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BackgroundB cells are important in the pathogenesis of primary Sjögren’s syndrome (pSS) with hypergammaglobulinemia, SSA/SSB autoantibodies and an increased risk of B cell lymphoma [1]. Patients with pSS positive for SSA/SSB autoantibodies are more prone to systemic disease manifestations and adverse outcomes. Only few studied have investigated peripheral blood mononuclear cells at single cell resolution and primarily reported differences in the T cell compartment while the B cells were only briefly considered [2,3].ObjectivesWe aimed to determine the role of peripheral B cell subtype composition, gene expression and B cell receptor (BCR) usage in patients with pSS stratified for SSA/SSB antibodies.MethodsOver 230 000 B cells were isolated by negative selection from peripheral blood of 24 pSS patients (n=6 SSA+, n=8 SSA- and n=10 SSA and SSB+ (SSAB)) and four healthy controls. Single cell gene expression and targeted BCR libraries were generated and processed for RNA and VDJ sequencing.ResultsGene expression-based clustering and cell annotation defined 16 B cell subtypes. We show that pSS patients have a heterogeneous circulating B cell composition, with SSAB patients presenting the highest and lowest proportion of naïve and memory B cells, respectively. Interferon-induced genes were upregulated across all B cell subtypes with the highest levels in the SSAB group. Differential usage of Immunoglobulin Heavy Chain (IGH) genes, showed that memory B cells from SSAB patients displayed a higher proportion of IGHM expressing cells and cells with unmutated VDJ transcripts, including IGHV1-69 and IGHV4-34, compared to other pSS patient groups and controls, indicating altered somatic hypermutation and class switching processes. Comparison with previous studies revealed heterogeneous clonotype pools, with little overlap in CDR3 sequences. Joint principal component analysis using scRNA-seq and scVDJ-seq data including B cell subtype composition, IFN-score, IGH constant gene usage, IGHV usage and VDJ mutation status allowed unsupervised stratification pSS patients that correlated to disease manifestations and antibody status.ConclusionCombining single cell gene expression and VDJ sequence data we describe heterogeneity and molecular characteristics in B cells from pSS patients with different autoantibody profiles. Our results may provide clues to intrinsic differences in B cells that affect the phenotype and outcome, and allow stratification of pSS patients at improved resolution.References[1] Nocturne G, Mariette X. B cells in the pathogenesis of primary Sjogren syndrome. Nat Rev Rheumatol 2018;14:133-45.[2] Hong X, et al. Single-Cell RNA Sequencing Reveals the Expansion of Cytotoxic CD4(+) T Lymphocytes and a Landscape of Immune Cells in Primary Sjogren’s Syndrome. Front Immunol 2020;11:594658.[3] Hou X, et al. Analysis of Gene Expression and TCR/B Cell Receptor Profiling of Immune Cells in Primary Sjogren’s Syndrome by Single-Cell Sequencing. J Immunol 2022;209:238-49.AcknowledgementsWe thank Rezvan Kiani Dehkordi for collecting patient and control samples.Disclosure of InterestsNone Declared.

Primary Sjögren's syndrome (SS) shares many features with systemic lupus erythematosus (SLE). Here we investigated the association of the three major polymorphisms in IRF5 and STAT4 found to be associated with SLE, in patients from Sweden and Norway with primary SS. These polymorphisms are a 5-bp CGGGG indel in the promoter of IRF5 , the single nucleotide polymorphism (SNP) rs10488631 downstream of IRF5 and the STAT4 SNP rs7582694, which tags the major risk haplotype of STAT4. We observed strong signals for association between all three polymorphisms and primary SS, with odds ratios (ORs) >1.4 and P -values <0.01. We also found a strong additive effect of the three risk alleles of IRF5 and STAT4 with an overall significance between the number of risk alleles and primary SS of P =2.5 × 10 −9 . The OR for primary SS increased in an additive manner, with an average increase in OR of 1.78. For carriers of two risk alleles, the OR for primary SS is 1.43, whereas carriers of five risk alleles have an OR of 6.78. IRF5 and STAT4 are components of the type I IFN system, and our findings emphasize the importance of this system in the etiopathogenesis of primary SS.

We performed a candidate gene association study in 540 patients with primary Sjögren's Syndrome (SS) from Sweden ( n =344) and Norway ( n =196) and 532 controls ( n =319 Swedish, n =213 Norwegian). A total of 1139 single-nucleotide polymorphisms (SNPs) in 84 genes were analyzed. In the meta-analysis of the Swedish and Norwegian cohorts, we found high signals for association between primary SS and SNPs in three gene loci, not previously associated with primary SS. These are the early B-cell factor 1 ( EBF1 ) gene, P =9.9 × 10 −5 , OR 1.68, the family with sequence similarity 167 member A–B-lymphoid tyrosine kinase ( FAM167A–BLK ) locus, P =4.7 × 10 −4 , OR 1.37 and the tumor necrosis factor superfamily ( TNFSF4 = Ox40L ) gene, P =7.4 × 10 −4 , OR 1.34. We also confirmed the association between primary SS and the IRF5/TNPO3 locus and the STAT4 gene. We found no association between the SNPs in these five genes and the presence of anti-SSA/anti-SSB antibodies. EBF1 , BLK and TNFSF4 are all involved in B-cell differentiation and activation, and we conclude that polymorphisms in several susceptibility genes in the immune system contribute to the pathogenesis of primary SS.

Objective To investigate whether the risk allele for systemic lupus erythematosus (SLE) in the signal transducer and activator of transcription factor 4 (STAT4) gene, defined by the single nucleotide polymorphism (SNP) rs10181656(G), is associated with vascular events and/or presence of prothrombotic anti-phospholipid antibodies (aPL) in patients with SLE. Methods Two independent groups of unrelated patients with SLE of Swedish ethnicity (n=424 and 154) were genotyped, and occurrence of previous manifestations of ischaemic heart disease (IHD), ischaemic cerebrovascular disease (ICVD) and venous thromboembolic events (VTE) was tabulated. aPL values were measured by ELISA. Matched controls (n=492 and 194) were genotyped. Results The STAT4 risk allele was more frequent in patients with SLE with previous arterial events (combined OR (ORc)=1.5, 95% CI 1.1 to 2.0) compared to patients without such events. The association was mainly attributable to an accumulation of the risk allele among patients with ICVD (ORc=2.3, CI 1.6 to 3.3). There was no association with IHD or VTE. The presence of two or more aPLs was associated with the risk allele (ORc=1.6, 95% CI 1.2 to 2.0). In multivariable-adjusted logistic regression analyses treatment for hypertension, at least one STAT4 risk allele, older age, IgG anti-cardiolipin antibodies and longer SLE duration remained independently associated with previous ICVD (p≤0.02 for all). Conclusion Patients with SLE with the STAT4 risk allele had a strikingly increased risk of ICVD, comparable in magnitude to that of hypertension. The results imply that a genetic predisposition is an important and previously unrecognised risk factor for ICVD in SLE, and that aPLs may be one underlying mechanism.

BackgroundSjögren’s syndrome (SS) is a chronic, heterogeneous disease with hallmark features of auto-inflammation and autoantibody production. We previously identified association between the DDX6-CXCR5 locus and SS surpassing genome-wide significance.ObjectivesThis study aims to determine the mechanism by which this association contributes to disease.MethodsFine mapping and imputation allowed enrichment of existing genetic datasets from a total of 1916 SS cases and 3684 controls with 971 testable variants in the DDX6-CXCR5 interval. Candidate variants were prioritized using statistical and bioinformatics approaches. Electromobility shift assays (EMSAs) and pull-downs (PDs) followed by mass spectrometry (MS) were used to determine allelic-specific differences in binding using lysates from HSB-2 (T), Jurkat (T), Reh (B), Ramos (B), Daudi (B), THP-1 (monocyte), and HEK 293T (epithelial) cells.ResultsBioinformatic analysis of the top associated variants after imputation (rs7125066 and rs7119038) in the DDX6-CXCR5 region did not yield evidence of regional functionality. However, 46 other candidates that span the region of association were identified through imputation. Chromatin methylation pattern data from the Roadmap database showed several variants in this region were within transcription start sites or enhancer elements depending on the cell type and state. Using RegulomeDB, Haploreg, and other databases, rs4938572, rs12365699, rs57494551, and rs10892294 showed strong evidence affecting binding and/or expression of one or more target genes in the region and were selected for further study. Using EMSAs, statistically significant increase in binding for the risk allele as compared to the non-risk allele was found for rs4938572 (p<0.01) for cell lysates from HSB-2, Reh, THP-1, Ramos, Jurkat, and Daudi but not HEK 293T cells. While decreased binding of risk allele for rs10892294 and rs12365699 were found for Ramos cells only, rs57494551 showed decreased binding for both Ramos and THP-1 cell lysate. The risk allele of rs57494551 also decreases binding using THP-1 cell lysate. Preliminary MS analysis showed several immune related transcription factors likely binding this region, including: IRF8, GTFTI, RFX5, IKZF3, PAX5, and IKZF1. Cell type-specific expression of the genes in the region shows the expression of DDX6 spans many immune cells subsets while the CXCR5 expression is more restrictedConclusionsThe genetic variants in the DDX6-CXCR5 locus that were selected based on bioinformatics data resulted in a difference in the direction of binding, with the risk allele all residing on the same risk haplotype. The change in binding of protein.to each associated variant likely would alter expression of both protein coding genes in a cell/context specific manner. Ongoing studies will assess the regulatory role of these sequences using luciferase assays and CRISPR/Cas9 based genetic modification of target SNPs in various cell types.Disclosure of InterestNone declared

BackgroundIn the widely used American-European Consensus Group (AECG) classification criteria for primary Sjögren's syndrome (pSS), pre-existing lymphoma is one of the exclusion criteria for pSS diagnosis (1). This is in contrast to the American College of Rheumatology (ACR)/Sjögren's International Collaborative Clinical Alliance (SICCA) 2012 classification criteria (2). The rationale and consequences of excluding or not excluding pSS in patients with pre-existing lymphoma have not specifically been described.ObjectivesTo compare patients with pre-existing lymphoma and a later pSS diagnosis, and patients with pSS and a subsequent lymphoma, derived from a large-population-based cohort of individuals with pSS and lymphoma.MethodsPatients with an International Classification of Diseases (ICD) diagnosis code for “Sjögren's syndrome/sicca syndrome” (SS) and prior or subsequent lymphoma diagnosis were identified by linking the Swedish Patient Register 1964–2007 with the Cancer Register 1990–2007 (n=205). Clinical data and lymphoma tissues were reviewed and the diagnoses evaluated. All 71 patients fulfilling the AECG and/or the proposed ACR-EULAR criteria for pSS (3) with a confirmed lymphoma were included in the study.ResultsWe identified 11 patients with pre-existing lymphoma, defined as lymphoma diagnosed before (n=4, median time four years to pSS diagnosis) or within six months after pSS diagnosis (n=7, median time three months to lymphoma diagnosis), and 60 pSS patients with subsequent lymphoma.The pSS characteristics were similar in patients with pre-existing lymphoma and pSS patients with subsequent lymphoma. There were no differences in reported duration of sicca symptoms (median time from sicca onset until pSS diagnosis 5 years in pre-existing lymphoma group vs. 4 years in pSS patients with subsequent lymphoma, p=0.1), sex distribution, median ages at sicca onset, pSS and lymphoma diagnoses, presence of autoantibodies, laboratory findings, overall survival after lymphoma diagnosis, and extraglandular pSS manifestations. However, lymphadenopathy at pSS diagnosis was more common in patients with pre-existing lymphoma (p<0.0001).There were some differences in lymphoma characteristics. Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) (72% vs. 22%, p<0.002), and lymphoma involvement of the salivary glands (82% vs. 32%, p<0.005) were more common in patients with pre-existing lymphoma than in pSS patients with subsequent lymphoma.ConclusionsThis study suggests that pre-existing lymphoma should not be used as a general exclusion criterion for pSS, which may erroneously remain undiagnosed. On the contrary, the combination of sicca symptoms and MALT lymphoma in the salivary glands could instead be a reason for adequate pSS investigation.ReferencesVitali C, et al.: Ann Rheum Dis 2002.Shiboski SC, et al.: Arthritis Care Res 2012.Shiboski CH, et al.: Abstract ACR 2015.Disclosure of InterestNone declared

The type I interferon system genes IKBKE and IFIH1 are associated with the risk of systemic lupus erythematosus (SLE). To identify the sequence variants that are able to account for the disease association, we resequenced the genes IKBKE and IFIH1 . Eighty-six single-nucleotide variants (SNVs) with potentially functional effect or differences in allele frequencies between patients and controls determined by sequencing were further genotyped in 1140 SLE patients and 2060 controls. In addition, 108 imputed sequence variants in IKBKE and IFIH1 were included in the association analysis. Ten IKBKE SNVs and three IFIH1 SNVs were associated with SLE. The SNVs rs1539241 and rs12142086 tagged two independent association signals in IKBKE , and the haplotype carrying their risk alleles showed an odds ratio of 1.68 ( P -value=1.0 × 10 −5 ). The risk allele of rs12142086 affects the binding of splicing factor 1 in vitro and could thus influence its transcriptional regulatory function. Two independent association signals were also detected in IFIH1 , which were tagged by a low-frequency SNV rs78456138 and a missense SNV rs3747517. Their joint effect is protective against SLE (odds ratio=0.56; P -value=6.6 × 10 −3 ). In conclusion, we have identified new SLE-associated sequence variants in IKBKE and IFIH1 , and proposed functional hypotheses for the association signals.

ObjectivesTo analyse the influence of epidemiology and ethnicity on the clinical systemic presentation at diagnosis of primary Sjögren syndrome (SjS).MethodsThe Big Data Sjögren Database included 10 475 worldwide patients from 22 countries fulfilling the 2002 criteria. Age at diagnosis, gender and ethnicity (77% White, 14% Asian, 6% Hispanic, 1% Black/African American, 2% others) were correlated with systemic involvement at diagnosis (retrospectively scored in 9974 patients using ESSDAI/clinESSDAI)ResultsMen had higher mean ESSDAI (8.0 vs 5.9, p<0.001) and clinESSDAI (8.4 vs 6.1, p<0.001) in comparison with women; the domains more active in men included lymphadenopathy (p<0.001), glandular (p<0.001), pulmonary (p=0.001), PNS (p<0.001) and CNS (p<0.001). Highest scores were also reported in patients with young-onset disease (<35 years) with respect to older onset (>65 years) for ESSDAI (6.5 vs 5.6, p=0.002) and clinESSDAI (6.4 vs 5.8, p=0.034). Highest ESSDAI scores were reported in Black/African American, followed by White, Asian and Hispanic patients (6.7 vs 6.3 vs 5.4 vs 4.9, respectively, p<0.001). BAA patients showed the highest frequency of activity in lymphadenopathy, articular, PNS, CNS and biological domains, Whites in glandular, cutaneous and muscular, Asians in pulmonary, renal and haematological and Hispanics in the constitutional domain.ConclusionsThis study provides the first evidence for a strong influence of epidemiology and ethnicity on the systemic phenotype at diagnosis of primary SjSDisclosure of InterestNone declared

BackgroundSjögren's syndrome (SS) is a complex autoimmune disease with both environmental and genetic factors playing important roles in its etiology.ObjectivesThe goal of this study was to replicate 8 suggestive loci that failed to exceed the genome-wide significance (GWS) threshold of 5x10E-8 in our previously published work: TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2, and PHIP.MethodsOur previous study included 1638 SS cases and 6754 population controls. In the current study, we genotyped an additional 282 SS cases and 2576 population controls yielding a total of 1920 SS cases and 9330 controls. Analysis was done using logistic regression accounting for ancestry and gender.ResultsTwo regions exceeded the GWS threshold. Association has been previously established with SS to a risk haplotype spanning the TNFAIP3 coding region described in lupus; however, we identified a novel independent effect ∼230kb 5' of TNFAIP3 (rs6933404 Pmeta=6.85x10E-9, OR=1.28) originally described in rheumatoid arthritis. Association was observed for the haplotype spanning the TNFAIP3 coding region peaking at rs58721818 (P=4.77x10E-4), which is not correlated with rs6933404 (r2=0.02; D'=0.49). Bioinformatics data provided limited support that rs6933404 is the functional variant in this region; however, another variant on this haplotype also surpassing GWS, rs6927172, modifies 8 transcription factor binding sites, and is located within an enhancer element in CD4+CD25–IL17+ PMA-Ionomycin stimulated Th17 primary cells by the Epigenetics Road Map Project. A variant in the region of PRDM1 also surpassed the GWS threshold (rs526531 Pmeta=1.70x10E-8, OR=1.25). Two additional variants on this haplotype exceeded GWS, but no clear candidate functional variant has emerged based on bioinformatics data. Of the 6 remaining suggestive regions, PTTG1, DGKQ, and FCGR2A continue trending towards GWS.ConclusionsThese data now establish 2 new SS risk loci, TNFAIP3 and PRDM1. TNFAIP3, which codes for the protein A20, is a negative regulator of NF-kB. PRDM1, which codes for the protein BLIMP1, is a transcription factor that regulates interferon-beta locus and plasma cell differentiation. Additional studies are needed to determine how these association signals function in the human genome and contribute to SS etiology.Disclosure of InterestNone declared

BackgroundSjögren's syndrome (SS) is a common, autoimmune exocrinopathy characterized by symptoms of dry eyes and mouth present in 0.7% of the European American population. Gene expression profiling (GEP) has previously demonstrated overexpression of transcripts induced by interferons (IFN) in SS patients.ObjectivesIn this study, we sought to identify and characterize underlying genetic contributions to dysregulation of IFN pathways in SS.MethodsIFN signature genes of interest were selected from GEP studies performed in 115 anti-Ro positive SS cases and 73 controls using microarray data and evaluated for cis-expression quantitative trait loci (eQTL) in 178 subjects by integration with genome-wide association study (GWAS) data. Gene splicing patterns were evaluated using RNA-sequencing (RNA-seq) performed in 57 SS cases and 27 controls on the Illumina platform.ResultsGEP showed that OAS1, an IFN-inducible gene involved in inhibition of virus replication, was significantly overexpressed in SS patients. Multiple cis-eQTL were identified in OAS1 with the most significant peaking at rs10774671, strengthening prior evidence of this variant for disease association ((p=8.47×10-5) obtained in our large GWAS dataset consisting of 765 cases and 3825 controls. We further replicated this genetic association in an independent set of 514 cases and 3466 controls followed by meta-analysis (p=2.59×10-9; OR=0.75). The risk allele “A” of rs10774671 is a splice site consensus variant located at the junction between intron-5 and exon-6 of OAS1, and thus may switch the primary isoform, p46, to various alternatives. Transcripts measured by RNA-seq were reconstructed and the abundance of isoforms was compared across samples according to the genotype of rs10774671. The risk allele “A”, which demolishes the splicing consensus sequence, was correlated with higher expression of p42, p48, and p44 isoforms, but a lower expression of the normally spiced OAS1 isoform, p46. Functional characterization of different OAS1 isoforms indicated that the alternatively spliced isoforms resulted in impaired protein expression and lack of response to type I IFNs.ConclusionsWe identified OAS1 as a novel candidate SS locus. The risk allele “A” has been reported to result in decreased Oas1 enzyme activity. These results suggest a mechanism in which alternatively spliced OAS1 transcripts lead to reduced viral clearance resulting in perpetuation of type I IFN signaling in SS.Disclosure of InterestNone declared