Relationship of relapse-over-expressed immune genes to the ribosomal gene signature.

<p>(A) Association of increased <i>HLA-DRB1</i> and <i>HLA-DRB5</i> expression with history of relapse. Proportions were compared using Fisher’s exact test. Y-axes are in normalized units. Expression levels >400 normalized units were considered high. (B) Positive correlation of the patient ribosomal gene signature with expression of seven of eight immune genes over-expressed in relapse patients. Spearman r values are shown. Y-axes are in normalized units.</p

Association of RGS, HLA DR gene expression or combination thereof with relapse in ADAMTS13-deficient TTP.

<p>^a Odds ratios (OR) estimated by modified median unbiased estimation (MMUE) due to zero cells;</p><p>^b Confidence intervals (CI) estimated by exact, bootstrapped MMUE;</p><p>^c Fisher’s exact test, 2-tailed.</p><p>Association of RGS, HLA DR gene expression or combination thereof with relapse in ADAMTS13-deficient TTP.</p

Relationship between peripheral blood markers and number of episodes.

<p>Expression of five immune transcripts overexpressed in patients with history of relapse correlates with number of past TTP episodes. Serum ADAMTS13 activity does not correlate with number of past episodes (upper left panel). Y-axis is percent of full activity for ADAMTS13 as measured by FRET assay (upper left panel). Y-axis is gene expression in normalized units for the five immune genes (all other panels). Spearman R values are shown.</p

Direct comparison of gene expression of peripheral blood samples between patients with or without a history of relapse.

<p>(A) Average normalized ribosomal gene signature expression values in patients with or without a history of relapse. Error bars depict standard error of the mean. Expression values were compared using Student’s t-test. (B) Relative expression levels of genes differentially expressed between relapse and non-relapse patients presented following clustering. Colors indicate relative increased (red) or decreased (green) expression.</p

Type I IFN and ribosomal gene expression signature associations.

<p>(A) Association of the type I IFN gene signature with antibodies to RNA binding proteins (RBP) but not relapse history. (B) Association of the ribosomal gene signature (RGS) with relapse history but not antibodies to RBP. Proportions were compared using Fisher’s exact test. Odds ratios (OR) were calculated as described in Materials and Methods.</p

Characteristics of TTP remission cohort^a.

<p>^a Individuals with a history of clinically diagnosed TTP (<10% ADAMTS13 activity during a prior acute episode), in remission for at least 2 months, who fail to meet criteria for any known rheumatic disease;</p><p>^b t-test;</p><p>^c Fisher’s exact test;</p><p>^d Kolmogorov-Smirnov test.</p><p>Characteristics of TTP remission cohort<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117614#t001fn001" target="_blank">^a</a>.</p

Expression of CD52, CD244 and CD161 protein on the cell surface of CD3^-CD56^dim NK and CD3⁺ T cells isolated from relapsed ADAMTS13-deficient TTP patients in remission.

<p>Mean fluorescence intensity (MFI) of CD52, CD244 and CD161 on CD3^-CD56^dim and CD3^-CD56^bright NK cells (A) and on CD3⁺ T cells (B) derived from relapsed (R) or non-relapsed (NR) TTP patients. Representative histograms of fluorescence intensity of markers exhibiting significant differences between relapse (thick, dashed lines) and non-relapsed (thin, solid lines) patients are shown in C. (relapse n = 9, non-relapse n = 13, <i>Mann-Whitney test</i>; **p<0.01).</p

Functional characterizations of <i>OAS1</i> isoforms.

<p>(A) Protein expression of OAS1 isoforms was evaluated in EBV-transformed B cells from SS patients (four independent samples from each genotype group) using anti-OAS1 antibody targeting the shared epitope of all the isoforms. The stimulated cells were treated with universal type I IFN (1500U/ml) for 24hrs. The p44 isoform was not detectable using western-blot due to its low expression. The right panel shows quantified band intensity normalized to the GAPDH in each sample. (B) The transcript levels of each <i>OAS1</i> isoform from the same sets of cells described above were determined using real-time PCR. Consistent with the RNA-seq results, the SS-associated risk allele A of rs10774671 was correlated with decreased levels of p46 and increased expression of the p42, p48, and p44 isoforms (significance levels are shown at the bottom). The transcript levels of all the isoforms significantly increased after IFN stimulation (two-tailed <i>t</i> test); however, only p46 had increased protein production after IFN stimulation. (Significance level: ** <i>P</i><0.01; *** <i>P</i><0.001) (C) Individual isoforms of <i>OAS1</i> tagged with Xpress epitope were cloned and transfected into HEK 293T cells for 48hrs. The p48 and p44 isoforms had impaired protein expression compared to p46 and p42, although their transcript levels were equivalent as determined by real-time PCR (n = 4; normalized to <i>HMBS</i>). (D) The full-length and truncated <i>OAS1</i> p48 and p44 isoforms were cloned into HEK 293T cells. Western-blot indicated the lack of expression of the full-length p48 and p44 isoforms, whereas the truncation of both isoform transcripts (T2 and T4) was able to restore protein expression. (E) The 3' alternatively spliced terminus of different <i>OAS1</i> isoforms were linked to the 3'-end of GFP to observe their influence on GFP protein expression in HEK 293T cells. The 3'-terminus from the p48 and p44 isoforms resulted in decreased expression of GFP.</p

Composition of independent cohorts used in the genetic association analyses.

<p>Composition of independent cohorts used in the genetic association analyses.</p

Study design.

<p>To evaluate genetic factors involved in the dysregulation of type I IFN signaling in SS, we first compared transcriptional profiles between anti-Ro/SSA positive SS cases and controls to identify genes that make up the IFN signature in SS. We then performed genetic association analysis for variants in the regions of the differentially expressed genes. By integrating transcriptome data with genotype data, <i>cis</i>-eQTL analysis was performed for SS-associated SNPs to evaluate their role in gene dysregulation. This genomic convergence approach resulted in increased power to identify and prioritize disease susceptibility genes for further genetic replication and functional studies.</p