Exome sequencing in motor neuron disease: bioinformatic analyses and biological validation of novel variants

Lead Research Organisation: King's College London
Department Name: Clinical Neuroscience

Abstract

There are no effective treatments for motor neuron disease (MND, also known as amyotrophic lateral sclerosis, ALS) because so little is known about what causes it. In most instances ALS appears out of the blue (called sporadic ALS) but in 10% of people the disease runs in families (familial ALS), usually due to a single defective gene, passed down from generation to generation in a dominantly inherited fashion.

Mutations in seven genes are known to cause dominantly-inherited adult-onset ALS but they account for only 55% of all familial and 10% of sporadic ALS patients. The cause of the disease in the remaining 45% of families and 90% of sporadic ALS patients is unknown but a genetic basis is strongly implicated even in the absence of a family history.

Advances in two DNA technologies have revolutionised our gene hunting efforts. Only ~2% of the human genetic code (DNA) provides the blueprint for making proteins, which are the building blocks of all cells. Mutations in the protein-coding sequence, known as "The Exome" account for most human diseases. Recently developed DNA capture methods mean that we can pull out 90-95% of the protein-making genes for further analysis. This would have taken us many decades but "Next Generation sequencing" has transformed the time and cost of large-scale sequencing. For example, the Human Genome Mapping Project which was published in the year 2000 spelt out one person's entire genetic code. It took 15 years and cost ~£300 million. This can now be done in 1 week for around £2,000. This grant application aims to harness the extraordinary power of these two new research tools in a global gene hunting effort to rapidly identify the disease-causing spelling mistakes.

Through an MRC and MND Association funded grant we sequenced the Exome (protein coding DNA) on 420 people with familial MND and compared it to the sequence to ~26,000 healthy control exomes. This has yielded five promising new ALS genes. In this project we are seeng to extend our study to test the potentially toxic effects of these gene mutations in cells grown in the laboratory. Those that show the greatest effect will be injected into fruit flies, zebrafish and chick embryos to see whether the effects are also seen in an intact nervous system. These experiments will rapidly tell us whether the mutant proteins are toxic and provide a glimpse as to precisely how they damage nerve cells. By modelling the disease process in cells and small animals (fruit flies, zebrafish, chicken embryos) it will also give us an opportunity to work with Pharmaceutical companies to screen for drugs that could reverse motor nerve degeneration.

We also intend to collaborate with other researchers from around the world who are using similar techniques to combine the Exome sequence of >1,000 familial ALS cases. The 50 top candidate genes will then be sequenced in 1,000 sporadic MND DNA samples from the MNDA BioBank which we can then validate in white blood cell lines that we have banked on these patients. We will also screen 50 top candidate genes in DNA from ALS brain tissues we have stored in the MRC funded London Degenerative Diseases Brain Bank. This will allow us to rapidly discover how any mutation affects nerve cells in post mortem tissues from MND and FTD patients.

This project aims to discover new genes that cause MND and determine their frequency in familial and sporadic disease. These genes can immediately be tested to aid in the diagnosis of people with motor nerve problems and for families concerned about genetic risk. Gene therapy trials are already underway for SOD1 mediated MND and the discovery of other genes will increase the opportunity to design therapies targeted at people with specific gene defects. It will also allow us to model the harmful effects of ALS gene mutations in cells and small animals to help understand disease mechanisms and screen for drugs that reverse this devastating disease process.

Technical Summary

Currently we can identify mutations in ~55% of familial and 10% of sporadic patients with ALS. Our group has contributed to the discovery of TARDBP and FUS genes and identified the Chr. 9p locus containing the G4C2 expansion mutation in C9ORF72. These genes are now screened in diagnostic and predictive tests around the world.

Further work is required to identify the genes for ~45% of families and 90% of sporadic patients who are increasingly worried about heritable risks. The identification of additional FALS genes will permit comprehensive gene testing and provide crucial insights into cellular pathways that contribute to motor neuron degeneration. This will enhance our understanding of disease mechanisms and identify new therapeutic targets. It may also advance the development of genotype-specific therapies, such as anti-sense oligonucleotides for C9ORF72 cases.

This application is to continue the work of an MRC and MNDA funded Bioinformatician and Geneticist/Cell Biologist. It is made in conjunction with an application to the MNDA who if we are successful have agreed to fund two PhD students to tackle the bottle-neck of functional studies.

We have identified 5 new ALS gene candidates, which are strongly associated with ALS. Pilot functional studies of 3 genes in transfected cells reveal that mutations can cause protein mislocalisation and aggregation but further work is required to prove pathogenicity.

Shaw leads an international consortium working with the Broad Institute to compare 1,000 FALS exomes with their database of ~50,000 exomes to identify additional ALS genes. We will multi-plex sequence the top 50 candidates in: 1,000 sporadic ALS cases from the MNDA Biobank (lymphoblasts available for RNA and protein) and 200 ALS brains from our MRC funded Brain Bank, to rapidly assess the effects of mutations. We will undertake functional studies of the most promising pathogenic candidates in cellular and in vivo models (Drosophila, zebrafish, chick).

Planned Impact

MND patients and their families will rapidly benefit from the discovery of new MND genes. As these gene tests are adopted by diagnostic laboratories and made available to patients from around the world they will aid in the early diagnosis of MND. This will lead to earlier treatments, such as riluzole and reduced anxiety over diagnostic uncertainty. It will also reduce the discomfort and expense of further diagnostic tests such as lumbar punctures, repeated electromyograms, muscle biopsy and MRI scans.

Those family members at risk in dominant kindreds and those with sporadic disease who are concerned about genetic risk to their children and siblings will be able to predictive gene testing. When a gene defect is discovered we can offer prenatal testing and even preimplantation genetic diagnosis (PGD). As an example I am enormously proud that a healthy boy was born in June 2013 to a family who carry a SOD1 mutation. I have cared for several members of this family who have developed and died from MND over the past 15 years. This is to my knowledge the first child to be born free of MND by PGD in Europe.

It will also others who undertake functional studies. We will be able to develop additional cellular and animal models to improve our understanding of disease mechanisms and identify new therapeutic targets. Although genes and gene testing for mutations cannot be patented additional genes will hopefully increase the engagement of industry in compound screening and developing gene therapies.

The UK has shown international leadership in MND research over many years and there are many successful clinical and Biological research laboratories (Sheffield, Oxford, UCL, Newcastle and Birmingham). The Shaw lab is recognised globally for its contribution to the genetic and biology of MND which has attracted many visiting scholars and international PhD students. If this project is funded by the MRC the MNDA will fund two PhD studentships which will build capacity and expand expertise and engagement in the field of Neurodegeneration more broadly.

Publications

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Barker HV (2017) RNA Misprocessing in -Linked Neurodegeneration. in Frontiers in cellular neuroscience

 
Description Exome sequencing in motor neuron disease: bioinformatic analyses and biological validation of novel variants
Amount £717,979 (GBP)
Funding ID MR/L021803/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 11/2014 
End 10/2017
 
Description King's Biomedical Sciences Institute PhD Studentship
Amount £100,000 (GBP)
Organisation King's College London 
Sector Academic/University
Country United Kingdom
Start 03/2014 
End 10/2017
 
Description MRC Case Studentship
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 10/2018
 
Description Project grant
Amount £493,407 (GBP)
Organisation Motor Neurone Disease Association (MND) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description Studentship
Amount £250,000 (GBP)
Organisation British Society for the History of Science (BSHS) 
Department Darwin Trust of Edinburgh
Sector Academic/University
Country United Kingdom
Start 09/2014 
End 09/2017
 
Description Exome sequencing in motor neuron disease: bioinformatic analyses and biological validation of novel variants 
Organisation Motor Neurone Disease Association (MND)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution ALS research excellence
Collaborator Contribution financial
Impact Publication of new ALS genes
Start Year 2015
 
Description From ALS exomes to Functional assays: turning candidates into confirmed genes 
Organisation Medical Research Council (MRC)
Department Medical Research Foundation
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Scientific expertise on ALS pathogenesis
Collaborator Contribution financial support
Impact None yet as started 5 months ago
Start Year 2015
 
Description Generation and characterisation of induced pluripotent stem cells from ALS lymphoblasts to model disease pathogenesis and advance drug discovery 
Organisation Motor Neurone Disease Association (MND)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Research excellence
Collaborator Contribution Financial and access to lymphoblast cell lines
Impact Nil yet as just started
Start Year 2015
 
Description Alinda Fernandes gave Lewisham Health Lectures at Lewisham Library - Talk Title: Alzheimer's Disease and Dementia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The talk was received very good feedback and triggered a series of stimulating conversations
Year(s) Of Engagement Activity 2017
 
Description Alinda Fernandes poster presentation at MNDA Symposium Boston (2017) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The poster was presented to mainly scientists during the poster session which enabled new collaborations.
Year(s) Of Engagement Activity 2017
 
Description Eva So Poster presentation at the Edmond J. Safra Memorial Lecture and Reception 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentation to the audience and interactive communication and discussion about research work. Event included presentation and talks highlighting the modern day approach to research in Parkinson's and other neurodegenerative diseases and the frontline advances being made by different researchers, followed by question and answer session with the audience and direct communication at the poster reception.
Year(s) Of Engagement Activity 2017
 
Description Graham Cocks Poster submission - Poster presentation on CRISPR work for the 4th Genome editing conference, Oxford (GEOX 2018) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of a poster to international scientists.
Year(s) Of Engagement Activity 2018
 
Description Graham Cocks' participation 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact This involved guiding a group of MNDA donors through a tour of research undertaken here at the Wohl.
Year(s) Of Engagement Activity 2018
 
Description Gresham Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Audience of 100 present and 5,659 views

Many emails from viewers from around the world
Year(s) Of Engagement Activity 2012
URL http://www.youtube.com/watch?v=GsfMU_yhFOA
 
Description Jenny Greig Legacy fundraising event 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact People interested in supporting the MNDA came for a day of presentations to see the facility and the work that takes place here.
Year(s) Of Engagement Activity 2018
 
Description Jenny Greig poster presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentation at Stem Cell conference at Edinburgh University
Year(s) Of Engagement Activity 2017
 
Description Jenny Greig poster presentation at MNDA conference in Boston 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at MNDA conference in Boston
Year(s) Of Engagement Activity 2017
 
Description Openin night of a film 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Spoke abou tMND at a screening of the film I am breathing

Contact by email from members of he public
Year(s) Of Engagement Activity 2013
URL http://www.youtube.com/watch?v=_wh6l9SHyD4
 
Description Public Lecture on MND 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Presentation and videotaped

Contact from public by email
Year(s) Of Engagement Activity 2012
 
Description Public lecture 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public lecture on the genetics and biology of motor neuron disease
Year(s) Of Engagement Activity 2016
 
Description Public lecture on motor neuron disease research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact I spoke at a dinner hosted by the MND Association for major donors at the Royal Institution
Year(s) Of Engagement Activity 2016
 
Description Simon Topp's participation in MNDA fundraiser outreach - Promoting the research done at KCL in the field of Amyotrophic Lateral Sclerosis, to representatives from the UK's primary Motor Neurone Disease charity 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact A group of fundraisers (primarily consisting of spouses, children, and friends of patients) from the Motor Neurone Disease Association charity visited KCL and met with researchers in small groups, with the aim of discovering more about the research work done here on MND (Amyotrophic Lateral Sclerosis). I gave a 10 minute presentation to 8 groups in succession on the use of Whole Exome Sequencing to discover new genes causative for MND/ALS, and highlighted how KCL had been closely involved in the discovery of 10 of the 45 genes identified to date, averaging over one per year for the last 6 years. This event was recent and as yet there has been no formal feedback, but responses on the day were very positive.
Year(s) Of Engagement Activity 2018