Background: Amyotrophic Lateral Sclerosis, in which cortical and spinal motor neurons degenerate, is a late onset neurodegenerative condition that accounts for ~1 in 400 UK deaths, typically within 3-5 years from the initial manifestations of disease. It forms part of a broad spectrum of clinically, genetically as well as pathologically heterogeneous disorders that include behavioural variant frontotemporal lobar degeneration (bvFTLD). A large intronic hexanucleotide G4C2 repeat expansion of >30 copies was recently identified, in 2011, in the previously uncharacterised chromosome 9 open reading frame 72 (C9ORF72) gene which is now thought to explain up to 43% of familial ALS (~20-30% of familial FTLD) and around 7% of sporadic cases. Rationale & Hypothesis: The principle aim of the PhD was to perform gene expression profiling of peripheral tissues in ALS. In the first instance whole blood was trialled. However, this proved unreliable, owing to the shear abundance of erythrocyte derived alpha and beta haemoglobin transcripts that are contained within the sample and the variability in the efficiency of its removal using the Ambion® GLOBINClear or NuGEN Ovation® WB reduction strategies. Instead disease related changes in transcription/alternative splicing were detected in a large bank (n=820) of patient and control lymphoblastoid cell lines (LCL’s) with the main purpose of: 1) elucidating further the mechanism(s) of neurotoxicity associated with the C9ORF72 G4C2 repeat expansion and, 2) establishing within this specific genetic subtype, modifiers of a fast (<2yrs) versus slow (≥4yrs) disease progression in order to identify potential new areas of therapeutic research. Methodology: Biotinylated, sense-strand cDNA targets of ~40 -70nt were hybridized onto Human Exon 1.0ST GeneChip® Arrays. A GC-RMA normalisation procedure was carried out in Partek® Genomics Suite and differentially expressed or alternatively spliced transcripts were detected at the 5% significance level (p<0.05) with a fold-change threshold of ≥ ±1.20 applied. Findings: Overall a marginal increase in gene transcription was observed with respect to C9ORF72 (59.3%, n=650/1,096) and nonC9ORF72-related_SALS patients (63.9%, n=1,148/1,796) compared to neurologically healthy controls. DAVID enriched gene ontology terms included translation, which was specific to carriers of the G4C2 repeat, in addition to RNA processing, DNA metabolism, RNP complex biogenesis and the cell cycle which reflect more common features of the broader ALS phenotype. A number of key validation targets, including several RNA binding partners of the G4C2 repeat (FUS, RPL22, NUDT2, PURA, EIF4H and HNRNPA0/F) were subsequently confirmed in a qRT-PCR assay. Isoform A/B specific transcripts of the C9ORF72 gene, itself, were found not to be differentially expressed across the LCL’s in the ECACC discovery and replication cohorts. Conclusions: Whether pathogenicity of the G4C2 expanded allele arises as a consequence of haploinsufficiency or through an aberrant gain of function mechanism has yet to be determined; although emerging evidence favours a role of RNA toxicity. In light of this model, an up-regulation in the expression of C9ORF72 binding partners and other, RNA processing & splicing related transcripts fits with the hypothesis that the cells are attempting to compensate for the sequestration of these proteins into toxic RNA foci in the cytoplasm which leads to disruption of their normal physiological function. Small sample sizes meant limited conclusions could be drawn from the analysis of C9ORF72 specific modifiers of survival in ALS. Clinical data points towards a possible effect of gender which is supported in the literature but other factors such as correlations with expansion length would need to be considered in conducting future work
