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Gene expression profile in CD4+ and CD8+ T cells from identical twins discordant for Multiple sclerosis

ABSTRACT: To gain insight into the etiopathogenesis of Multiple sclerosis (MS) we investigated gene expression changes in CD4+ and CD8+ T lymphocytes from monozygotic twins (MZ) discordant for relapsing remitting MS. We studied 4 monozygotic twin pairs discordant for disease, with the affected co-twin free of disease modifying therapies (F/M = 3/1, mean age 36.25±3.9). Following leukapheresis, CD4+ and CD8+ T cells were separated and studied by Affymetrix GeneChip®

ORGANISM(S): Homo sapiens

PROVIDER: GSE16461 | GEO | 2010/12/31

SECONDARY ACCESSION(S): PRJNA116037

REPOSITORIES: GEO

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Project description:To gain insight into the etiopathogenesis of Multiple sclerosis (MS) we investigated gene expression changes in CD4+ and CD8+ T lymphocytes from monozygotic twins (MZ) discordant for relapsing remitting MS. We studied 4 monozygotic twin pairs discordant for disease, with the affected co-twin free of disease modifying therapies (F/M = 3/1, mean age 36.25±3.9). Following leukapheresis, CD4+ and CD8+ T cells were separated and studied by Affymetrix GeneChip® For gene expression profiling, peripheral blood mononuclear cell (PBMC) were obtained from leukopheresis by density centrifugation over Ficoll-hypaque according to standard procedures. Briefly, heparinized blood was diluted with 1 volume of RPMI medium and gently layered over the Fycoll. Following centrifugation at 660 g for 30 minutes, cells at the interface of the gradient was collected and washed 3 times. Highly purified CD4+ and CD8+ (purity >95%) cell subsets were then sorted by flow cytometry on MoFlo high speed cell sorter (Beckman Coulter, Fullerton, CA) and frozen in Trizol (Invitrogen). Total RNA was extracted using TRIzol® (Invitrogen), according to the manufacturer's instructions. RNA quality and purity were assessed with the use of the RNA 6000 Nano assay on Agilent 2100 Bioanalyzer (Agilent). Total RNA from each sample was prepared using GeneChip® one Cycle cDNA Synthesis kit and GeneChip® IVT Labeling kit (Affymetrix), according to the manufacturer’s protocols. Briefly, starting from 2.5µg total RNA, biotinylated cRNA was produced by one cycle of reverse transcription and in vitro transcription (IVT). Biotinylated cRNA was fragmented and hybridized for 16 h at 45°C onto GeneChip® Human Genome U133 Plus 2.0 arrays (Affymetrix). The statistical analysis was performed couple by couple using the Rosetta Resolver® system (Rosetta Biosoftware). The fold-change of each gene was calculated by comparing gene expression in the affected twin with that in the unaffected twin and the genes with the same trend in at least 3 twin pairs out of the 4 studied were selected as candidates to be validated by real-time RT-PCR.

Project description:We aim to investigate circulating genome-wide microRNA (miRome) profiles in Moyamoya disease (MMD)-discordant monozygotic (MZ) twins with the RNF213 founder mutation (rs112735431).A disease discordant monozygotic twin-based study design may unmask potential confounders from previously published circulating microRNA signature in MMD. Circulating genome-wide microRNA (miRNome) profiling was performed in MMD-discordant monozygotic twins, non-twin-MMD patients, and non-MMD healthy volunteers by microarray followed by qPCRvalidation, using blood samples. Differential plasma-microRNAs were further quantified in endothelial cells differentiated from iPS cell lines (iPSECs) derived from another independent non-twin cohort. Lastly, their target gene expression in the iPSECs was analyzed. Microarray detected 309 plasma-microRNAs in MMD-discordant monozygotic twins that were also detected in the non-twin cohort. Principal component analysis of the plasma-microRNA expression level demonstrated distinct 2 groups separated by MMD and healthy control in the twin- and non-twin cohorts. Of these, differential up-regulations of hsa-miR-6722-3p/-328-3p were validated in the plasma of MMD (Imposed threshold: absolute log2 expression fold change (logFC) > 0.26 for the twin cohort; absolute logFC > 0.26, p < 0.05, and q < 0.15 for the non-twin cohort). In MMD derived iPSECs, hsa-miR-6722-3p/-328-3p showed a trend of up-regulation with a 3.0- or higher expression fold change. Bioinformatics analysis revealed that 41 target genes of miR- 6722-3p/-328-3p were significantly down-regulated in MMD derived iPSECs and were involved in STAT3, IGF-1-, and PTEN-signaling, suggesting a potential microRNA- gene expression interaction between circulating plasma and endothelial cells. In conclusion, our MMD-discordant monozygotic twin-based study confirmed a novel circulating microRNA signature in MMD as a potential diagnostic biomarker minimally confounded by genetic heterogeneity. The novel circulating microRNA signature can contribute for the future functional microRNA analysis to find new diagnostic and therapeutic target of MMD.

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Gene expression profile in CD4+ and CD8+ T cells from identical twins discordant for Multiple sclerosis

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