Multiple Sclerosis Risk Allele in <i>CLEC16A</i> Acts as an Expression Quantitative Trait Locus for <i>CLEC16A</i> and <i>SOCS1</i> in CD4+ T Cells

<div><p>For multiple sclerosis, genome wide association studies and follow up studies have identified susceptibility single nucleotide polymorphisms located in or near <i>CLEC16A</i> at chromosome 16p13.13, encompassing among others <i>CIITA</i>, <i>DEXI</i> and <i>SOCS1</i> in addition to <i>CLEC16A</i>. These genetic variants are located in intronic or intergenic regions and display strong linkage disequilibrium with each other, complicating the understanding of their functional contribution and the identification of the direct causal variant(s). Previous studies have shown that multiple sclerosis-associated risk variants in <i>CLEC16A</i> act as expression quantitative trait loci for <i>CLEC16A</i> itself in human pancreatic β-cells, for <i>DEXI</i> and <i>SOCS1</i> in thymic tissue samples, and for <i>DEXI</i> in monocytes and lymphoblastoid cell lines. Since T cells are major players in multiple sclerosis pathogenesis, we have performed expression analyses of the <i>CIITA-DEXI-CLEC16A-SOCS1</i> gene cluster in CD4+ and CD8+ T cells isolated from multiple sclerosis patients and healthy controls. We observed a higher expression of <i>SOCS1</i> and <i>CLEC16A</i> in CD4+ T cells in samples homozygous for the risk allele of <i>CLEC16A</i> rs12927355. Pair-wise linear regression analysis revealed high correlation in gene expression in peripheral T cells of <i>CIITA</i>, <i>DEXI</i>, <i>CLEC16A</i> and <i>SOCS1</i>. Our data imply a possible regulatory role for the multiple sclerosis-associated rs12927355 in <i>CLEC16A</i>.</p></div

The genotype of rs12927355 associates with increased expression of <i>CLEC16A</i> and <i>SOCS1</i> in CD4+T cells.

<p>The plots show gene expression of <i>CIITA</i>, <i>DEXI</i>, <i>CLEC16A</i> and <i>SOCS1</i> relative to <i>TBP</i> in CD4+ T cells (n = 50) from MS patients (n = 27) and HCs (n = 23). The samples were sorted according to <i>CLEC16A</i> genotype of two MS-associated SNPs (A) rs12927355 (risk allele = G): GG: n = 35, AG: n = 14 and AA: n = 1, and (B) rs4780346 (risk allele = A): AA: n = 4 and AG: n = 19, GG: n = 27. Mann-Whitney U-test was performed to compare the groups. Significant <i>P</i>-values are shown in the figure. The median value in each group is indicated as a horizontal line.</p

No difference in 16p13.13 T cell expression between MS patients and healthy controls.

<p>The plots show gene expression of <i>CIITA</i>, <i>DEXI</i>, <i>CLEC16A</i> and <i>SOCS1</i> relative to <i>TBP</i> in (A) CD4+ T cells (MS: n = 28; HC: n = 26) and (B) CD8+ T cells (MS n = 17; HC: n = 23). Mann-Whitney U-test was performed to compare the groups. The median value in each group is indicated as a horizontal line.</p

Characteristics of MS patients and controls.

<p>¹ At inclusion in this study.</p><p>Abbreviations: EDSS = expanded disability status scale, S.D. = standard deviation, N/A = not applicable.</p><p>Characteristics of MS patients and controls.</p

Characteristics of individual MS patients and summaries of patients and controls.

<p>¹Age category: 1 = 25–29, 2 = 30–34, 3 = 35–39, 4 = 40–44, 5 = 45–49, 6 = 60–64.</p><p>²At inclusion in this study.</p><p>³Oligoclonal bands present in cerebrospinal fluid taken at time of diagnosis.</p><p>⁴Contrast enhancing lesions on MRI.</p><p>Abbreviations: EDSS = Expanded Disability Status Scale, MSSS = Multiple Sclerosis Severity Score, OCB = oligoclonal bands, MRI = Magnetic Resonance Imaging, S.D. standard deviation</p><p>Characteristics of individual MS patients and summaries of patients and controls.</p

Principal component analyses.

<p>For samples in analyses a PCA was performed on overall methylation levels of CpG-sites that passed both quality controls and SNP filtering in (<b>A</b>) whole blood (Red), CD4+ T cells (Blue) and CD8+ T cells (Magenta) for all cases (squares) and controls (triangles). (<b>B</b>) PCA of DNA methylation data from whole blood only. (<b>C</b>) PCA of DNA methylation data from CD4+ T cells only. (<b>D</b>) PCA of DNA methylation data from CD8+ T cells only.</p

Pie charts of overall methylation levels for the three sample types.

<p><b>A</b>. Pie-charts of DNA hyper- and hypomethylation for all CpG sites with p-values less then or equal to 0.05. <b>B</b>. Pie-charts of DNA hyper- and hypomethylation for all CpG-sites with p-values above 0.05. Abbreviations: Hypo – hypomethylation, Hyper – hypermethylation, CD4 – CD4+ T cell data, CD8 – CD8+ T cell data, WB – whole blood data.</p

Top 40 results sorted by p-values from linear regression analysis models of DNA methylation in whole blood samples.

<p>¹Probe ID on 450K chip.</p><p>²Gene annotated to probe.</p><p>³p-value for specified probe in whole blood.</p><p>⁴Effect size of beta difference for specified probe. Positive values indicate hypomethylation of MS samples (i.e. controls DNA methylation higher than MS patients)</p><p>⁵Standard deviation for specified probe.</p><p>Formatting legend</p><p>“Bold probeID” Specific probe occurs in all three data top-40 (see Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117403#pone.0117403.t002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117403#pone.0117403.t003" target="_blank">3</a>)</p><p>“<i>Bold Italic Gene</i>” Gene occurs in all three data top-40 (see Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117403#pone.0117403.t002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117403#pone.0117403.t003" target="_blank">3</a>)</p><p>”Bold Effectsize” Hypermethylation of probe in MS patients</p><p>Results shown are restricted to methylation differences of at least 5% (absolute beta difference). Full lists are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117403#pone.0117403.s003" target="_blank">S1 Table</a>.</p><p>Top 40 results sorted by p-values from linear regression analysis models of DNA methylation in whole blood samples.</p

Genes Involved in the Osteoarthritis Process Identified through Genome Wide Expression Analysis in Articular Cartilage; the RAAK Study

<div><p>Objective</p><p>Identify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process.</p><p>Methods</p><p>Genome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions.</p><p>Results</p><p>Among the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. <i>TNFRSF11B</i> and <i>FRZB</i>). Also several inflammatory genes such as <i>CD55</i>, <i>PTGES</i> and <i>TNFAIP6</i>, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of <i>NGF</i> in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score) but were independent of joint-site or sex.</p><p>Conclusion</p><p>We provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways.</p></div

Representative slides of immunohistochemical staining.

<p>A) H&E staining. B) Toluidine blue staining. C) SERPINE1. D) CD55 (magnification 20x; insets show larger overview at magnification 4x; white scale bars indicate 50 µm and 200 µm, respectively). The left panels show preserved cartilage area (P) and the right panels show the OA affected cartilage area (OA).</p