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

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 seeking 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.

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).

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.