Clinical and demographic characteristics of the sets of Finnish patient samples in this study.

1<p>Because of differences in laboratory methods and reference values, only patients from Helsinki were included.</p><p>Na  =  not available.</p><p>Percentage (%) of patients with each phenotype is shown except for mean age, which is shown in years (yrs).</p

Clinical and demographic characteristics of the Swedish sample sets in this study.

1<p>Ro/SSA positive patients without signs of systemic inflammation  = 21, DLE  = 2, SCLE  = 8, SLE  = 31, SS  = 23, UCTD  = 6 from the study of Popovic et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014212#pone.0014212-Popovic1" target="_blank">[19]</a>.</p>2<p>SS patients, data provided by Prof. Marie Wahren-Herlenius, MD, PhD, Dept. of Medicine, Rheumatology Unit, Karolinska Institutet, Solna, Sweden.</p>3<p>Mean age at initial testing for Ro/SSA-autoantibodies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014212#pone.0014212-Popovic1" target="_blank">[19]</a>.</p><p>Na  =  data not available.</p><p>Percentage (%) of patients is shown except for mean age, which is shown in years (yrs).</p

Single marker association results in Finnish SLE patients with renal involvement.

<p>The frequency of the associated allele in controls and cases is shown, as well as its uncorrected <i>P</i>-value, and <i>P</i>-value corrected for multiple testing (in parentheses) as well as odds ratio (OR) with 95% confidence interval (CI). The marker rs12928810 has success below the study threshold (<85%) and was excluded from analysis. Renal involvement is defined as fulfilment of the ACR renal criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014212#pone.0014212-Tan1" target="_blank">[18]</a>.</p

Single marker association results in individual Finnish and Swedish patients positive for Ro/SSA-autoantibody as well as in a combined dataset¹.

<p>The frequency of the associated allele in controls and cases is shown, as well as its uncorrected <i>P</i>-value, and <i>P</i>-value corrected for multiple testing (in parentheses) as well as odds ratio (OR) with 95% confidence interval (CI).</p>1<p>The Finnish sample set consists of sporadic patients with DLE (n = 36), SCLE (n = 25) and SLE (n = 40) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014212#pone.0014212-Koskenmies1" target="_blank">[15]</a>. Ro/SSA information was not available for SLE family probands. The Swedish sample set consists of 21 Ro-positive patients without signs of systemic inflammation, 6 patients with UCTD, 2 DLE, 8 SCLE, 31 SLE, and 66 SS patients.</p

Linkage disequilibrium (LD) patterns across the <i>ITGAM-ITGAX</i> locus and haplotype association analysis results.

<p>A) LD plot of the genotyped <i>ITGAM</i> region on chromosome 16p11 (HapMap CEU data from build 36). Darker colour denotes higher LD (D'). Asterisks indicate single nucleotide polymorphism (SNP) markers genotyped in all datasets in the study. B) Haplotype associations for markers conferring risk for cutaneous DLE, SLE with discoid rash and unstratified SLE. Haplotype 2 is associated with increased, and haplotypes 1 and 3 with decreased risk of cutaneous DLE, SLE with discoid rash, and SLE. Haplotype 2 carries the minor allele A (in bold) of rs1143679, shown in previous studies to tag risk haplotypes for SLE. Significant <i>P</i>-values are indicated in bold. The order of SNPs in haplotypes is as follows: rs1143679 - rs9936831 - rs9937837 - rs9888879 - rs12928810 - rs9888739 - rs11860650 - rs4548893 - rs11574637. C) LD plot of the genotyped SNPs in the DLE dataset. Similar LD patterns were observed in unstratified SLE and in SLE patients stratified for discoid rash and renal involvement as well as in Ro/SSA-positive patients (data not shown).</p

Single marker association results in Finnish DLE, in SLE with discoid rash and in SLE patients.

<p>The frequency of the associated allele in controls and cases is shown, as well as its uncorrected <i>P</i>-value and <i>P</i>-value corrected for multiple testing (in parentheses) as well as odds ratio (OR) with 95% confidence interval (CI).</p><p>Abbreviations: DLE, discoid lupus erythematosus; SLE, systemic lupus erythematosus.</p>1<p>The marker has success below the study threshold (<85%) and was excluded from analysis in SLE.</p

Differentially expressed transcripts between 115 anti-Ro/SSA positive SS cases and 56 controls identified through transcriptome profiling.

<p>(A) We identified 73 genes (represented by 83 probes on the heatmap) differentially expressed between anti-Ro/SSA positive SS cases and healthy controls (absolute FC >2 and <i>q</i><0.05). Among the differentially expressed genes, 57 were type I IFN-regulated genes (black bar on right) and formed an IFN signature where most genes were overexpressed in SS patients (yellow indicates overexpressed genes compared to controls). (B) The 57 differentially expressed type I IFN-regulated genes were re-clustered in anti-Ro/SSA positive SS cases using <i>k</i>-means (<i>k</i> = 3) algorithm and heterogeneity of the IFN signature levels in anti-Ro/SSA positive SS cases was observed.</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

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

Results of <i>cis</i>-eQTL analysis in <i>OAS1</i> region.

<p>(A) After imputation, 453 variants near <i>OAS1</i> were tested for association with <i>OAS1</i> transcript expression using linear regression. The association of each variant with the transcript level of <i>OAS1</i> (represented by 3 probes on the microarray; see B) are plotted based on the most significant -log₁₀(<i>P</i>_<i>eQTL</i>) values. We identified <i>cis</i>-eQTLs within and near <i>OAS1</i>, with the top association at rs10774671 (<i>P</i>_<i>eQTL</i> = 6.05×10⁻¹⁴). The variant rs10774671 was also the most significant genotyped SS-associated SNP in the genetic association analysis (<i>P</i>_<i>assoc</i> = 8.47×10⁻⁵; The top imputed SS-associated variant rs4767023 is also marked on the plot). The <i>r</i>² coded by colors indicating LD with rs10774671 are given in the figure. Variants above the dashed line were associated with <i>OAS1</i> transcript expression with <i>q</i><0.01. No eQTL was observed for <i>OAS2</i> or <i>OAS3</i>. (B) The genomic structures of the isoforms of <i>OAS1</i> (p46: NM_016816; p42: NM_002534; p48: NM_001032409; and p44, as described previously and identified in our RNA-seq analysis) are shown. The location of rs10774671 and the splicing consensus sequence AG in p46, p48, and p44 are indicated. One probe on the microarray specifically detects the p42 isoform (Probe 3). (C) The <i>cis</i>-eQTL analysis was performed through integration of the microarray expression data of <i>OAS1</i> with the genotype data of rs10774671. The SS-associated risk allele A of rs10774671 was associated with higher expression level of the p42 isoform as determined by Probe 3. The A allele was associated with lower expression of total <i>OAS1</i> as measured by Probe 1 and Probe 2. The <i>cis</i>-eQTL analysis results were determined using both a linear model and ANOVA. The mean value and the standard error of the mean (Mean±SEM) in each group are plotted in red.</p