Characterisation of biological pathways dysregulated in amyotrophic lateral sclerosis (ALS) cases with a hexanucleotide repeat expansion of C9ORF72.

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Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and tragic neurodegenerative condition. It has been recently been discovered that an expansion of the gene C9ORF72 causes a relatively large proportion of ALS. The function of C9ORF72 in disease and health is currently unknown. The aim of this project is to determine disease mechanisms associated with the C9ORF72 expansion. This will be a major advance towards the development of therapeutic agents for ALS more widely. In order to become functional, genes (as DNA) must be expressed in a RNA form. It has been postulated that the C9ORF72 expansion causes disease by disruption of the assembly of RNA in the cell nucleus. This is supported by pilot work carried out by the applicant. The ideal method for studying a disease of RNA assembly is gene expression profiling (GEP); this is a technique which measures the levels of various species of RNA in a sample. GEP will be carried out on post-mortem tissue from C9ORF72 patients and controls within our locally held tissue bank; and on cells from the blood of C9ORF72 patients and controls obtained from the MNDA National DNA bank and associated lymphoblastoid cell line (LCL) resource. Furthermore technology specialised for identifying misassembled RNA will be applied to skin cells from C9ORF72 patients and controls within our locally held biobank; this will be carried out in the MRC High-Throughput Sequencing (HTS) hub in Liverpool (see letter of support). The identity of samples with the C9ORF72 expansion within the MNDA National DNA bank and associated LCL resource will be obtained through collaboration with Professor Al-Chalabi (see letter of support). This resource contains a large number of samples and therefore is suitable for a GEP study, not only of patients with the C9ORF72 expansion but of such patients with either rapidly progressive or slowly progressive disease. This may facilitate the discovery of modifiers of disease which are potential therapeutic targets. The presence of disease modifiers is supported pilot work by the applicant showing that disease associated with the C9ORF72 expansion is significantly variable. Data about DNA changes in the same group of patients has already been obtained through collaboration with Professor Al-Chalabi and will be correlated with the GEP data. Thus disease modifiers will be understood at the DNA and the RNA level. A modifier of disease associated with C9ORF72 which has aready been proposed is the size of the expansion which has been shown to be variable between patients. This is a significant disease modifier in other similar diseases. The expansion will be sized using DNA samples from the MNDA National DNA bank using a technique called Southern blotting. The relationship between the size of the expansion and various aspects of disease such as survival time will be examined.Cell models are a useful tool for studying disease mechanisms. As well as being used in GEP skin cells from patients with the C9ORF72 expansion will be studied independently to determine how they differ to skin cells from controls. This technique has been used successfully in the study of ALS and other neurological diseases. This will be guided in part by the results of the other analyses described above to provide a validation of those discoveries. Another observation about C9ORF72 ALS is that the protein encoded by the C9ORF72 gene is present at a reduced concentration in patients with the expansion. This may be an additional disease mechanism co-existing with an effect on RNA assembly. This will be investigated by observing the effect of preventing expression of the C9ORF72 protein in a model consisting of nerve cells grown in the lab.This work will be carried out primarily by the applicant but with assistance from the Functional Genomics, Computational Biology and Cell Biology teams within SITraN (Sheffield Institute for Translational Neuroscience).