Gene expression profiles and protein-protein interaction networks in amyotrophic lateral sclerosis patients with C9orf72 mutation

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that involves the death of neurons [1, 2]. ALS is often called Lou Gehrig’s disease, after the famous baseball player who was diagnosed with it. The occurrence of ALS is two per 100,000 people, and it is estimated that more than 20,000 Americans may be living with ALS at any given time. It is worldwide with no racial, ethnic or socioeconomic boundaries and can affect anyone. It affects the nerve cells in the brain and the spinal cord and is characterized by stiff muscles, muscle twitching and gradually worsening weakness due to muscles decreasing in size. Despite intensive research, the clinical and pathophysiological mechanisms of ALS still remain unclear. There are two types of ALS, sporadic (SALS) and familial (FALS). 90–95 % of ALS cases are sporadic for which the cause is unknown and 5–10 % are familial, which are inherited [3]. Findings on ALS patients have drawn in numerous genes related to ALS pathogenesis and have identified diverse processes, such as excitotoxicity, oxidative stress, cytoskeletol abnormalities, impaired axonal transport, mitochondrial dysfunction and protein aggregation. Presently, ten genes such as SOD1, ALSIN, SETX, SPG11, FUS, VAPB, TARDBP, OPTN, ATXN2 and C9orf72 were found to affect the pathogenesis of ALS [4]. Though SALS and FALS are clinically and pathophysiologically similar, there is a requirement of finding the genes involved in FALS and the molecular mechanism pathways involved in the mutation of these genes. Till date studies were done to know the molecular mechanisms and underlying pathogenesis of ALS disease related to SALS with Superoxide dismutase 1 (SOD1) mutation [5, 6]. As the study of SOD1 has led to great advances in proper illustration of molecular mechanisms underlying in ALS disease, identifying the mutations in other genes and the pathways involved in these mechanisms is utmost important. Till date there is no cure for ALS, however the drug riluzole – the only prescribed drug approved by Food and Drug Administration (FDA) to treat ALS which prolongs the life by 2–3 months but do not relieve the symptoms [7, 8]. Improving diagnoses and treatment of this disease is utmost essential, as currently there exists no cure for ALS.

C9orf72 mutation is currently the major genetic cause of ALS disease accounting for approximately 34.2 % of the familial ALS cases. C9orf72 mutation is a hexanucleotide repeat expansion of the six letter string of nucleotides GGGGCC. Although nothing about C9orf72 function [9, 10] and the mechanisms by which expanded repeats cause neurodegeneration are known, C9orf72 RNAs levels were found to be reduced as reported in tissue samples from patients with C9orf72 expansions [11‐13]. The leading candidate mechanisms by which expanded repeats cause neurodegeneration are RNA-mediated toxicity, loss of C9orf72 gene function, or a combination of the two. In a normal person there would be few repeats of this nucleotide, but in the person with C9orf72 mutation this repeat can occur in the order of hundreds that result in the formation of RNA foci in white blood cells, fibroblasts, glia and multiple neuronal cell types. These RNA foci are not present in the sporadic ALS and familial ALS caused by other mutations. This RNA-mediated toxicity is found to play a crucial role in a variety of repeat expansion disorders by accumulation of expanded transcripts into nuclear RNA foci [14]. These RNA repeats fold into stable structures and sequester RNA binding proteins to set off a molecular cascade leading to neurodegeneration [14]. A loss of C9orf72 gene function is observed in patients with expansion by reported reductions of C9orf72 transcript levels [11‐13] Although this reduction to neuronal death is not established, loss of C9orf72 gene function during embryonic development is associated with motor deficits in zebrafish [15].

Lagier-Tourenne et al. have demonstrated the ASO mediated reduction of C9orf72 RNAs by aggravating the loss of C9orf72 function [16]. In this view of finding different molecular mechanisms and pathways related to C9orf72 mutation in ALS disease, we have used systems biology approach for the gene expression data to identify Gene Ontologies (GO) and pathways related to familial ALS. Genome-wide RNA profiling in fibroblasts from patients with C9orf72 expansion was studied in the RNA microarray data [9] used for the present study. This microarray data retrieved from Gene expression omnibus (GEO) [17] was screened to identify differentially expressed genes (DEGs) between ALS and control fibroblasts before and after ASO mediated disease therapy in order to find potential genes for the pathogenesis of ALS. Gene ontology(GO) enrichment analysis and Kyoto encyclopedia of Genes and Genomes(KEGG) pathway analysis was performed, and a protein-protein interaction (PPI) network was constructed by mapping the DEGs to the human PPI data. Our research laid a bridge between the DEGs, the KEGG pathways and the protein-protein interaction networks which will help for the development of novel targets for ALS therapeutic intervention.