrals/neurosciences/myasthenia.aspx
 
Description Lecture in science week 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Audience from the general public of around 125 people attended seminar presentation. Question and answer session after talk

Several pupils and schools asked for work experience placements
Year(s) Of Engagement Activity 2010,2011,2012
 
Description Oxford CMS Research Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Approx 250 pateints + familes attend a research open day for lectures about research, patient questions to clinicians, and visits to the laboratories.

Good patient feedback with lots of questions and it gives us an opportunity to update the patients on what we have achieved.
Year(s) Of Engagement Activity 2006,2008,2010,2012,2013
 
Description Patient information pamphlet 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact The pamphlet is used by families to inform them about their disorder and to take to their schools to inform them about the disorder that the children have. This enables the schools to understand the childs condition and to put in place appropriate safeguards and facilities to ensure the patient is safe and to enable adaptations to allow a full potential for learning to be in place.

Should help school, social workers to understand patient disabilities and their special needs when at school.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2017
URL http://www.ouh.nhs.uk/services/referrals/neurosciences/myasthenia.aspx
 
Description Patient video 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Video to inform patients about their condition, problems they might encounter in everyday management of their condition, such as when at school, and information on the research that is being carried out into their condition

Very positive feedback from patient groups
Year(s) Of Engagement Activity 2011,2012,2013,2014
 
Description Talks to patient group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact Updating patient support groups on the research activities.


Patients are always interested in their disorders and usually ask lots of questions.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
 
Description Work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact Individual students spend a week in the laboratory learning about scientific research.

School comes back each year requesting further pupil experience.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012