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1.  Association of STAT4 Polymorphism with Severe Renal Insufficiency in Lupus Nephritis 
PLoS ONE  2013;8(12):e84450.
Lupus nephritis is a cause of significant morbidity in systemic lupus erythematosus (SLE) and its genetic background has not been completely clarified. The aim of this investigation was to analyze single nucleotide polymorphisms (SNPs) for association with lupus nephritis, its severe form proliferative nephritis and renal outcome, in two Swedish cohorts. Cohort I (n = 567 SLE cases, n =  512 controls) was previously genotyped for 5676 SNPs and cohort II (n = 145 SLE cases, n = 619 controls) was genotyped for SNPs in STAT4, IRF5, TNIP1 and BLK.
Case-control and case-only association analyses for patients with lupus nephritis, proliferative nephritis and severe renal insufficiency were performed. In the case-control analysis of cohort I, four highly linked SNPs in STAT4 were associated with lupus nephritis with genome wide significance with p = 3.7×10−9, OR 2.20 for the best SNP rs11889341. Strong signals of association between IRF5 and an HLA-DR3 SNP marker were also detected in the lupus nephritis case versus healthy control analysis (p <0.0001). An additional six genes showed an association with lupus nephritis with p <0.001 (PMS2, TNIP1, CARD11, ITGAM, BLK and IRAK1). In the case-only meta-analysis of the two cohorts, the STAT4 SNP rs7582694 was associated with severe renal insufficiency with p  = 1.6×10−3 and OR 2.22. We conclude that genetic variations in STAT4 predispose to lupus nephritis and a worse outcome with severe renal insufficiency.
doi:10.1371/journal.pone.0084450
PMCID: PMC3873995  PMID: 24386384
2.  Epitopes of Microbial and Human Heat Shock Protein 60 and Their Recognition in Myalgic Encephalomyelitis 
PLoS ONE  2013;8(11):e81155.
Myalgic encephalomyelitis (ME, also called Chronic Fatigue Syndrome), a common disease with chronic fatigability, cognitive dysfunction and myalgia of unknown etiology, often starts with an infection. The chaperonin human heat shock protein 60 (HSP60) occurs in mitochondria and in bacteria, is highly conserved, antigenic and a major autoantigen. The anti-HSP60 humoral (IgG and IgM) immune response was studied in 69 ME patients and 76 blood donors (BD) (the Training set) with recombinant human and E coli HSP60, and 136 30-mer overlapping and targeted peptides from HSP60 of humans, Chlamydia, Mycoplasma and 26 other species in a multiplex suspension array. Peptides from HSP60 helix I had a chaperonin-like activity, but these and other HSP60 peptides also bound IgG and IgM with an ME preference, theoretically indicating a competition between HSP60 function and antibody binding. A HSP60-based panel of 25 antigens was selected. When evaluated with 61 other ME and 399 non-ME samples (331 BD, 20 Multiple Sclerosis and 48 Systemic Lupus Erythematosus patients), a peptide from Chlamydia pneumoniae HSP60 detected IgM in 15 of 61 (24%) of ME, and in 1 of 399 non-ME at a high cutoff (p<0.0001). IgM to specific cross-reactive epitopes of human and microbial HSP60 occurs in a subset of ME, compatible with infection-induced autoimmunity.
doi:10.1371/journal.pone.0081155
PMCID: PMC3842916  PMID: 24312270
3.  Genes identified in Asian SLE GWASs are also associated with SLE in Caucasian populations 
Recent genome-wide association studies (GWASs) conducted in Asian populations have identified novel risk loci for systemic lupus erythematosus (SLE). Here, we genotyped 10 single-nucleotide polymorphisms (SNPs) in eight such loci and investigated their disease associations in three independent Caucasian SLE case–control cohorts recruited from Sweden, Finland and the United States. The disease associations of the SNPs in ETS1, IKZF1, LRRC18-WDFY4, RASGRP3, SLC15A4, TNIP1 and 16p11.2 were replicated, whereas no solid evidence of association was observed for the 7q11.23 locus in the Caucasian cohorts. SLC15A4 was significantly associated with renal involvement in SLE. The association of TNIP1 was more pronounced in SLE patients with renal and immunological disorder, which is corroborated by two previous studies in Asian cohorts. The effects of all the associated SNPs, either conferring risk for or being protective against SLE, were in the same direction in Caucasians and Asians. The magnitudes of the allelic effects for most of the SNPs were also comparable across different ethnic groups. On the contrary, remarkable differences in allele frequencies between Caucasian and Asian populations were observed for all associated SNPs. In conclusion, most of the novel SLE risk loci identified by GWASs in Asian populations were also associated with SLE in Caucasian populations. We observed both similarities and differences with respect to the effect sizes and risk allele frequencies across ethnicities.
doi:10.1038/ejhg.2012.277
PMCID: PMC3746253  PMID: 23249952
systemic lupus erythematosus; genetic-association study; Asian; Caucasian
4.  IRF5 activation in monocytes of SLE patients is triggered by circulating autoantigens independent of type I IFN 
Arthritis and Rheumatism  2012;64(3):788-798.
Objective
Genetic variants of interferon regulatory factor 5 (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF5 regulates the expression of proinflammatory cytokines and type I interferons (IFN) believed to be involved in SLE pathogenesis. The aim of this study was to determine the activation status of IRF5 by assessing its nuclear localization in immune cells of SLE patients and healthy donors, and to identify SLE triggers of IRF5 activation.
Methods
IRF5 nuclear localization in subpopulations of peripheral blood mononuclear cells (PBMC) from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. IRF5 activation and function were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream and cytokine expression by Q-PCR and ELISA.
Results
IRF5 was activated in a cell type-specific manner; monocytes of SLE patients had constitutively elevated levels of nuclear IRF5 compared to NK and T cells. SLE serum was identified as a trigger for IRF5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens comprised of apoptotic/necrotic material triggered IRF5 nuclear accumulation in monocytes. Production of cytokines IFNα, TNFα and IL6 in monocytes stimulated with SLE serum or autoantigens was distinct yet correlated with the kinetics of IRF5 nuclear localization.
Conclusion
This study provides the first formal proof that IRF5 activation is altered in monocytes of SLE patients that is in part contributed by the SLE blood environment.
doi:10.1002/art.33395
PMCID: PMC3288585  PMID: 21968701
5.  RNA-Seq for Enrichment and Analysis of IRF5 Transcript Expression in SLE 
PLoS ONE  2013;8(1):e54487.
Polymorphisms in the interferon regulatory factor 5 (IRF5) gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE). IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s), it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1) SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2) an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3) an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.
doi:10.1371/journal.pone.0054487
PMCID: PMC3548774  PMID: 23349905
6.  Mutations in genes encoding complement inhibitors CD46 and CFH affect the age at nephritis onset in patients with systemic lupus erythematosus 
Arthritis Research & Therapy  2011;13(6):R206.
Introduction
Inherited deficiencies of several complement components strongly predispose to systemic lupus erythematosus (SLE) while deficiencies of complement inhibitors are found in kidney diseases such as atypical hemolytic uremic syndrome (aHUS).
Methods
The exons of complement inhibitor genes CD46 and CFH (factor H) were fully sequenced using the Sanger method in SLE patients with nephritis originating from two cohorts from southern and mid Sweden (n = 196). All identified mutations and polymorphisms were then analyzed in SLE patients without nephritis (n = 326) and in healthy controls (n = 523).
Results
We found nonsynonymous, heterozygous mutations in CFH in 6.1% patients with nephritis, in comparison with 4.0% and 5.4% in patients without nephritis and controls, respectively. No associations of SLE or nephritis with common variants in CFH (V62I/Y402H/E936D) were found. Furthermore, we found two nonsynonymous heterozygous mutations in CD46 in SLE patients but not in controls. The A353V polymorphism, known to affect function of CD46, was found in 6.6% of nephritis patients versus 4.9% and 6.1% of the non-nephritis SLE patients and controls. The presence of mutations in CD46 and CFH did not predispose to SLE or nephritis but was associated with earlier onset of nephritis. Furthermore, we found weak indications that there is one protective and one risk haplotype predisposing to nephritis composed of several polymorphisms in noncoding regions of CD46, which were previously implicated in aHUS.
Conclusions
SLE nephritis is not associated with frequent mutations in CFH and CD46 as found in aHUS but these may be modifying factors causing earlier onset of nephritis.
doi:10.1186/ar3539
PMCID: PMC3334659  PMID: 22171659
7.  Genetic variants and disease-associated factors contribute to enhanced IRF-5 expression in blood cells of systemic lupus erythematosus patients 
Arthritis and rheumatism  2010;62(2):562-573.
Objective
Genetic variants of the interferon (IFN) regulatory factor 5 (IRF5) gene are associated with systemic lupus erythematosus (SLE) susceptibility. The contribution of these variants to IRF-5 expression in primary blood cells of SLE patients has not been addressed, nor has the role of type I IFN. The aim of this study was to determine the association between increased IRF-5 expression and the IRF5 risk haplotype in SLE patients.
Methods
IRF-5 transcript and protein levels in 44 Swedish patients with SLE and 16 healthy controls were measured by quantitative real-time PCR, minigene assay, and flow cytometry. The rs2004640, rs10954213, rs10488631 and the CGGGG indel were genotyped in these patients. Genotypes of these polymorphisms defined a common risk and protective haplotype.
Results
IRF-5 expression and alternative splicing were significantly upregulated in SLE patients versus healthy donors. Enhanced transcript and protein levels were associated with the risk haplotype of IRF5; rs10488631 gave the only significant independent association that correlated with increased transcription from non-coding exon 1C. Minigene experiments demonstrated an important role for rs2004640 and the CGGGG indel, along with type I IFNs in regulating IRF-5 expression.
Conclusions
This study provides the first formal proof that IRF-5 expression and alternative splicing are significantly upregulated in primary blood cells of SLE patients. The risk haplotype is associated with enhanced IRF-5 transcript and protein expression in SLE patients.
doi:10.1002/art.27223
PMCID: PMC3213692  PMID: 20112383
8.  The type I interferon system in the etiopathogenesis of autoimmune diseases 
Upsala Journal of Medical Sciences  2011;116(4):227-237.
Many patients with systemic autoimmune diseases have signs of a continuous production of type I interferon (IFN) and display an increased expression of IFN-α-regulated genes. The reason for the on-going IFN-α synthesis in these patients seems to be an activation of plasmacytoid dendritic cells (pDCs) by immune complexes (ICs), consisting of autoantibodies in combination with DNA or RNA-containing autoantigens. Such interferogenic ICs are internalized via the FcγRIIa expressed on pDCs, reach the endosome, and stimulate Toll-like receptor (TLR)-7 or -9, which subsequently leads to IFN-α gene transcription. Variants of genes involved in both the IFN-α synthesis and response have been linked to an increased risk to develop systemic lupus erythematosus (SLE) and other autoimmune diseases. Among these autoimmunity risk genes are IFN regulatory factor 5 (IRF5), which is involved in TLR signaling, and the signal transducer and activator of transcription 4 (STAT4) that interacts with the type I IFN receptor. Several other gene variants in the IFN signaling pathway also confer an increased risk to develop an autoimmune disease. The observations that IFN-α therapy can induce autoimmunity and that many autoimmune conditions have an on-going type I IFN production suggest that the type I IFN system has a pivotal role in the etiopathogenesis of these diseases. Possible mechanisms behind the dysregulated type IFNsystem in autoimmune diseases and how the IFN-α produced can contribute to the development of an autoimmune process will be reviewed.
doi:10.3109/03009734.2011.624649
PMCID: PMC3207297  PMID: 22066971
Autoimmune; immune complex; interferon; plasmacytoid dendritic cell; systemic lupus erythematosus
9.  Association of NCF2, IKZF1, IRF8, IFIH1, and TYK2 with Systemic Lupus Erythematosus 
PLoS Genetics  2011;7(10):e1002341.
Systemic lupus erythematosus (SLE) is a complex trait characterised by the production of a range of auto-antibodies and a diverse set of clinical phenotypes. Currently, ∼8% of the genetic contribution to SLE in Europeans is known, following publication of several moderate-sized genome-wide (GW) association studies, which identified loci with a strong effect (OR>1.3). In order to identify additional genes contributing to SLE susceptibility, we conducted a replication study in a UK dataset (870 cases, 5,551 controls) of 23 variants that showed moderate-risk for lupus in previous studies. Association analysis in the UK dataset and subsequent meta-analysis with the published data identified five SLE susceptibility genes reaching genome-wide levels of significance (Pcomb<5×10−8): NCF2 (Pcomb = 2.87×10−11), IKZF1 (Pcomb = 2.33×10−9), IRF8 (Pcomb = 1.24×10−8), IFIH1 (Pcomb = 1.63×10−8), and TYK2 (Pcomb = 3.88×10−8). Each of the five new loci identified here can be mapped into interferon signalling pathways, which are known to play a key role in the pathogenesis of SLE. These results increase the number of established susceptibility genes for lupus to ∼30 and validate the importance of using large datasets to confirm associations of loci which moderately increase the risk for disease.
Author Summary
Genome-wide association studies have revolutionised our ability to identify common susceptibility alleles for systemic lupus erythematosus (SLE). In complex diseases such as SLE, where many different genes make a modest contribution to disease susceptibility, it is necessary to perform large-scale association studies to combine results from several datasets, to have sufficient power to identify highly significant novel loci (P<5×10−8). Using a large SLE collection of 870 UK SLE cases and 5,551 UK unaffected individuals, we firstly replicated ten moderate-risk alleles (P<0.05) from a US–Swedish study of 3,273 SLE cases and 12,188 healthy controls. Combining our results with the US-Swedish data identified five new loci, which crossed the level for genome-wide significance: NCF2 (neutrophil cytosolic factor 2), IKZF1 (Ikaros family zinc-finger 1), IRF8 (interferon regulatory factor 8), IFIH1 (interferon-induced helicase C domain-containing protein 1), and TYK2 (tyrosine kinase 2). Each of these five genes regulates a different aspect of the immune response and contributes to the production of type-I and type-II interferons. Although further studies will be required to identify the causal alleles within these loci, the confirmation of five new susceptibility genes for lupus makes a significant step forward in our understanding of the genetic contribution to SLE.
doi:10.1371/journal.pgen.1002341
PMCID: PMC3203198  PMID: 22046141
10.  A candidate gene study of the type I interferon pathway implicates IKBKE and IL8 as risk loci for SLE 
Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease in which the type I interferon pathway has a crucial role. We have previously shown that three genes in this pathway, IRF5, TYK2 and STAT4, are strongly associated with risk for SLE. Here, we investigated 78 genes involved in the type I interferon pathway to identify additional SLE susceptibility loci. First, we genotyped 896 single-nucleotide polymorphisms in these 78 genes and 14 other candidate genes in 482 Swedish SLE patients and 536 controls. Genes with P<0.01 in the initial screen were then followed up in 344 additional Swedish patients and 1299 controls. SNPs in the IKBKE, TANK, STAT1, IL8 and TRAF6 genes gave nominal signals of association with SLE in this extended Swedish cohort. To replicate these findings we extracted data from a genomewide association study on SLE performed in a US cohort. Combined analysis of the Swedish and US data, comprising a total of 2136 cases and 9694 controls, implicates IKBKE and IL8 as SLE susceptibility loci (Pmeta=0.00010 and Pmeta=0.00040, respectively). STAT1 was also associated with SLE in this cohort (Pmeta=3.3 × 10−5), but this association signal appears to be dependent of that previously reported for the neighbouring STAT4 gene. Our study suggests additional genes from the type I interferon system in SLE, and highlights genes in this pathway for further functional analysis.
doi:10.1038/ejhg.2010.197
PMCID: PMC3060320  PMID: 21179067
systemic lupus erythematosus; type I interferon system; candidate gene study; single nucleotide polymorphism; IKBKE; IL8
11.  A large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus 
Nature genetics  2009;41(11):1228-1233.
Genome-wide association studies have recently identified at least 15 susceptibility loci for systemic lupus erythematosus (SLE). To confirm additional risk loci, we selected SNPs from 2,466 regions that showed nominal evidence of association to SLE (P < 0.05) in a genome-wide study and genotyped them in an independent sample of 1,963 cases and 4,329 controls. This replication effort identified five new SLE susceptibility loci (P < 5 × 10−8): TNIP1 (odds ratio (OR) = 1.27), PRDM1 (OR = 1.20), JAZF1 (OR = 1.20), UHRF1BP1 (OR = 1.17) and IL10 (OR = 1.19). We identified 21 additional candidate loci with P ≤ 1 × 10−5. A candidate screen of alleles previously associated with other autoimmune diseases suggested five loci (P < 1 × 10−3) that may contribute to SLE: IFIH1, CFB, CLEC16A, IL12B and SH2B3. These results expand the number of confirmed and candidate SLE susceptibility loci and implicate several key immunologic pathways in SLE pathogenesis.
doi:10.1038/ng.468
PMCID: PMC2925843  PMID: 19838195
12.  Potential role of IFNα in adult lupus 
Arthritis Research & Therapy  2010;12(Suppl 1):S3.
Patients with lupus have a continuous production of IFNα and display an increased expression of IFNα-regulated genes. The reason for the ongoing IFNα synthesis in these patients seems to be an activation of plasmacytoid dendritic cells (pDCs) by immune complexes (ICs), consisting of autoantibodies in combination with DNA-containing or RNA-containing autoantigens. The mechanisms behind the activation of pDCs by such ICs have to some extent been elucidated during the last years. Thus, interferogenic ICs are internalized via the FcγRIIa expressed on pDCs, reach the endosomes and stimulate Toll-like receptor (TLR) 7 or 9, which subsequently leads to IFNα gene transcription. Variants of genes involved in both the IFNα synthesis and response have been linked to an increased risk to develop lupus. Among these genes are interferon regulatory factor 5 (IRF5), which is involved in TLR signaling, and the signal transducer and activator of transcription 4 (STAT4) that interacts with the type I interferon receptor. Produced IFNα may at least partially be responsible for several of the observed alterations in the immune system of lupus patients and contribute to the autoimmune disease process, which will be discussed in the present review. How produced IFNα can contribute to some clinical manifestations will briefly be described, as well as the possible consequences of this knowledge in clinical practice for disease monitoring and therapy
doi:10.1186/ar2884
PMCID: PMC2991776  PMID: 20392290
13.  A risk haplotype of STAT4 for systemic lupus erythematosus is over-expressed, correlates with anti-dsDNA and shows additive effects with two risk alleles of IRF5 
Human Molecular Genetics  2008;17(18):2868-2876.
Systemic lupus erythematosus (SLE) is the prototype autoimmune disease where genes regulated by type I interferon (IFN) are over-expressed and contribute to the disease pathogenesis. Because signal transducer and activator of transcription 4 (STAT4) plays a key role in the type I IFN receptor signaling, we performed a candidate gene study of a comprehensive set of single nucleotide polymorphism (SNPs) in STAT4 in Swedish patients with SLE. We found that 10 out of 53 analyzed SNPs in STAT4 were associated with SLE, with the strongest signal of association (P = 7.1 × 10−8) for two perfectly linked SNPs rs10181656 and rs7582694. The risk alleles of these 10 SNPs form a common risk haplotype for SLE (P = 1.7 × 10−5). According to conditional logistic regression analysis the SNP rs10181656 or rs7582694 accounts for all of the observed association signal. By quantitative analysis of the allelic expression of STAT4 we found that the risk allele of STAT4 was over-expressed in primary human cells of mesenchymal origin, but not in B-cells, and that the risk allele of STAT4 was over-expressed (P = 8.4 × 10−5) in cells carrying the risk haplotype for SLE compared with cells with a non-risk haplotype. The risk allele of the SNP rs7582694 in STAT4 correlated to production of anti-dsDNA (double-stranded DNA) antibodies and displayed a multiplicatively increased, 1.82-fold risk of SLE with two independent risk alleles of the IRF5 (interferon regulatory factor 5) gene.
doi:10.1093/hmg/ddn184
PMCID: PMC2525501  PMID: 18579578
14.  Systemic lupus erythematosus and the type I interferon system 
Arthritis Research & Therapy  2003;5(2):68-75.
Patients with systemic lupus erythematosus (SLE) have ongoing interferon-α (IFN-α) production and serum IFN-α levels are correlated with both disease activity and severity. Recent studies of patients with SLE have demonstrated the presence of endogenous IFN-α inducers in such individuals, consisting of small immune complexes (ICs) containing IgG and DNA. These ICs act specifically on natural IFN-α-producing cells (NIPCs), often termed plasmacytoid dendritic cells (PDCs). Given the fact that the NIPC/PDC has a key role in both the innate and adaptive immune response, as well as the many immunoregulatory effects of IFN-α, these observations might be important for the understanding of the etiopathogenesis of SLE. In this review we briefly describe the biology of the type I IFN system, with emphasis on inducers, producing cells (especially NIPCs/PDCs), IFN-α actions and target immune cells that might be relevant in SLE. On the basis of this information and results from studies in SLE patients, we propose a hypothesis that explains how NIPCs/PDCs become activated and have a pivotal etiopathogenic role in SLE. This hypothesis also indicates new therapeutic targets in this autoimmune disease.
PMCID: PMC165035  PMID: 12718746
dendritic cells; interferon-α; lupus; systemic lupus erythematosus; type I interferon
15.  Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system 
Nucleic Acids Research  2002;30(14):e70.
We selected 125 candidate single nucleotide polymorphisms (SNPs) in genes belonging to the human type 1 interferon (IFN) gene family and the genes coding for proteins in the main type 1 IFN signalling pathway by screening databases and by in silico comparison of DNA sequences. Using quantitative analysis of pooled DNA samples by solid-phase mini-sequencing, we found that only 20% of the candidate SNPs were polymorphic in the Finnish and Swedish populations. To allow more effective validation of candidate SNPs, we developed a four-colour microarray-based mini-sequencing assay for multiplex, quantitative allele frequency determination in pooled DNA samples. We used cyclic mini-sequencing reactions with primers carrying 5′-tag sequences, followed by capture of the products on microarrays by hybridisation to complementary tag oligonucleotides. Standard curves prepared from mixtures of known amounts of SNP alleles demonstrate the applicability of the system to quantitative analysis, and showed that for about half of the tested SNPs the limit of detection for the minority allele was below 5%. The microarray-based genotyping system established here is universally applicable for genotyping and quantification of any SNP, and the validated system for SNPs in type 1 IFN-related genes should find many applications in genetic studies of this important immunoregulatory pathway.
PMCID: PMC135771  PMID: 12136118

Results 1-16 (16)