RNA Expression Profiling in Lymphoblastoid Cell Lines from Mutated and Non-Mutated Amyotrophic Lateral Sclerosis Patients

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the death of upper and lower motor neurons with unknown etiology. The difficulty to recover biological material from patients led to employ lymphoblastoid cell lines (LCLs) as a model for ALS, since many pathways, typically located in neurons, are also activated in these cells. To investigate the expression of coding and long noncoding RNAs in LCLs, a transcriptomic profiling of Sporadic ALS (SALS) and mutated patients (FUS, TARDBP, C9ORF72 and SOD1), and matched controls was realized. Thus, Differentially Expressed Genes (DEGs) were investigated among the different subgroups of patients. Peripheral Blood Mononuclear Cells (PBMCs) were isolated and immortalized into LCLs via Epstein-Barr Virus (EBV) infection, RNA was extracted and RNA-sequencing analysis was performed. Gene expression profiles of LCLs were genetic-background specific, indeed only 12 genes were commonly deregulated in all groups. Nonetheless, pathways enriched by DEGs in each group were also compared and a total of 89 KEGG terms were shared among all patients. Eventually, the similarity of affected pathways was also assessed when our data were matched with a transcriptomic profile realized in PBMCs of the same patients. Thus, we concluded that LCLs are a good model for the study of RNA deregulation in ALS. PBMCs were isolated from peripheral venous blood samples. Lymphocytes B were isolated from an aliquot of PBMCs and were immortalized into LCLs via Epstein-Barr Virus (EBV) infection. Samples were homogenized and total RNA was isolated by Trizol® reagent. Sequencing libraries were prepared by the Illumina TruSeq RNA Library Prep, version 2, Protocol D, using 500-ng total RNA (Illumina, California, USA). FastQ files were generated using the llumina bcl2fastq2 software (Version 2.17.1.14 -http://support.illumina.com/downloads/bcl2fastq-conversion-software-v217.html) from raw sequencing reads gener-ated by the Illumina NextSeq 500 sequencer (Illumina, California, USA). STAR/RSEM software was used to cal-culate gene and transcript intensities using Gencode Release 19 (GRCh37.p13) as a reference and the "stranded" op-tion. The mRNA differential expression analysis was carried out using the R package EBSeq. The R package DESeq2 was used to perform differential expression analysis on lncRNAs. With |log2(disease sample/healthy con-trol)|1 and an FDR of 0.1, genes were considered differentially expressed and retained for further analysis.