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ObjectiveSjögren’s disease (SjD) is an autoimmune disease characterised by inflammatory destruction of exocrine glands. Patients with autoantibodies to Ro/SSA (SjDRo+) exhibit more severe disease. Long non-coding RNAs (lncRNAs) are a functionally diverse class of non-protein-coding RNAs whose role in autoimmune disease pathology has not been well characterised.MethodsWhole blood RNA-sequencing (RNA-seq) was performed on SjD cases (n=23 Ro/SSA negative (SjDRo−); n=27 Ro/SSA positive (SjDRo+) and healthy controls (HCs; n=27). Bioinformatics and pathway analyses of differentially expressed (DE) transcripts (log2fold change ≥2 or ≤0.5; padjLINC01871was characterised by RNA-seq analyses of HSB-2 cells with CRISPR-targetedLINC01871deletion (LINC01871−/−) and in vitro stimulation assays.ResultsWhole blood RNA-seq revealed autoantibody-specific transcription profiles and disproportionate downregulation of DE transcripts in SjD cases relative to HCs. Sixteen DE lncRNAs exhibited correlated expression with the interferon (IFN)-regulated gene,RSAD2, in SjDRo+(r≥0.65 or ≤−0.6); four antisense lncRNAs exhibited IFN-regulated expression in immune cell lines.LINC01871was upregulated in all SjD cases. RNA-seq and pathway analyses ofLINC01871−/−cells implicated roles in cytotoxic function, differentiation and IFNγ induction.LINC01871was induced by IFNγ in a myeloid cell line and regulated by calcineurin/NFAT pathway and T cell receptor (TCR) signalling in primary human T cells.ConclusionLINC01871influences expression of many immune cell genes and growth factors, is IFNγ inducible, and regulated by calcineurin signalling and TCR ligand engagement. AlteredLINC01871expression may influence the dysregulated T cell inflammatory pathways implicated in SjD.

In primary Sjogren's syndrome (pSS), FcRL4+ B cells are present in inflamed salivary gland tissue, within or in close proximity to ductal epithelium. FcRL4 is also expressed by nearly all pSS-related mucosa-associated lymphoid tissue (MALT) B cell lymphomas, linking FcRL4 expression to lymphomagenesis. Whether glandular FcRL4+ B cells are pathogenic, how these cells originate, and how they functionally differ from FcRL4- B cells in pSS is unclear. This study aimed to investigate the phenotype and function of FcRL4+ B cells in the periphery and parotid gland tissue of patients with pSS. First, circulating FcRL4+ B cells from 44 pSS and 54 non–SS–sicca patients were analyzed by flow cytometry. Additionally, RNA sequencing of FcRL4+ B cells sorted from parotid gland cell suspensions of 6 pSS patients was performed. B cells were sorted from cell suspensions as mini bulk (5 cells/well) based on the following definitions: CD19+CD27−FcRL4- (‘naive’), CD19+CD27+FcRL4- (‘memory’), and CD19+FcRL4+ B cells. We found that, although FcRL4+ B cells were not enriched in blood in pSS compared with non-SS sicca patients, these cells generally exhibited a pro-inflammatory phenotype. Genes coding for CD11c (ITGAX), T-bet (TBX21), TACI (TNFRSF13B), Src tyrosine kinases and NF-κB pathway-related genes were, among others, significantly upregulated in glandular FcRL4+ B cells versus FcRL4- B cells. Pathway analysis showed upregulation of B cell activation, cell cycle and metabolic pathways. Thus, FcRL4+ B cells in pSS exhibit many characteristics of chronically activated, pro-inflammatory B cells and their gene expression profile suggests increased risk of lymphomagenesis. We postulate that these cells contribute significantly to the epithelial damage seen in the glandular tissue and that FcRL4+ B cells are an important treatment target in pSS.

In primary Sjögren’s syndrome (pSS), FcRL4+ B cells are present in inflamed salivary gland tissue, within or in close proximity to ductal epithelium. FcRL4 is also expressed by nearly all pSS-related mucosa-associated lymphoid tissue (MALT) B cell lymphomas, linking FcRL4 expression to lymphomagenesis. Whether glandular FcRL4+ B cells are pathogenic, how these cells originate, and how they functionally differ from FcRL4− B cells in pSS is unclear. This study aimed to investigate the phenotype and function of FcRL4+ B cells in the periphery and parotid gland tissue of patients with pSS. First, circulating FcRL4+ B cells from 44 pSS and 54 non–SS–sicca patients were analyzed by flow cytometry. Additionally, RNA sequencing of FcRL4+ B cells sorted from parotid gland cell suspensions of 6 pSS patients was performed. B cells were sorted from cell suspensions as mini bulk (5 cells/well) based on the following definitions: CD19+CD27−FcRL4− (‘naive’), CD19+CD27+FcRL4− (‘memory’), and CD19+FcRL4+ B cells. We found that, although FcRL4+ B cells were not enriched in blood in pSS compared with non-SS sicca patients, these cells generally exhibited a pro-inflammatory phenotype. Genes coding for CD11c (ITGAX), T-bet (TBX21), TACI (TNFRSF13B), Src tyrosine kinases and NF-κB pathway-related genes were, among others, significantly upregulated in glandular FcRL4+ B cells versus FcRL4− B cells. Pathway analysis showed upregulation of B cell activation, cell cycle and metabolic pathways. Thus, FcRL4+ B cells in pSS exhibit many characteristics of chronically activated, pro-inflammatory B cells and their gene expression profile suggests increased risk of lymphomagenesis. We postulate that these cells contribute significantly to the epithelial damage seen in the glandular tissue and that FcRL4+ B cells are an important treatment target in pSS.

Objective Sjogren's Syndrome (SS) patients are prone to develop malignant lymphomas and a correlation has been established between lymphoproliferations and the presence in patients’ blood of an unusual B cell population that downregulated complement receptor 2/CD21. We sought to determine from which B cell compartment lymphoproliferations in SS patients emerge and what are the mechanisms that promote these clonal B cell expansions. Methods Using a PCR-based approach that allows us to clone and express, in vitro, recombinant antibodies produced by single B cells, we tested the reactivity of antibodies expressed by CD19+CD10-CD27-IgM+CD21-/low cells from SS patients. Results We identified clonal expansions in CD21-/low B cells isolated from the blood of three SS patients. All three lymphoproliferations expressed B cell receptors (BCRs) that displayed somatic hypermutation lineage trees characteristic of strong selection by antigens, one of which was identified as a ribosomal self-antigen. When mutated BCR sequences expressed by SS expanded clones were reverted, in vitro, to their germline counterparts, one remained autoreactive. Conclusion Clonal lymphoproliferations in SS patients preferentially accumulate in the autoreactive CD21-/low B cell compartment, which is often amplified in these subjects, and (self)-antigen recognition may drive expansion while further refining BCR (self)-reactivity. This article is protected by copyright. All rights reserved.

Objective Autoantibodies reactive with Ro52 (tripartite motif−containing protein 21 [TRIM21]) are detected in 70% of patients with primary Sjogren's syndrome (SS). TRIM21 belongs to a 34-member C-IV family of TRIM proteins. Although autoantibodies against other TRIM proteins within the C-IV family have been detected in the sera of patients with primary SS, their clinical relevance remains unclear. This study was undertaken to investigate the frequency of anti-TRIM38 in patients with primary SS and evaluate its association with various clinical measures of the disease. Methods Serum samples from patients with primary SS (n = 235) and controls (n = 50) were analyzed for reactivity with in vitro−transcribed and −translated 35S-methionine−labeled TRIM38 protein. The associations of anti-TRIM38 with various laboratory and clinical measures of primary SS were evaluated. Reactivity of anti-TRIM38 with different structural domains of TRIM38 was analyzed. Affinity-purified anti-TRIM38 antibodies were used to immunoprecipitate TRIM21. Results TRIM38-reactive autoantibodies were detected in the sera of 24 of the 235 patients with primary SS and 2 of the 50 controls. Anti-TRIM38 positivity was significantly associated with the presence of anti-Ro60, anti-Ro52, anti-La, rheumatoid factor, and hypergammaglobulinemia. Clinically, anti-TRIM38 was associated with significantly higher ocular surface staining scores, lower Schirmer's test scores, and minor labial salivary gland biopsy focus scores of ≥3.0. Anti-TRIM38 antibodies mainly recognized the cortactin-binding protein 2 (CortBP-2; amino acids 128–238) and the B30.2/SPRY (amino acids 268–465) domains on TRIM38. Affinity-purified antibodies to TRIM38–CortBP-2 and TRIM38–B30.2/SPRY domains reacted with TRIM21. Conclusion Our data demonstrate that anti-TRIM38 specificity arising in a subset of patients with primary SS is associated with increased severity of the disease.

Background Sjogren’s syndrome (SS) is a chronic, heterogeneous disease with hallmark features of auto-inflammation and autoantibody production. Upregulation of type I and II interferon-stimulated genes (ISGs), known as the “Interferon (IFN) Signature” is correlated with anti-Ro titers and has been observed both in the salivary glands and peripheral blood of SS patients. Within the 2 p25.2 genomic interval, the long non-coding RNA (lncRNA) negative regulator of the interferon response (NRIR) has been identified as inducible by type I IFN and is responsible for the downregulation of the ISGs CMPK2 and RSAD2[.1 Objectives We sought to identify additional unannotated ISGs lncRNAs that are differentially expressed (DE) in SS patients utilising correlated expression of RSAD2. Methods We evaluated and compared the transcriptome of anti-Ro(+) patients (n=27) and healthy controls (n=27) using RNA-seq with DE defined as q Results One of the most overexpressed type I ISGs in the SS Ro(+) is RSAD2 (FC=8.05, p=3.29x10E-07). Because of its role in the type I IFN pathway, pairwise correlation coefficients between all DE transcripts and RSAD2 for SS Ro(+) patients were calculated. In total, we found 223 transcripts exhibiting correlation with RSAD2 expression (Pearson’s r>+0.70 or Conclusions Given the importance of the IFN signature to disease pathogenesis in the autoantibody positive patients, it is critical that we better understand how this complex pathway is coordinately regulated. Since one critical function of lncRNAs is to regulate the genome, characterising the mechanisms by which these 14 identified by this study regulate the ISG coordinately could result in new diagnostic and/or therapeutic options. Reference [1] Kambara H, et al. Nucleic Acids Res2014;42(16):10668–80. Disclosure of Interest None declared

Background Despite concerted efforts to characterize dysregulated transcriptional responses observed in Sjogren9s syndrome and related autoimmune disorders (both in whole blood and target tissues), the functional roles of non-coding RNAs (ncRNAs), many of which have been identified as critical players in transcriptional regulation of disease, remain poorly defined. Objectives In the present study, we describe ongoing efforts to functionally characterize the upregulated ncRNA identified by RNA-seq and in silico approaches, AC092580.4 (FC=2.54), which we hypothesize plays a role in T and NK cell responses. Methods To study the immunomodulation of the ncRNA AC092580.4, we carried out a time-course experiment (0–36 hrs) using either PMA/I (500x dil) or universal Type I Interferon. Relative gene expression changes were determined using the Livak method by qPCR using optimized primers for GZMA and AC092580.4 normalized to GAPD. Healthy PBMCs were subjected to stimulation by PHA (1mg/mL; 3 days), PMA/I (500x dil; 3 days), or anti-CD3/CD28 (50uL/1x106cells; 1 day). An average 150-bp RNA-seq reads were generated for each sample; alignment was carried out using STAR (hg38) and comparisions of stimulated vs unstimulated cells were done using DEseq. Pearson9s correlation (r) was calculated for all 3,748 differentially expressed (DE) transcripts to identify transcripts co-expressed with AC092580.4. Results Of the transcripts showing DE in our SS RNA-seq study, we identified 8 as having significantly correlated expression with AC092580.4 in the SSRo- expression matrix (r>0.70 or Conclusions In the present study, we have initiated stimulation studies with to understand the immune relevance of AC092580.4 and co-expressed targets. AC092580.4 shows transcriptional induction by potent inducers of T cell responses (PMA/I, PHA, CD3/CD28) but is downregulated by type I IFN. Transcripts showing co-expressed with AC092580.4 by whole-blood RNA-seq show divergent expression patterns according to the specific stimulus, suggesting a complex regulatory network governing dysregulated T and NK responses. Disclosure of Interest None declared

Exploiting genotyping, DNA sequencing, imputation and trans-ancestral mapping, we used Bayesian and frequentist approaches to model the IRF5-TNPO3 locus association, now implicated in two immunotherapies and seven autoimmune diseases. Specifically, in systemic lupus erythematosus (SLE), we resolved separate associations in the IRF5 promoter (all ancestries) and with an extended European haplotype. We captured 3230 IRF5-TNPO3 high-quality, common variants across 5 ethnicities in 8395 SLE cases and 7367 controls. The genetic effect from the IRF5 promoter can be explained by any one of four variants in 5.7 kb (P-valuemeta = 6 × 10-49; OR = 1.38-1.97). The second genetic effect spanned an 85.5-kb, 24-variant haplotype that included the genes IRF5 and TNPO3(P-valuesEU = 10-27-10-32, OR = 1.7-1.81). Many variants at the IRF5 locus with previously assigned biological function are not members of either final credibleset of potential causal variants identified herein. In addition to the known biologically functional variants, we demonstrated that the risk allele of rs4728142, a variant in the promoter among the lowest frequentist probability and highest Bayesian posterior probability, was correlated with IRF5 expression and differentially binds the transcription factor ZBTB3. Our analytical strategy provides a novel framework for future studies aimed at dissecting etiological genetic effects. Finally, both SLE elements of the statistical model appear to operate in Sjögren's syndrome and systemic sclerosis whereas only the IRF5-TNPO3 gene-spanning haplotype is associated with primary biliary cirrhosis, demonstrating the nuance of similarity and difference in autoimmune disease risk mechanisms at IRF5-TNPO3. © The Author 2014.

Supported by grants from the NIH (5P01-AI-083194 to Drs. Lessard, Rasmussen, Gaffney, Alarcon-Riquelme, Criswell, Jacob, Kimberly, Vyse, Harley, Sivils, Langefeld, and Tsao; 5P01-AR-049084 to Drs. Kimberly and Langefeld; and R01-AR-043814 and R21-AR-065626 to Dr. Tsao), the Oklahoma Medical Research Foundation (to Drs. Lessard, Gaffney, and Sivils), the Alliance for Lupus Research (to Drs. Criswell and Tsao), and the Wake Forest School of Medicine Center for Public Health Genomics (to Dr. Langefeld). Dr. Criswell is recipient of a Kirkland Scholar Award. Sample collection and phenotyping of the subjects utilized in this study was funded by the Ministry for Health and Welfare, Republic of Korea (Korea Healthcare Technology R&D Project grant HI13C2124 to Dr. Bae, and HI13C1754 to Dr. Song). The out-of-study Korean control data were provided by the Korean Biobank Project, which is supported by the Korea Centers for Disease Control and Prevention at the Korea National Institute of Health.

Objective More than 80% of autoimmune disease predominantly affects females, but the mechanism for this female bias is poorly understood. We suspected that an X chromosome dose effect accounts for this, and we undertook this study to test our hypothesis that trisomy X (47,XXX; occurring in ∼1 in 1,000 live female births) would be increased in patients with female-predominant diseases (systemic lupus erythematosus [SLE], primary Sjogren's syndrome [SS], primary biliary cirrhosis, and rheumatoid arthritis [RA]) compared to patients with diseases without female predominance (sarcoidosis) and compared to controls. Methods All subjects in this study were female. We identified subjects with 47,XXX using aggregate data from single-nucleotide polymorphism arrays, and, when possible, we confirmed the presence of 47,XXX using fluorescence in situ hybridization or quantitative polymerase chain reaction. Results We found 47,XXX in 7 of 2,826 SLE patients and in 3 of 1,033 SS patients, but in only 2 of 7,074 controls (odds ratio in the SLE and primary SS groups 8.78 [95% confidence interval 1.67–86.79], P = 0.003 and odds ratio 10.29 [95% confidence interval 1.18–123.47], P = 0.02, respectively). One in 404 women with SLE and 1 in 344 women with SS had 47,XXX. There was an excess of 47,XXX among SLE and SS patients. Conclusion The estimated prevalence of SLE and SS in women with 47,XXX was ∼2.5 and ∼2.9 times higher, respectively, than that in women with 46,XX and ∼25 and ∼41 times higher, respectively, than that in men with 46,XY. No statistically significant increase of 47,XXX was observed in other female-biased diseases (primary biliary cirrhosis or RA), supporting the idea of multiple pathways to sex bias in autoimmunity.

Autoantibodies reactive with Ro52 (tripartite motif-containing protein 21 [TRIM21]) are detected in 70% of patients with primary Sjögren's syndrome (SS). TRIM21 belongs to a 34-member C-IV family of TRIM proteins. Although autoantibodies against other TRIM proteins within the C-IV family have been detected in the sera of patients with primary SS, their clinical relevance remains unclear. This study was undertaken to investigate the frequency of anti-TRIM38 in patients with primary SS and evaluate its association with various clinical measures of the disease.Serum samples from patients with primary SS (n = 235) and controls (n = 50) were analyzed for reactivity with in vitro-transcribed and -translated (35) S-methionine-labeled TRIM38 protein. The associations of anti-TRIM38 with various laboratory and clinical measures of primary SS were evaluated. Reactivity of anti-TRIM38 with different structural domains of TRIM38 was analyzed. Affinity-purified anti-TRIM38 antibodies were used to immunoprecipitate TRIM21.TRIM38-reactive autoantibodies were detected in the sera of 24 of the 235 patients with primary SS and 2 of the 50 controls. Anti-TRIM38 positivity was significantly associated with the presence of anti-Ro60, anti-Ro52, anti-La, rheumatoid factor, and hypergammaglobulinemia. Clinically, anti-TRIM38 was associated with significantly higher ocular surface staining scores, lower Schirmer's test scores, and minor labial salivary gland biopsy focus scores of ≥3.0. Anti-TRIM38 antibodies mainly recognized the cortactin-binding protein 2 (CortBP-2; amino acids 128-238) and the B30.2/SPRY (amino acids 268-465) domains on TRIM38. Affinity-purified antibodies to TRIM38-CortBP-2 and TRIM38-B30.2/SPRY domains reacted with TRIM21.Our data demonstrate that anti-TRIM38 specificity arising in a subset of patients with primary SS is associated with increased severity of the disease.

Objective To compare the performance of the American–European Consensus Group (AECG) and the newly proposed American College of Rheumatology (ACR) classification criteria for Sjogren9s Syndrome (SS) in a well-characterised sicca cohort, given ongoing efforts to resolve discrepancies and weaknesses in the systems. Methods In a multidisciplinary clinic for the evaluation of sicca, we assessed features of salivary and lacrimal gland dysfunction and autoimmunity as defined by tests of both AECG and ACR criteria in 646 participants. Global gene expression profiles were compared in a subset of 180 participants. Results Application of the AECG and ACR criteria resulted in classification of 279 and 268 participants with SS, respectively. Both criteria were met by 244 participants (81%). In 26 of the 35 AECG+/ACR participants, the minor salivary gland biopsy focal score was ≥1 (74%), while nine had positive anti-Ro/La (26%). There were 24 AECG−/ACR+ who met ACR criteria mainly due to differences in the scoring of corneal staining. All patients with SS, regardless of classification, had similar gene expression profiles, which were distinct from the healthy controls. Conclusions The two sets of classification criteria yield concordant results in the majority of cases and gene expression profiling suggests that patients meeting either set of criteria are more similar to other SS participants than to healthy controls. Thus, there is no clear evidence for increased value of the new ACR criteria over the old AECG criteria from the clinical or biological perspective. It is our contention, supported by this report, that improvements in diagnostic acumen will require a more fundamental understanding of the pathogenic mechanisms than is at present available.

Sjogren’s syndrome (SS) is a common, progressive autoimmune exocrinopathy distinguished by dry eyes and mouth and affects ~0.7% of European population. Overexpression of transcripts induced by interferons (IFN), termed as an ‘IFN signature’, has been found in SS patients. Four microarray studies have been published in SS that identified dysregulated genes within type I IFN signaling in either salivary glands or peripheral blood of SS patients. The mechanism of this type I IFN activation is still obscure, but several possible explanations have been proposed, including virus infection-initiated and immune-complex-initiated type I IFN production by plasmacytoid dendritic cells (pDCs). Genetic predisposition to increased type I IFN signaling is supported by candidate gene studies showing evidence for association of variants within IFN-related genes. Once activated, IFN signaling may contribute to numerous aspects of SS pathophysiology, including lymphocyte infiltration into exocrine glands, autoantibody production, and glandular cell apoptosis. Thus, dysregulation of IFN pathways is an important feature that can be potentially used as a serum biomarker for diagnosis and targeting of new treatments in this complex autoimmune disease.

Sjögren's syndrome is a common autoimmune disease (affecting ?0.7% of European Americans) that typically presents as keratoconjunctivitis sicca and xerostomia. Here we report results of a large-scale association study of Sjögren's syndrome. In addition to strong association within the human leukocyte antigen (HLA) region at 6p21 (P meta = 7.65 × 10 -114), we establish associations with IRF5-TNPO3 (P meta = 2.73 × 10 -19), STAT4 (P meta = 6.80 × 10 -15), IL12A (P meta = 1.17 × 10 -10), FAM167A-BLK (P meta = 4.97 × 10 -10), DDX6-CXCR5 (P meta = 1.10 × 10 -8) and TNIP1 (P meta = 3.30 × 10 -8). We also observed suggestive associations (P meta less than 5 × 10 -5) with variants in 29 other regions, including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2 and PHIP, among others. These results highlight the importance of genes that are involved in both innate and adaptive immunity in Sjögren's syndrome. © 2013 Nature America, Inc. All rights reserved.

Background Sjogren9s syndrome (SS) is a chronic autoimmune disorder in which exocrine dysfunction can lead to chronic, debilitating dryness. Expression studies in SS have identified the dysregulated expression of coding and non-coding transcripts enriched in innate and adaptive immune response pathways. Objectives In our recent SS RNA-seq study, we identified 3748 transcripts showing differential expression (DE; FC>2 or Methods Normalized expression data from a whole blood SS RNA-seq study was obtained for 57 cases and 11 healthy controls who underwent multidisciplinary clinical evaluation for the 2002 AECG classification criteria. Objective dryness measures (WUSF, LG, & Sch) were normalized by log 2 transformation and correlation analysis (Spearman for WUSF and LG; Pearson for Sch) was performed for each clinical measure against all of 3748 DE transcripts. Both r or σ and a FDR-corrected p -value, or q -value, were calculated. Significantly correlated transcripts were defined by q Results For WUSF, the significant positive correlation between WUSF rate and the expression of 2 non-coding transcripts was observed: the small Cajal body-specific RNA 5 ( SCARNA5 ; σ=0.50, q =0.018) and the uncharacterized antisense lncRNA RP11–137H2.4 (σ=0.50, q =0.018). For LG, positive correlation was observed for 31 transcripts (0.42 q IFIT3 , OAS3 , and IRF7) , although for the pseudogene, ZDHHC4P1 , and its neighboring IFI gene EPSTI1 both showed significant positive correlation. For LG, the only negatively correlated transcript was the sodium bicarbonate transporter SCL4A10 (σ= -0.43, q =0.045). For Sch, 3 transcripts ( CARD16 , HMGB2 , and BLC2A1 ) were negatively correlated (-0.51 q IQCH was positively correlated (σ=0.49, q =0.019). Conclusions We identified SS-associated transcripts whose expression correlates with clinical measures of dryness. For WUSF, the ncRNA SCARNA5 is situated within an intron of the Crohn9s disease-associated gene autophagy-related 16-like 1 ( ATG16L1 ). Although IFI genes have previously shown correlation with WUSF, the ZDHHC4P1 pseudogene could regulate neighboring SS-associated IFI gene EPSTI1 . For Sch, CARD16 is a caspase inhibitor that influences apoptotic responses that induces NF-kB activation in inflammation, while BLC2A1 has been shown to slow apoptoic responses. Further transcript characterization will allow us to assess their potentialas biomarkers or surrogates of objective clinical measures for SS. Disclosure of Interest None declared

Background Sjogren9s syndrome (SS) is a complex autoimmune disease with both environmental and genetic factors playing important roles in its etiology. Objectives The 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 . Methods Our 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. Results Two 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 59 of TNFAIP3 (rs6933404 P meta =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 ( r 2 =0.02; D9=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 P meta =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. Conclusions These 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 Interest None declared

Although war can impose powerful stresses on family relationships and functions, and its horrors have been described since Euripides wrote The Trojan Women, the subject has received little scientific study. The American Civil War had significant effects on the family and increasing industrialization further disrupted family ties and transformed women's roles. Twentieth-century wars have had both immediate and delayed effects on the family, ranging from anxiety and grief about losses and separations to increased antisocial behavior and juvenile delinquency. Subsequent problems stem from readjustments to civilian life, high divorce rates and women assuming more powerful roles in the family and society. Analysis of a random sample of child guidance clinic records 1923-1983 revealed an increase in children's academic problems and aggressive behaviors during and after World War II. Increases in anxiety and obsessive and aggressive behaviors were evident following the Vietnam conflict along with the adverse effects of post traumatic stress disorder on family life. Although US participation in the Gulf War was limited, disruptive effects were seen in the children of affected military families, and for Iraqi children the consequences were devastating. War generally accelerates dormant changes in family life not yet fully apparent or appreciated

Background: Sjögren syndrome is a common, chronic autoimmune disease that typically produces inflammation and poor function of the salivary and lacrimal glands. Other organs can be affected, including the nervous system. Sensory peripheral neuropathy is a common manifestation of the disease. Methods: Eight-eight patients attending a dry eyesYdry mouth clinic were diagnosed to have primary Sjögren syndrome and underwent a neurological examination. Anti-Ro (or SSA) and anti-La (or SSB) were determined using immunodiffusion as well as Inno-Lia and BioPlex ANA screen. Serum vitamin B12 levels were determined using an enzyme-linked microtiter plate assay. Results: Twenty-seven (31%) of the 88 patients had peripheral neuropathy as defined by loss of light touch, proprioception, or vibratory sensation. Anti-Ro and anti-La were found by immunodiffusion in 12 patients, and 8 of these 12 had neuropathy (W2 = 8.46, P = 0.0036, odds ratio = 6.0 compared to those without precipitating anti-Ro and anti- La). Of the 27 patients with only anti-Ro by immunodiffusion, 13 (48.1%) had neuropathy (W2 = 5.587, P = 0.018, compared to those without anti-Ro). There was no relationship of the other, more sensitive measures of anti-Ro and anti-La to neuropathy. In addition, we found no association of serum vitamin B12 levels to neuropathy among these patients with Sjögren syndrome. Conclusions: Sensory peripheral neuropathy is common among patients with Sjögren syndrome and is associated with the presence of anti-Ro and anti-La when determined by immunodiffusion. Copyright © 2012 by Lippincott Williams and amp; Wilkins.

While Sjogren’s syndrome (SS) is more common than related autoimmune disorders, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), scientific and medical research in SS has lagged behind significantly. This is especially true in the field of SS genetics, where efforts to date have relied heavily on candidate gene approaches. Within the last decade, the advent of the genome-wide association (GWA) scan has altered our understanding of disease pathogenesis in hundreds of disorders through the successful identification of novel risk loci. With strong evidence for a genetic component in SS as evidenced by familial aggregation of SS as well as similarities between SS and SLE and RA, the application of GWA approaches would likely yield numerous novel risk loci in SS. Here we review the fundamental scientific principles employed in GWA scans as well as the limitations of this tool, and we discuss the application of GWA scans in determining genetic variants at play in complex disease. We also examine the successful application of GWA scans in SLE, which now has more than 40 confirmed risk loci, and consider the possibility for a similar trajectory of SS genetic discovery in the era of GWA scans. Ultimately, the GWA studies that will be performed in SS have the potential to identify a myriad of novel genetic loci that will allow scientists to begin filling in the gaps in our understanding of the SS pathogenesis.

Recent advances in the field of genetics have dramatically changed our understanding of autoimmune disease. Candidate gene and, more recently, genome-wide association (GWA) studies have led to an explosion in the number of loci and pathways known to contribute to autoimmune phenotypes. Since the 1970s, researchers have known that several alleles in the MHC region play a role in the pathogenesis of many autoimmune diseases. More recent work has identified numerous risk loci involving both the innate and adaptive immune responses. However, much remains to be learned about the heritability of autoimmune conditions. Most regions found through GWA scans have yet to isolate the association to the causal allele(s) responsible for conferring disease risk. A role for rare variants (allele frequencies of

Dramatic advances in identifying the genetic basis of many human diseases are transforming our fundamental understanding of etiology and pathogenesis. Over the past decade, large global efforts to characterize sequence variation in the human genome have provided the foundation for this extraordinary progress. Success in mapping disease genes has also been fueled by revolutionary advances in our technical capacity for genotyping and analyzing complex genetic datasets. These advances include the technical capacity for genotyping millions of known variants and have ushered in a new era of powerful, large-scale, and highly successful genome screens for many diseases. Scanning the human genome for association of variants with disease is unbiased and not limited by prior selection of a putative candidate gene for testing. As a result, the genes that are associated with disease can be surprising, oftentimes linking previously unsuspected molecular pathways to numerous disease phenotypes. In many cases, an association may be located between genes and have no known or obvious functional effect. Thus, a dramatic shift in our knowledge of the genetic architecture of human disease is underway. Still, much remains to be learned.

Background Sjogren9s 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. Objectives In this study, we sought to identify and characterize underlying genetic contributions to dysregulation of IFN pathways in SS. Methods IFN 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. Results GEP 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. Conclusions We 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 Interest None declared

Background Sjogren9s syndrome (SS) is a common autoimmune disorder characterized by immune-mediated exocrine gland destruction and systemic inflammation contributing to clinical heterogeneity. The complex regulatory mechanisms governing these responses are poorly understood. We previously performed an RNA-sequencing (RNA-seq) study and identified >2,600 differentially expressed (DE) transcripts associated with SS. Objectives This study sought to validate, replicate, and functionally characterize one upregulated long non-coding RNA (lncRNA) mapped to chromosome 2p25.1 to better understand its role in SS pathogenesis. Methods Technical validation and replication of the 2p25.1 lncRNA upregulation was assessed by qPCR. Bioinformatic analysis using GAMMA-seq was used to identify co-expression patterns of this lncRNA with other transcripts. Cellular expression patterns of the 2p25.1 lncRNA were determined by FACS in 9 distinct immune cell subsets in a healthy control followed by RNA isolation and qPCR to assess expression. Statistical comparisons were made using t-tests and Pearson correlations. Results RNA-seq showed significant upregulation of the 2p25.1 lncRNA when comparing 27 healthy controls and 57 SS patients ( P adj =3.69x10 -5 ; Fold Change=2.4). Technical validation by qPCR confirmed this finding ( P =0.0096), and correlation with RNA-seq results was observed ( r =0.869). Transcript expression in an independent sample set of 36 SS patients and 21 controls confirmed the lncRNA upregulation ( P =0.0183). Co-expression patterns showed T, NK, and dendritic cell activation, development, and proliferation. Expression levels of the 2p25.1 lncRNA were highest in the CD8 + T cells (RU=3.34), followed by CD56int NK cells (RU=2.08), CD56hi NK cells (RU=0.83), and CD4 + T cells (RU=0.81). Expression was not detected in CD141 + and CD1c + CD11c + myeloid DCs, monocytes, B cells, or pDCs. Conclusions We have identified, technically validated, and independently replicated the upregulation of a novel SS lncRNA at 2p25.1. Furthermore, we have established that this transcript is highly expressed in CD4 + and CD8 + T cells, and NK cells. This study establishes the 2p25.1 lncRNA as the first associated with SS and lays the groundwork for further functional characterization in the pathogenesis of this complex disorder. Disclosure of Interest None declared

Evidence exists that the olfactory property of alcohol affects cerebral processes to influence the pattern of alcohol drinking in an experimental animal. In this study, the level of activity was quantified in the genetically selected alcohol-preferring P rats during exposure to the odor of 4 concentrations of alcohol. A Mini-Mitter ® transmitter was implanted intraperitoneally in each animal to record individual locomotor activity continuously, in terms of counts per unit time. In the first experiment, each rat was exposed between 1730 and 1930 h to the odor of 5%, 15%, 30%, or 60% alcohol placed in 2 petri dishes flanking both sides of each cage. The control condition was identical, except that the petri dishes were empty. In the second experiment, each rat was offered water and its individually preffered solution of alcohol in the drinking tubes during 1730 to 1930 h on alternate days of exposure to the odor of alcohol. During the odor condition alone, the 30% concentration of alcohol increased the activity counts significantly from the control level of 228.8 ± 23.9 to 303.5 ± 28.1 over the first 30 min of the 2-h test period. Plots of activity every 30 min for each alcohol solution, calculated as percent of control, also showed that the odor of 30% alcohol evoked significantly greater activity during the first and fourth intervals, in contrast to the exposure to 5%, 15%, and 60% alcohol, which failed to shift activity beyond the control level. When the P rats were exposed to 30% alcohol 24 h after 2 h of alcohol drinking, their profile of activity was concordant with that prior to alcohol drinking. These results demonstrate that the odor of a specific concentration of alcohol (i.e., 30%) selectively enhances the activity of the P rat. This could reflect an association of the sensory quality of alcohol with an anticipation of drinking. Finally, these experiments provide the first quantitative evidence that an olfactory cue associated with the unique odor of a specific solution of alcohol may determine the nature of the drinking response.

Background Sjogren’s syndrome (SS) is a common, clinically heterogeneous autoimmune disease characterized by exocrine gland dysfunction that involves both innate and adaptive immune responses. A complex genetic architecture has been hypothesized; however, genetic studies to date have been limited to candidate gene approaches. Objectives We sought to perform the first genome-wide association scan (GWAS) in an unbiased manner to identify SS risk loci. Methods We used high-density Illumina OMNI1-Quad genotyping arrays in a discovery cohort of 395 European-derived SS cases and 1975 healthy controls for the GWAS discovery phase. Stringent quality control (QC) criteria, adjustments for population stratification, and standard GWA statistical methods were used to compare allele frequencies between cases and controls. For replication, an independent set of 1243 SS cases and 4779 healthy controls were genotyped using 2 custom array (CA) platforms. Meta-analysis between the GWAS and CAs were done using METAL (P meta ). Results The most significantly associated region with SS was the MHC, with 6324 overlapping SNPs between the GWAS and CAs exceeding the genome-wide significance (GWS) threshold of P meta =7.65x10E-114. This study also identified 3 novel associations not previously reported as risk loci for SS. In the region of IL12A, 7 variants passed GWS, peaking at rs485497 (P meta =1.17x10E-10). Near CXCR5, 4 variants exceeded GWS with rs7119038 (P meta =1.10x10E-8) the most significant. One variant, rs6579837 (P meta =3.30x10E-8), in TNIP1 also surpassed GWS. Three regions previously implicated were now identified as surpassing GWS for the first time in the current study: IRF5 (rs3757387 P meta =2.73x10E-19), STAT4 (rs10553577 P meta =6.80x10E-15), and BLK (rs2736345 P meta =4.97x10E-10). Conclusions We present the first GWAS of SS identifying and confirming IL12A, CXCR5, and TNIP1 as novel susceptibility loci. We also observed IRF5, STAT4, and BLK for the first time at GWS establishing them as risk loci for SS. Collectively these genes illustrate the importance of both the innate and adaptive immune responses in the etiology of SS. Disclosure of Interest None Declared

Background Sjogren’s syndrome (SS) is a complex disorder that is classically defined by autoimmune processes that result in exocrine gland dysfunction. There is no single clinical diagnostic test for SS, and for research purposes, multiple classification criteria have been proposed over the past decades; efforts to resolve discrepancies and weaknesses between the systems are ongoing. The most widely used criteria are the 2002 American-European Consensus Group (AECG) criteria; recently (2012), a new classification system has been proposed with provisional endorsement of the American College of Rheumatology (ACR). Objectives To compare the performance of these two sets of criteria in a large carefully characterized sicca cohort. Methods In a multidisciplinary clinic for the evaluation of sicca, we assessed features of salivary and lacrimal gland dysfunction and autoimmunity as defined by tests of both AECG and ACR criteria, classifying 646 participants. Global gene expression profiles were also compared in a subset of 180 participants. Results Among the 646 subjects, 279 were classified as SS by AECG, 268 were so classified by ACR while 244 met both sets of criteria. The two sets of criteria were not significantly different (p=0.19; concordance=0.81); the ACR criteria had a sensitivity of 0.87 and specificity of 0.93. Thirty-five subjects were classified as SS by AECG only, of whom 26 (74%) had a salivary gland biopsy focal score >1, while 9 (26%) had positive anti-Ro/La. There were 24 ACR+/AECG-, who met ACR criteria mainly due to differences in the scoring of the corneal staining. All patients with SS, regardless of classification, had a similar gene expression profile, which was distinct from healthy controls. Conclusions The two sets of classification criteria yield concordant results in the majority of cases and gene expression profiling suggests that patients meeting only one set of criteria are more similar to other SS subjects than to healthy controls. Thus, there is no clear evidence for increased value of the new criteria over the old from the data analysis or biological perspective. Moreover, the new criteria require evaluation in a specialty setting, while both the Schirmer’s and WUSF tests can be performed in a standard medical office without the need for sophisticated equipment or specialists. This comparison shows that modifying classification using clinical criteria is not likely to lead to consequential improvements in our ability to identify patients with SS. We believe that such improvements in diagnostic acumen will require a more fundamental understanding of the pathogenic mechanisms than is at present available. Disclosure of Interest None Declared

The plight of millions of children in the United States in the 1980s finally began to be acknowledged by government officials and politicians in the early 1990s as political candidates struggled with issues that could be used for political gain. Children have no voting power, and those that are in grievous distress too often are living with a disturbed, disadvantaged, or demoralized parent(s) who is/are so alienated that he/she/they do not vote. Consequently, the status and well-being of the children in our society have depended on shifting humanitarian sentiments and our national leaders’ often fragile social consciences. The tragedy of children who are hungry, homeless, and/or ill-cared for (if not harmed) has stirred some advocates, who point to the sad statistics about the lack of well-being of children in America and hope that the 1990s will be the decade of our children.

In our everyday clinical experiences, many of the concerns about the well-being of the family have appeared in all-too-human form as adults and children in distress. Their problems, the rapidly changing social scene, and our long-standing interest in the epidemiology of the mental disorders combined to stimulate us to undertake a series of family studies. Three other major factors coalesced to increase our interest. One was the influence of James Grier Miller, president of the University of Louisville and a professor in its Department of Psychiatry, and the publication of his definitive General Living Systems (1978), which supplied the theory needed for our studies. The second was the development of instruments for community studies of mental disorder, based on the new American Psychiatric Association diagnostic and statistical manual (DSM-III), that were being used effectively in the Epidemiologic Catchment Area (ECA) studies (Freedman, 1984). The third was that after those studies were completed, there would be need for the field of psychiatric epidemiology to make a conceptual and methodological leap forward.

Concern about the family throughout the century, culminating in the crisis in the family in the 1980s, prompted us to conduct a review of records of children seen at our Bingham Child Guidance Clinic since the early 1920s. We thought that information about the children’s sociodemographic characteristics and presenting problems and about their families’ (and especially their parents’) problems over most of this century would enlarge our historical perspective and add a clinical component to our extensive literature reviews and epidemiological study. We were particularly interested in change in the family over time. Therefore, we reviewed a random sample of records of children seen at the clinic from 1923 to 1988 to determine possible changes in children’s and families’ presenting problems and in family factors that influenced the children over the greater part of this century.

Many of the concerns about the family described in Chapter 1 have mounted during the past century. In 1889, Commissioner of Labor Carroll D. Wright shocked the nation with his report that the number of divorces had increased from 10,000 in 1867 to 25,000 in 1886, a rise of 157% compared to the population increase of 100% from 1870 to 1890 (Ellwood, 1910). Ellwood stated: “Already in 1885, this country had more divorces than all the rest of the Christian civilized world put together” (p. 114). By the 1880s, desertion and divorce in the cities were major social problems. The first family organization, the National Divorce Reform League (later called the National League for the Protection of the Family) was the forerunner of some current family organizations.

As we have seen, the family crisis of the 1980s stimulated interest in the family and concerns about its well-being. To grasp the meaning of the changes in the structure and functions of the family that have been occurring, as well as the concerns and pathologies, it will be helpful to look at the history of the family. We shall do so not in terms of Santayana’s famous aphorism—that a person who does not learn the lessons of the past is doomed to repeat it—but more in accord with Benedetto Croce’s (1960) thesis that the past has contemporary significance because it is within us.

The preparation of a preliminary outline, listing all data apt to be required, is a basic requirement for field estimating and cost control programs. Preparation of a formal classification of items of work should follow, considering adequate description division and proper indexing. Method of tabulation of indirect and overhead expense should be coordinated between various departments and projects. Periodic recapitulation of quantity and cost data can be keyed for efficient reference. Formal reports indicate independent courses of activity and are prepared at intervals to indicate trend. A labor productivity analysis will indicate variances between different projects and areas, based on a common standard. Recommended data for field estimates includes project records and reports, field manpower and equipment estimates, information from vendors and subcontractors, initial project estimate, data from previous projects, and handbook information. Application of field estimates includes construction alternates, analysis of extra work and revisions, and project cost estimates. A coordinated cost control program will assure adequate permanent property records and adequate information for productivity analysis, field estimating, and future estimating requirements.

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 ( IRF8 ; rs11644034; p meta-Euro = 2.08 × 10 −10 ), transmembrane protein 39A ( TMEM39A ; rs1132200; p meta-all = 8.62 × 10 −9 ), and 17q21 (rs1453560; p meta-all = 3.48 × 10 −10 ) between IKAROS family of zinc finger 3 ( AIOLOS ; IKZF3 ) and zona pellucida binding protein 2 ( ZPBP2 ). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10 −8 meta-Euro −5 ) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6 . The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.

The authors describe the establishment of a unit for children that emphasizes the integration of the pediatric model of acute, short-term inpatient care with the psychological and developmental perspective of the psychiatric model. Of the 145 children admitted during the first year, more than 33% manifested aggressive or hyperactive behavior and 25%, depression or suicidal behavior. Eighty-five percent were discharged to their homes or previous residences after an average length of stay of 24 days. The authors suggest that similar units established in children's hospitals or general hospitals could help meet the urgent need for acute inpatient psychiatric care of children in this country.