OBJECTIVES:To calculate a polygenic risk score (PRS) based on single nucleotide variants (SNVs) previously associated with primary Sjögren's disease (SjD) with genome-wide significance and determine the genetic risk for SjD stratified by antibodies, sex and age at diagnosis. METHODS:Patients with SjD (n = 1065) were genotyped using Illumina OmniExpressExome chip. Control genotype data were available (n = 7742). Two PRSs were constructed, one including HLA gene variants (n = 21 SNVs), and one without HLA (n = 18 SNVs). High PRS quartile (Q4) individuals were compared with low PRS (Q1-3). RESULTS:A high PRS was associated with SSA antibody-positive SjD (OR 9.16, 95% CI 7.75-10.85, P = 3.7 × 10-146), and strengthened in SjD positive for both SSA/SSB antibodies (OR 13.67, 95% CI 10.88-17.32, P = 4.6 × 10-108). High PRS classified SSA/SSB antibody-positive SjD with very good accuracy (AUC 0.86). PRS without HLA showed a weaker association with SSA/SSB positive SjD (OR 2.09, 95% CI 1.71-2.55, P = 6.4 × 10-13). Antibody negative SjD displayed a PRS similar to controls. Patients in the high PRS quartile were significantly younger at diagnosis, 48.9 ± 14.9 vs 53.4 ± 13.4 years in the low PRS quartiles (Q1-3), P = 2.2 × 10-6, and presented higher frequencies of ANA, SSA and SSA/SSB antibodies, P < 1 × 10-5. CONCLUSION:A high PRS is associated with SSA/SSB antibody positivity and early disease onset, both largely attributed to the weight of the HLA alleles. Integration of PRS with other biomarkers applied to clinical phenotypes could be a useful tool for disease risk stratification and treatment decisions.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a heterogenous clinical picture. This study aimed to link genetic SLE predisposition with relevant clinical manifestations using a two-step approach. First, we identified datasets best corresponding to the 11 American College of Rheumatology 1982 (ACR-82) classification criteria for SLE using an ICD-10 code-based search in a large, public database (FinnGen consortium). Mendelian Randomization analysis of these datasets linked genetic SLE predisposition to several SLE-like manifestations: rosacea, OR 1.09(1.03-1.16), polyarthropathies, OR 1.10(1.06-1.14), pleural effusions, OR 1.09(1.04-1.14), and hemolytic anemia, OR 1.32(1.10-1.58). Second, validation was conducted in a clinical SLE cohort comprising 1,487 genotyped Scandinavian patients with detailed medical records. Based on the public datasets, genetic risk scores (GRS) for each relevant manifestation were constructed for each patient. Associations between each GRS and the corresponding ACR-82 criterion were evaluated using sex- and disease duration-adjusted logistic regression. Five of the 11 ACR-82 criteria were associated with their corresponding GRS: arthritis, OR 1.15(1.02-1.31), nephritis, OR 1.15(1.04-1.29), neurology, OR 1.24(1.04-1.47), hematology, OR 1.12(1.00-1.24), and immunology, OR 1.37(1.22-1.56), indicating that our method of using publicly available datasets to construct manifestation-specific GRSs may be useful in predicting SLE outcomes. ### Competing Interest Statement CS reports the following competing interests: Bristol-Myers Squibb(BMS) (employee). PP reports the following competing interests: Olink/Thermo Fisher Scientific(employee).LR reports the following competing interests: Ampel Biosolutions (Advisor or Review Panel Member); AstraZeneca (Advisor or Review Panel Member, Speaker/Honoraria); Bayer (Consultant); BMS (Advisor or Review Panel Member); UCB (Advisor or Review Panel Member). DL reports the following competing interests: AstraZeneca (Advisor or Review Panel Member). ### Funding Statement This study was supported by the Gustaf Prim Foundation, the Swedish Society for Medical Research (S20-0127), the Swedish Research Council for Medicine and Health, the Swedish Rheumatism Association, King Gustaf V 80‐Year Foundation, the Swedish Society of Medicine, the Agnes and Mac Rudberg Foundation, the Ingegerd Johansson donation, the Gustafsson Foundation, the Selander Foundation and the County Council of Uppsala. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The protocol for this study was approved by the Regional Ethical Review Board, Uppsala (DNR 2009/013 and 2020-05065) and the local ethics committees. Coordinating Ethics Committee of the Helsinki and Uusimaa Hospital District have provided ethical approval for the FinnGen project. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

OBJECTIVE:Complete genetic deficiency of the complement component C2 is a strong risk factor for monogenic systemic lupus erythematosus (SLE), but whether heterozygous C2 deficiency adds to the risk of SLE or primary Sjögren's syndrome (SS) has not been studied systematically. This study was undertaken to investigate potential associations of heterozygous C2 deficiency and C4 copy number variation with clinical manifestations in patients with SLE and patients with primary SS. METHODS:The presence of the common 28-bp C2 deletion rs9332736 and C4 copy number variation was examined in Scandinavian patients who had received a diagnosis of SLE (n = 958) or primary SS (n = 911) and in 2,262 healthy controls through the use of DNA sequencing. The concentration of complement proteins in plasma and classical complement function were analyzed in a subgroup of SLE patients. RESULTS:Heterozygous C2 deficiency-when present in combination with a low C4A copy number-substantially increased the risk of SLE (odds ratio [OR] 10.2 [95% confidence interval (95% CI) 3.5-37.0]) and the risk of primary SS (OR 13.0 [95% CI 4.5-48.4]) when compared to individuals with 2 C4A copies and normal C2. For patients heterozygous for rs9332736 with 1 C4A copy, the median age at diagnosis was 7 years earlier in patients with SLE and 12 years earlier in patients with primary SS when compared to patients with normal C2. Reduced C2 levels in plasma (P = 2 × 10-9 ) and impaired function of the classical complement pathway (P = 0.03) were detected in SLE patients with heterozygous C2 deficiency. Finally, in a primary SS patient homozygous for C2 deficiency, we observed low levels of anti-Scl-70, which suggests a risk of developing systemic sclerosis or potential overlap between primary SS and other systemic autoimmune diseases. CONCLUSION:We demonstrate that a genetic pattern involving partial deficiencies of C2 and C4A in the classical complement pathway is a strong risk factor for SLE and for primary SS. Our results emphasize the central role of the complement system in the pathogenesis of both SLE and primary SS.

Objective Lupus nephritis (LN) is a common and severe manifestation of SLE. The genetic risk for nephritis and progression to end-stage renal disease (ESRD) in patients with LN remains unclear. Herein, we aimed to identify novel genetic associations with LN, focusing on subphenotypes and ESRD. Methods We analysed genomic data on 958 patients with SLE (discovery cohort: LN=338) with targeted sequencing data from 1832 immunological pathway genes. We used an independent multiethnic cohort comprising 1226 patients with SLE (LN=603) as a replication dataset. Detailed functional annotation and functional epigenomic enrichment analyses were applied to predict functional effects of the candidate variants. Results A genetic variant (rs56097910) within the MERTK gene was associated with ESRD in both cohorts, meta-analysis OR=5.4 (2.8 to 10.6); p=1.0×10-6. We observed decreased methylation levels in peripheral blood cells from SLE patients with ESRD, compared with patients without renal SLE (p=2.7×10-4), at one CpG site (cg16333401) in close vicinity to the transcription start site of MERTK and located in a DNAse hypersensitivity region in T and B cells. Rs56097910 is linked to altered MERTK expression in kidney tissue in public eQTL databases. Two loci were replicated for association with proliferative LN: PRDM1 (rs6924535, pmeta=1.6×10-5, OR=0.58) and APOA1BP (NAXE) (rs942960, pmeta=1.2×10-5, OR=2.64). Conclusion We identified a novel genetic risk locus, MERTK, associated with SLE-ESRD using the data from two large SLE cohorts. Through DNA methylation analysis and functional annotation, we showed that the risk could be mediated through regulation of gene expression. Our results suggest that variants in the MERTK gene are important for the risk of developing SLE-ESRD and suggest a role for PRDM1 and APOA1BP in proliferative LN.

OBJECTIVE:Copy number variation of the C4 complement components, C4A and C4B, has been associated with systemic inflammatory autoimmune diseases. This study was undertaken to investigate whether C4 copy number variation is connected to the autoimmune repertoire in systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), or myositis. METHODS:Using targeted DNA sequencing, we determined the copy number and genetic variants of C4 in 2,290 well-characterized Scandinavian patients with SLE, primary SS, or myositis and 1,251 healthy controls. RESULTS:A prominent relationship was observed between C4A copy number and the presence of SSA/SSB autoantibodies, which was shared between the 3 diseases. The strongest association was detected in patients with autoantibodies against both SSA and SSB and 0 C4A copies when compared to healthy controls (odds ratio [OR] 18.0 [95% confidence interval (95% CI) 10.2-33.3]), whereas a weaker association was seen in patients without SSA/SSB autoantibodies (OR 3.1 [95% CI 1.7-5.5]). The copy number of C4 correlated positively with C4 plasma levels. Further, a common loss-of-function variant in C4A leading to reduced plasma C4 was more prevalent in SLE patients with a low copy number of C4A. Functionally, we showed that absence of C4A reduced the individuals' capacity to deposit C4b on immune complexes. CONCLUSION:We show that a low C4A copy number is more strongly associated with the autoantibody repertoire than with the clinically defined disease entities. These findings may have implications for understanding the etiopathogenetic mechanisms of systemic inflammatory autoimmune diseases and for patient stratification when taking the genetic profile into account.

Objective To investigate how genetics influence the risk of smoking-related systemic lupus erythematosus (SLE) manifestations. Methods Patients with SLE (n discovery cohort =776, n replication cohort =836) were genotyped using the 200K Immunochip single nucleotide polymorphisms (SNP) Array (Illumina) and a custom array. Sixty SNPs with SLE association (p<5.0×10 −8 ) were analysed. Signal transducer and activator of transcription 4 (STAT4) activation was assessed in in vitro stimulated peripheral blood mononuclear cells from healthy controls (n=45). Results In the discovery cohort, smoking was associated with myocardial infarction (MI) (OR 1.96 (95% CI 1.09 to 3.55)), with a greater effect in patients carrying any rs11889341 STAT4 risk allele (OR 2.72 (95% CI 1.24 to 6.00)) or two risk alleles (OR 8.27 (95% CI 1.48 to 46.27)). Smokers carrying the risk allele also displayed an increased risk of nephritis (OR 1.47 (95% CI 1.06 to 2.03)). In the replication cohort, the high risk of MI in smokers carrying the risk allele and the association between the STAT4 risk allele and nephritis in smokers were confirmed (OR 6.19 (95% CI 1.29 to 29.79) and 1.84 (95% CI 1.05 to 3.29), respectively). The interaction between smoking and the STAT4 risk allele resulted in further increase in the risk of MI (OR 2.14 (95% CI 1.01 to 4.62)) and nephritis (OR 1.53 (95% CI 1.08 to 2.17)), with 54% (MI) and 34% (nephritis) of the risk attributable to the interaction. Levels of interleukin-12-induced phosphorylation of STAT4 in CD8+ T cells were higher in smokers than in non-smokers (mean geometric fluorescence intensity 1063 vs 565, p=0.0063). Lastly, the IL12A rs564799 risk allele displayed association with MI in both cohorts (OR 1.53 (95% CI 1.01 to 2.31) and 2.15 (95% CI 1.08 to 4.26), respectively). Conclusions Smoking in the presence of the STAT4 risk gene variant appears to increase the risk of MI and nephritis in SLE. Our results also highlight the role of the IL12−STAT4 pathway in SLE-cardiovascular morbidity.

ObjectiveIdiopathic inflammatory myopathies (IIMs) are a heterogeneous group of complex autoimmune conditions characterized by inflammation in skeletal muscle and extramuscular compartments, and interferon (IFN) system activation. We undertook this study to examine the contribution of genetic variation to disease susceptibility and to identify novel avenues for research in IIMs.MethodsTargeted DNA sequencing was used to mine coding and potentially regulatory single nucleotide variants from ~1,900 immune‐related genes in a Scandinavian case–control cohort of 454 IIM patients and 1,024 healthy controls. Gene‐based aggregate testing, together with rare variant– and gene‐level enrichment analyses, was implemented to explore genotype–phenotype relations.ResultsGene‐based aggregate tests of all variants, including rare variants, identified IFI35 as a potential genetic risk locus for IIMs, suggesting a genetic signature of type I IFN pathway activation. Functional annotation of the IFI35 locus highlighted a regulatory network linked to the skeletal muscle–specific gene PTGES3L, as a potential candidate for IIM pathogenesis. Aggregate genetic associations with AGER and PSMB8 in the major histocompatibility complex locus were detected in the antisynthetase syndrome subgroup, which also showed a less marked genetic signature of the type I IFN pathway. Enrichment analyses indicated a burden of synonymous and noncoding rare variants in IIM patients, suggesting increased disease predisposition associated with these classes of rare variants.ConclusionOur study suggests the contribution of rare genetic variation to disease susceptibility in IIM and specific patient subgroups, and pinpoints genetic associations consistent with previous findings by gene expression profiling. These features highlight genetic profiles that are potentially relevant to disease pathogenesis.

Objectives Systemic lupus erythematosus (SLE) is an autoimmune disease with extensive heterogeneity in disease presentation between patients, which is likely due to an underlying molecular diversity. Here, we aimed at elucidating the genetic aetiology of SLE from the immunity pathway level to the single variant level, and stratify patients with SLE into distinguishable molecular subgroups, which could inform treatment choices in SLE. Methods We undertook a pathway-centred approach, using sequencing of immunological pathway genes. Altogether 1832 candidate genes were analysed in 958 Swedish patients with SLE and 1026 healthy individuals. Aggregate and single variant association testing was performed, and we generated pathway polygenic risk scores (PRS). Results We identified two main independent pathways involved in SLE susceptibility: T lymphocyte differentiation and innate immunity, characterised by HLA and interferon, respectively. Pathway PRS defined pathways in individual patients, who on average were positive for seven pathways. We found that SLE organ damage was more pronounced in patients positive for the T or B cell receptor signalling pathways. Further, pathway PRS-based clustering allowed stratification of patients into four groups with different risk score profiles. Studying sets of genes with priors for involvement in SLE, we observed an aggregate common variant contribution to SLE at genes previously reported for monogenic SLE as well as at interferonopathy genes. Conclusions Our results show that pathway risk scores have the potential to stratify patients with SLE beyond clinical manifestations into molecular subsets, which may have implications for clinical follow-up and therapy selection.

The genetic background of lupus nephritis (LN) has not been completely elucidated. We performed a case-only study of 2886 SLE patients, including 947 (33%) with LN. Renal biopsies were available from 396 patients. The discovery cohort (Sweden, n = 1091) and replication cohort 1 (US, n = 962) were genotyped on the Immunochip and replication cohort 2 (Denmark/Norway, n = 833) on a custom array. Patients with LN, proliferative nephritis, or LN with end-stage renal disease were compared with SLE without nephritis. Six loci were associated with LN (p < 1 × 10−4, NFKBIA, CACNA1S, ITGA1, BANK1, OR2Y, and ACER3) in the discovery cohort. Variants in BANK1 showed the strongest association with LN in replication cohort 1 (p = 9.5 × 10−4) and proliferative nephritis in a meta-analysis of discovery and replication cohort 1. There was a weak association between BANK1 and LN in replication cohort 2 (p = 0.052), and in the meta-analysis of all three cohorts the association was strengthened (p = 2.2 × 10−7). DNA methylation data in 180 LN patients demonstrated methylation quantitative trait loci (meQTL) effects between a CpG site and BANK1 variants. To conclude, we describe genetic variations in BANK1 associated with LN and evidence for genetic regulation of DNA methylation within the BANK1 locus. This indicates a role for BANK1 in LN pathogenesis.

BACKGROUND:Sex chromosomes are in some species largely undifferentiated (homomorphic) with restricted sex determination regions. Homomorphic but different sex chromosomes are found in the closely related genera Populus and Salix indicating flexible sex determination systems, ideal for studies of processes involved in sex chromosome evolution. We have performed genome-wide association studies of sex and analysed sex chromosomes in a population of 265 wild collected Salix viminalis accessions and studied the sex determining locus.RESULTS:A total of 19,592 markers were used in association analyses using both Fisher's exact tests and a single-marker mixed linear model, which resulted in 48 and 41 sex-associated (SA) markers respectively. Across all 48 SA markers, females were much more often heterozygous than males, which is expected if females were the heterogametic sex. The majority of the SA markers were, based on positions in the S. purpurea genome, located on chromosome 15, previously demonstrated to be the sex chromosome. Interestingly, when mapping the genotyping-by-sequencing sequence tag harbouring the two SA markers with the highest significance to the S. viminalis genomic scaffolds, five regions of very high similarity were found: three on a scaffold that represents a part of chromosome 15, one on a scaffold that represents a part of chromosome 9 and one on a scaffold not anchored to the genome. Based on segregation differences of the alleles at the two marker positions and on differences in PCR amplification between females and males we conclude that females had multiple copies of this DNA fragment (chromosome 9 and 15), whereas males only had one (chromosome 9). We therefore postulate that the female specific sequences have been copied from chromosome 9 and inserted on chromosome 15, subsequently developing into a hemizygous W chromosome linked region.CONCLUSIONS:Our results support that sex determination in S. viminalis is controlled by one locus on chromosome 15. The segregation patterns observed at the SA markers furthermore confirm that S. viminalis females are the heterogametic sex. We also identified a translocation from chromosome 9 to the W chromosome.

Toll-like receptors revisited; a possible role for TLR1 in lupus nephritisSeveral studies in systemic lupus erythematosus (SLE) have shown a possible role of endosomal toll-like receptors (TLRs) in lupus nephritis (LN), but the role of those interacting with ligands in the plasma membrane remains unclear. 1 Herein, we revisit the genetic contribution of TLRs in SLE inspired by a patient with LN who carries a rare TLR1 variant.We analysed coding and regulatory regions of the TLR1-10 genes in 855 patients with SLE (online supplemental data 1).Six variants (rs142003616, rs76600635, rs72493538, rs41305843, rs113706342, rs41311400) within TLR1, one (rs10006364) within TLR2, one (rs79088436) within TLR5 and two (rs55695972, rs117985012) within TLR6 were significantly enriched in LN but only rs142003616 (TLR1) remained significant after Bonferroni correction (p<0.039,online supplemental table 1).To assess its biological significance, we employed in-silico functional annotation.The calculated deleteriousness score, CADD PHRED, for rs142003616 (5.56) points at the variant's potential functional importance.The rare risk allele is predicted to create a strong binding site for the core binding factor (CBF). 2 CBF, also known as runt-related transcription factors (RUNX), are also associated with SLE, psoriasis and rheumatoid arthritis. 3To evaluate rs142003616 functional potential, we lastly performed a reporter assay that demonstrated a significantly higher expression of the reporter with the G allele in Jurkat (p<0.0001) and Daudi cells (p<0.001), and a strong enhancer potential without allelic difference in THP-1 (figure 1).Of interest, despite the higher prevalence of proliferative LN overall (215 of 292 LN, 75%), it was less associated with rs142003616 in comparison to membranous LN (p=0.047,Fisher's exact test).Table 1 summarises the characteristics of patients with LN carrying the minor allele of rs142003616.One of them was a 39-year-old woman (#6 table 1) admitted on July 17, 2023 by guest.

Synthetic Toll‐like receptor (TLR) 7 agonists have been suggested as immune modulators in a range of conditions. In contrast, self‐derived TLR7 activators, such as RNA‐containing immune complexes (RNA‐IC), can contribute to autoimmune diseases due to endogenous immune activation. The exact difference in immune cell response between synthetic and endogenous TLR7 triggers is only partly known. An understanding of these differences could aid in the development of new therapeutic agents and provide insights into autoimmune disease mechanisms. We therefore compared the stimulatory capacity of two TLR7 agonists, RNA‐IC and a synthetic small molecule DSR‐6434, on blood leucocytes, plasmacytoid dendritic cells (pDCs) and B cells from healthy individuals. IFN‐α, IL‐6, IL‐8 and TNF levels were measured by immunoassays, and gene expression in pDCs was analysed by an expression array. DSR‐6434 triggered 20‐fold lower levels of IFN‐α by pDCs, but higher production of IL‐6, IL‐8 and TNF, compared to RNA‐IC. Furthermore, IFN‐α and TNF production were increased with exogenous IFN‐α2b priming, whereas IL‐8 synthesis by B cells was reduced for both stimuli. Cocultivation of pDCs and B cells increased the RNA‐IC‐stimulated IFN‐α and TNF levels, while only IL‐6 production was enhanced in the DSR‐6434‐stimulated cocultures. When comparing pDCs stimulated with RNA‐IC and DSR‐6434, twelve genes were differentially expressed (log 2 fold change >2, adjusted P ‐value <.05). In conclusion, RNA‐IC, which mimics an endogenous TLR7 stimulator, and the synthetic TLR7 agonist DSR‐6434 trigger distinct inflammatory profiles in immune cells. This demonstrates the importance of using relevant stimuli when targeting the TLR7 pathway for therapeutic purposes.

Background Sex chromosomes have evolved independently multiple times in eukaryotes and are therefore considered a prime example of convergent genome evolution. Sex chromosomes are known to emerge after recombination is halted between a homologous pair of chromosomes, and this leads to a range of non-adaptive modifications causing gradual degeneration and gene loss on the sex-limited chromosome. However, the proximal causes of recombination suppression and the pace at which degeneration subsequently occurs remain unclear. Results Here, we use long- and short-read single-molecule sequencing approaches to assemble and annotate a draft genome of the basket willow,Salix viminalis, a species with a female heterogametic system at the earliest stages of sex chromosome emergence. Our single-molecule approach allowed us to phase the emerging Z and W haplotypes in a female, and we detected very low levels of Z/W single-nucleotide divergence in the non-recombining region. Linked-read sequencing of the same female and an additional male (ZZ) revealed the presence of two evolutionary strata supported by both divergence between the Z and W haplotypes and by haplotype phylogenetic trees. Gene order is still largely conserved between the Z and W homologs, although the W-linked region contains genes involved in cytokinin signaling regulation that are not syntenic with the Z homolog. Furthermore, we find no support across multiple lines of evidence for inversions, which have long been assumed to halt recombination between the sex chromosomes. Conclusions Our data suggest that selection against recombination is a more gradual process at the earliest stages of sex chromosome formation than would be expected from an inversion and may result instead from the accumulation of transposable elements. Our results present a cohesive understanding of the earliest genomic consequences of recombination suppression as well as valuable insights into the initial stages of sex chromosome formation and regulation of sex differentiation.

Interferons (IFNs) are cytokines that are central to the host defence against viruses and other microorganisms. If not properly regulated, IFNs may contribute to the pathogenesis of inflammatory autoimmune, or infectious diseases. To identify genetic polymorphisms regulating the IFN system we performed an unbiased genome-wide protein-quantitative trait loci (pQTL) mapping of cell-type specific type I and type II IFN receptor levels and their responses in immune cells from 303 healthy individuals. Seven genome-wide significant (p < 5.0E-8) pQTLs were identified. Two independent SNPs that tagged the multiple sclerosis (MS)-protective HLA class I alleles A*02/A*68 and B*44, respectively, were associated with increased levels of IFNAR2 in B and T cells, with the most prominent effect in IgD-CD27+ memory B cells. The increased IFNAR2 levels in B cells were replicated in cells from an independent set of healthy individuals and in MS patients. Despite increased IFNAR2 levels, B and T cells carrying the MS-protective alleles displayed a reduced response to type I IFN stimulation. Expression and methylation-QTL analysis demonstrated increased mRNA expression of the pseudogene HLA-J in B cells carrying the MS-protective class I alleles, possibly driven via methylation-dependent transcriptional regulation. Together these data suggest that the MS-protective effects of HLA class I alleles are unrelated to their antigen-presenting function, and propose a previously unappreciated function of type I IFN signalling in B and T cells in MS immune-pathogenesis.

Background Interferon (IFN)-α and IFN-γ are important cytokines in the pathogenesis of systemic lupus erythematosus (SLE), and several of the genetic associations with SLE are found in genes that are fundamental for the IFN response (e.g. TYK2, STAT4, IRF5). This study aimed to define the genetic regulation of the IFN system, and to link disease-associated SNPs to alterations in the IFN system. Methods Peripheral blood mononuclear cells from 303 healthy individuals were stimulated with IFN-α or IFN-γ (figure 1). Basal levels of IFN-receptors (IFNAR2 and IFNGR1) and IFN-induced phosphorylation of STAT1 and STAT4, expression of CXCL9, CXCL10, HLA-ABC and HLA-DRPQ was determined in 6 cell subsets using flow cytometry. Each read-out was mapped as a pQTL using 3.4 million SNPs with a minor allele frequency ≥5% (Illumina Global Screening Array with subsequent genome-wide imputation) in an additive model correcting for covariates. pQTLs were probed for overlap with GWAS SLE-associated SNPs. Results We identified 8 genome-wide significant pQTLs (p A trans-pQTL for IFNGR1 level in monocytes (rs1801274 in FCGR2A; p=3e-23) and a suggestive significant pQTL (p Conclusions We demonstrate a cell-type and stimuli-specific genetic regulation of the IFN system. Two SNPs previously linked to SLE were associated with alterations in the IFN-γ-receptor expression or response. Further studies to determine the underlying mechanisms of these associations are ongoing.

Objective Patients with systemic lupus erythematosus (SLE) have an ongoing interferon (IFN) production due to an activation of plasmacytoid dendritic cells (pDCs), which can be triggered to type I IFN synthesis by RNA containing immune complexes (RNA-IC). Considering emerging data suggesting a role of type III IFN in the SLE disease process, we asked if RNA-IC can induce type III IFN production in pDC and how this production can be regulated. Methods Peripheral blood mononuclear cells (PBMCs) or immune cell subsets were isolated from healthy blood donors or SLE patients and stimulated with IC containing U1 snRNP and SLE-IgG (RNA-IC). Hydroxychloroquine (HCQ) and an interleukin receptor 1-associated kinase 4 inhibitor (IRAK4i) were added to cell cultures. Cytokine mRNA levels were determined with a microarray and protein levels with immunoassays. Single-cell RNA sequencing of pDCs using ddSEQ technology was performed. Results Type III IFN mRNA and protein was induced in RNA-IC-stimulated pDC-NK and pDC-B cell co-cultures. A subset of activated pDCs (3%) expressed both type III and type I IFN mRNA. IFN-λ2, IFN-α2b, interleukin (IL)-3, IL-6, or granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced IFN-λ1/3 production 2–5-fold. HCQ and an IRAK4i blocked the RNA-IC-triggered IFN-λ1/3 production ( p < 0.01). IFN-α2b and GM-CSF increased the proportion of SLE patients producing IFN-λ1/3 in response to RNA-IC from 11 to 33%. Conclusions Type III IFN production is triggered by RNA-IC in pDCs in a TLR-MyD88-dependent manner, enhanced by NK and B cells as well as several pro-inflammatory cytokines. These results support a contributing role for both type I and type III IFNs in SLE, which needs to be considered when targeting the IFN system in this disease.

The understanding of miRNA target interactions is still limited due to conflicting data and the fact that high-quality validation of targets is a time-consuming process. Faster methods like high-throughput screens and bioinformatics predictions are employed but suffer from several problems. One of these, namely the potential occurrence of downstream (i.e. secondary) effects in high-throughput screens has been only little discussed so far. However, such effects limit usage for both the identification of interactions and for the training of bioinformatics tools. In order to analyse this problem more closely, we performed time-dependent microarray screening experiments overexpressing human miR-517a-3p, and, together with published time-dependent datasets of human miR-17-5p, miR-135b and miR-124 overexpression, we analysed the dynamics of deregulated genes. We show that the number of deregulated targets increases over time, whereas seed sequence content and performance of several miRNA target prediction algorithms actually decrease over time. Bioinformatics recognition success of validated miR-17 targets was comparable to that of data gained only 12 h post-transfection. We therefore argue that the timing of microarray experiments is of critical importance for detecting direct targets with high confidence and for the usability of these data for the training of bioinformatics prediction tools.

Background Patients with SLE have increased morbidity and mortality due to cardiovascular disease. Here, we construct and validate a polygenic risk score (PRS) for myocardial infarction (MI) in SLE. Methods Patients with SLE (European decent, ≥4 ACR-criteria) were genotyped using a 200K Immunochip SNP array (discovery cohort, Sweden, n=776) and custom MassARRAY assays (replication cohort, Norway/Denmark, n=890). In the discovery cohort, 57 SNPs with previously established association with SLE development (p<5.0×10-8) were investigated for associations with MI using a cox regression model. Significant SNPs were included in a PRS, weighted by their ORs for MI development. The PRS was subsequently validated in the replication cohort. Results Four SLE-risk genes were found to be associated with a decreased time until the first MI; PTPN22 (OR 1.61, p=0.041), NCF2 (OR 2.47, p=2.1×10-3), STAT4 (OR 1.66, p=5.2×10-3) and IL12A (OR 1.45, p=0.047) and were included in a PRS. The PRS was associated with a higher cumulative prevalence of MI in both the discovery cohort (p=1.1×10-5, fig 1A) and replication cohort (p=7.7×10-3, fig 1B). Exploring the PRS further in the replication cohort, patients in the high, compared to the low, PRS-quartile were more often male (p=1.3×10-3), and displayed higher prevalence of the ACR-1982 nephritis and immunological criteria (p=4.1×10-4 and p=0.036) (fig1C). Analyzing combinations of the identified SNPs, we found the prevalence of MI to be further increased in patients homozygous for both NCF2+STAT4 (pdiscovery=1.6×10-3, preplication=0.015) or STAT4+IL12A (pdiscovery=3.0×10-5, preplication=0.036) (fig1D). Conclusion A high polygenic risk score for MI in SLE is associated with an increased prevalence of myocardial infarction. If confirmed in prospective studies, our results suggest that genetic profiling may be useful for predicting MI in patients with SLE.