39 research outputs found

    Differentially methylated probes for each cell population in comparison to whole blood.

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    <p>PBMC-Peripheral blood mononuclear cells. Differentially methylated probes were defined by a linear model using the M-values. M-value is the log2 ratio of the intensities of methylated probe versus unmethylated probe, a measurement of how much more a probe is methylated compared to unmethylated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041361#pone.0041361-Du2" target="_blank">[46]</a>. *To extract the probes with largest difference in methylation, a gamma fit model was applied to M-values in order to define the three calls: “unmethylated”, “marginal” and “methylated”. Significant probes sharing the same call in the two compared populations were removed. **Variation is based on the estimate of the log2-fold-change corresponding to the effect obtained from the linear model, absolute M-values are presented. The percentages are based on the call distribution.</p

    Differentially methylated CpG sites in candidate genes related to inflammatory diseases.

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    <p>A) Heatmap of 1865 probes representing 293 candidate genes for selected inflammatory diseases showing differential methylation in blood cell populations. Candidate genes for the diseases asthma, atopy, atopic dermatitis, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, Type 1 and Type 2 diabetes were selected from the Genome wide association study atlas (<a href="http://www.genome.gov/gwastudies/" target="_blank">http://www.genome.gov/gwastudies/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041361#pone.0041361-Hindorff1" target="_blank">[30]</a>. The heatmap is based on median M-values. The M-value is calculated as the log2 ratio of the intensities of methylated probe versus unmethylated probe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041361#pone.0041361-Du2" target="_blank">[46]</a>. Blue color indicates low DNA methylation while yellow represents high DNA methylation. WB  =  whole blood, CD4T  =  CD4<sup>+</sup> T cells, CD8T  =  CD8<sup>+</sup> T cells, CD56NK  =  CD56<sup>+</sup> NK cells, CD19B  =  CD19<sup>+</sup> B cells, CD14Mono  =  CD14<sup>+</sup> monocytes. B) The genomic distribution of the differentially methylated probes associated with inflammatory complex diseases according to the UCSC RefGene group (included in the Illumina annotation data). Intergenic  =  site not annotated in a gene, TSS  =  transcription start site at 200–1500 bp, 5′ region  = 5′UTR and 1st exon, Intragenic  =  gene body including introns and exons and, 3′ region  = 3′UTR. UTR – untranslated region.</p

    Schematic presentation of the isolation protocol and purity of the cell populations as measured by flow cytometry.

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    <p>Peripheral blood mononuclear cells (PBMC) and granulocytes were obtained by density gradient centrifugation and seven cell populations were purified by magnetic sorting. Upper panel shows forward and side scatter which confirmed cell morphology and granularity. The lower panel shows the overlay of cell surface markers for all the six donors. The purities of the cell populations were highly similar among all the six donors. Th cells  =  CD4<sup>+</sup> T cells, Tc cells  =  CD8<sup>+</sup> T cells, NK cells  =  CD56<sup>+</sup> NK cells, B cells  =  CD19<sup>+</sup> B cells, Monocytes  =  CD14<sup>+</sup> monocytes. Data analyses are based on the comparison of all cell populations to whole blood.</p

    Differentially methylated sites are harbored by cell-specific genes but the connection between methylation status and surface expression depend on the gene and lineage.

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    <p>The surface expression of CD3 and CD14 according to the methylation status of the coding gene is presented. Demethylation of CpG sites was observed in T cells expressing the CD3 marker (upper panel). For CD14 the difference between positive and negative cells involved cell-restricted marginal status in a context of demethylation (lower panel). Histograms (right) represent the log fluorescence of the marker (<i>x</i>-axis) and the cell counts (<i>y</i>-axis). Peaks within the gray shaded gates represent positive populations. For CD14 two gates are presented, the “<i>low</i>” which include monocytes and a fraction of neutrophils and the “<i>high</i>” population of monocytes. m  =  membrane; e*  =  positive or negative cell expression of the marker according to the Human Leucocyte Differentiation Antigens (HLDA) Workshop and CD nomenclature (<a href="http://www.hcdm.org/Home/tabid/36/Default.aspx" target="_blank">http://www.hcdm.org/Home/tabid/36/Default.aspx</a>).</p

    Clustering of cell populations in blood with regard to global DNA methylation.

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    <p>A) Hierarchical tree presenting the relationship between cell populations based on median M-values from six donors. B) Principal component analysis of each individual sample showing specific clustering based on cell population.</p

    Gene ontology enrichment for isolated cell populations.

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    <p>Gene ontology was performed using DAVID (<a href="http://david.abcc.ncifcrf.gov" target="_blank">http://david.abcc.ncifcrf.gov</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041361#pone.0041361-Huangda1" target="_blank">[47]</a>. The human genome was used as background and the level of significance was set to p<0.05. The top ten enriched pathways are described for genes showing significantly differentially methylated probes in comparison to whole blood where the cell population shows unmethylated state and whole blood shows methylated state according to the gamma fit model. Red color indicates peripheral blood mononuclear cells (PBMC) and green color indicates granulocytes.</p

    DNA methylation levels across the gene regions in purified cell populations for candidate genes.

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    <p>DNA methylation is defined by the M-values for each cell population at a given CpG site. The M-value is calculated as the log2 ratio of the intensities of methylated probe versus unmethylated probe and describes a measurement of how much more a probe is methylated compared to unmethylated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041361#pone.0041361-Du2" target="_blank">[46]</a>. Negative numbers represent unmethylated and positive numbers represent methylated. Every cell population correspond to a vertical bar which is listed from left to right as peripheral blood mononuclear cells (PBMC), CD4<sup>+</sup> T cells, CD8<sup>+</sup> T cells, CD56<sup>+</sup> NK cells, CD19<sup>+</sup> B cells, CD14<sup>+</sup> monocytes, granulocytes, neutrophils, eosinophils and whole blood. Lymphoid cells are colored in red bars, myeloid cells are colored in green bars and whole blood is represented by black bars. A) the asthma candidate genes lymphotoxin alpha (<i>LTA</i>) and tumor necrosis factor (<i>TNF</i>), and B) the Type 2 diabetes candidate gene transcription factor 7-like 2 (<i>TCF7L2</i>), black arrows indicate regions with cell type specific pattern for monocytes. TSS  =  transcription start site at 200–1500 bp; UTR  =  untranslated region, gene body including introns and exons.</p

    Gene-gene interactions between NPSR1 and NPS.

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    <p>Location of six <i>NPSR1</i> SNPs with significant interactions with <i>NPS</i> rs10830123 in the multiplicative model for epistasis in connection with their p-value for interaction and linkage disequilibrium. The most significant interaction signals in BAMSE (green) and MAGIC/ISAAC (orange) are marked in bold. Statistics on LD between the functional rs324981 and other interacting SNPs are provided as D’ and r<sup>2</sup>.</p
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