Similar TLR-7 and -9 mRNA level in pSS patients and controls.

<p>No significant disparities were detected in mRNA level, however a slightly lower level of TLR-9 was noticed in pSS. RNA was isolated from negatively isolated B cells, and cDNA was synthesized with High-Capacity RNA-to-cDNA Kit. Taqman gene expression assays were used to detect TLR-7 and -9. GAPDH was used for normalization. Median is indicated in the figure.</p

Altered distribution of B cells between subsets in pSS compared to healthy controls.

<p>A) B cells in PBMC were detected by gating CD19⁺ CD45⁺ cells. B cell subsets were defined by CD27 and IgD expression. Naïve B cells were defined as CD19⁺CD27⁻IgD⁺ (Q1), memory B cells as CD19⁺CD27⁺IgD⁻ (Q3), and pre-switched memory B cells as CD19⁺CD27⁺IgD⁺ (Q2). B) An increased number of naïve B cells was detected in pSS patients compared to healthy controls. No differences were found in amount of memory B cells between the groups, while a decreased level of pre-switched memory B cells were discovered in pSS patients. The bar indicates the median; *: p <0.05, **: p <0.01.</p

Similar numbers of CD19⁺ B cells in pSS patients and controls, but decreased amounts of CD27⁺ cells.

<p>No significant difference was identified in number of CD19⁺ B cells in PBMC comparing pSS patients and healthy controls, although a greater variation was detected in the patient group (A). Fewer CD19⁺ B cells expressed CD27 in pSS patients compared to healthy controls, yet the MFI values were significantly higher in pSS (C). The bar indicates the median; *: p <0.05, **: p <0.01.</p

The medications used did not have a significant effect on B cell subsets or TLR expression.

<p>No significant differences were detected in the distribution B cells between the different subsets (A), or in TLR-7 and -9 expression (B and C). Patients not receiving any medications had a slightly increased TLR-7 and -9 expression in all B cell subpopulations, compared to those on any medication (B and C). The median is indicated as a line in the bars. No medication; n = 12, hydroxychloroquine; n = 5, predisolone; n = 3, and hydroxychloroquine+prednisolone; n = 4.</p

Gating strategy for the detection of TLR-7 and -9 in B cell subsets.

<p>PBMC were isolated from whole blood and stained for surface markers before cells were fixed, permeabilised and stained for TLR-7 and -9. FSC and SSC were first used to gate out debris (A) and SSC-A and SSC-H was utilized to eliminate duplicates (B). Further gating was done on CD45 and CD19, to target B cells (C). To separate between the different B cell populations, we gated on CD27 and IgD (D), followed by TLR-7 and -9 expression on these subsets (E). Data from one representative patient is shown.</p

Composition of independent cohorts used in the genetic association analyses.

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

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

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