Objective

Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are related chronic autoimmune diseases of complex aetiology in which the interferon (IFN) pathway plays a key role. Recent studies have reported an association between IRF7 and SLE which confers a risk to autoantibody production. A study was undertaken to investigate whether the IRF7 genomic region is also involved in susceptibility to SSc and the main clinical features.

Methods

Two case-control sets of Caucasian origin from the USA and Spain, comprising a total of 2316 cases of SSc and 2347 healthy controls, were included in the study. Five single nucleotide polymorphisms (SNPs) in the PHRF1-IRF7-CDHR5 locus were genotyped using TaqMan allelic discrimination technology. A meta-analysis was performed to test the overall effect of these genetic variants on SSc.

Results

Four out of five analysed SNPs were Significantly associated with the presence of anticentromere autoantibodies (ACA) in the patients with SSc in the combined analysis (rs1131665: pFDR=6.14 × 10−4, OR=0.78; rs4963128: pFDR=6.14 × 10−4, OR=0.79; rs702966: pFDR=3.83 × 10−3, OR=0.82; and rs2246614: pFDR=3.83 × 10−3, OR=0.83). Significant p values were also obtained when the disease was tested globally; however, the statistical significance was lost when the ACA-positive patients were excluded from the study, suggesting that these associations rely on ACA positivity. Conditional logistic regression and allelic combination analyses suggested that the functional IRF7 SNP rs1131665 is the most likely causal variant.

Conclusions

The results show that variation in the IRF7 genomic region is associated with the presence of ACA in patients with SSc, supporting other evidence that this locus represents a common risk factor for autoantibody production in autoimmune diseases.

Objective

To observe the proportion of peripheral T follicular helper (Tfh) cells in patients with systemic lupus erythematosus (SLE) and to assess the role of steroids on Tfh cells from SLE patients.

Methods

Peripheral blood mononuclear cells (PBMCs) from 42 SLE patients and 22 matched healthy subjects were collected to assess proportions of circulating CXCR5+PD1+/CD4+ T cells (Tfh), CD4+CCR6+ T cells (Th17-like) and CD19+CD138+ plasma cells by flow cytometry. 8 of the patients had their blood redrawn within one week after receiving methylprednisolone pulse treatment. Disease activity was evaluated by SLE disease activity index. To test the effect of IL-21 and corticosteroids on Tfh cells in vitro, PBMCs harvested from another 15 SLE patients were cultured with medium, IL-21, or IL-21+ dexamethasone for 24 hours and 72 hours. PBMCs from an independent 23 SLE patients were cultured with different concentrations of dexamethasone for 24 hours.

Results

Compared to normal controls, percentages of circulating Tfh cells, but not Th17 cells, were elevated in SLE patients and correlated with disease activity. Proportions of Tfh cells in SLE patients were positively correlated with those of plasma cells and serum levels of antinuclear antibodies. After methylprednisolone pulse treatment, both percentages and absolute numbers of circulating Tfh cells were significantly decreased. In vitro cultures showed an increase of Tfh cell proportion after IL-21 stimulation that was totally abolished by the addition of dexamethasone. Both 0.5 and 1 µM dexamethasone decreased Tfh cells dose dependently (overall p = 0.013).

Conclusions

We demonstrated that elevated circulating Tfh cell proportions in SLE patients correlated with their disease activities, and circulating levels of plasma cells and ANA. Corticosteroids treatment down-regulated aberrant circulating Tfh cell proportions both in vivo and in vitro, making Tfh cells a new treatment target for SLE patients.

Objective

The genetic association of interferon regulatory factor 5 (IRF5) with systemic lupus erythematosus (SLE) susceptibility has been convincingly established. To gain understanding of the effect of IRF5 variation in individuals without SLE, a study was undertaken to examine whether such genetic variation predisposes to activation of the interferon α (IFNα) pathway.

Methods

Using a computer simulated approach, 14 single nucleotide polymorphisms (SNPs) and haplotypes of IRF5 were tested for association with mRNA expression levels of IRF5, IFNα and IFN-inducible genes and chemokines in lymphoblastoid cell lines (LCLs) from individuals of European (CEU), Han Chinese (CHB), Japanese (JPT) and Yoruba Nigerian (YRI) backgrounds. IFN-inducible gene expression was assessed in LCLs from children with SLE in the presence and absence of IFNα stimulation.

Results

The major alleles of IRF5 rs13242262 and rs2280714 were associated with increased IRF5 mRNA expression levels in the CEU, CHB+JPT and YRI samples. The minor allele of IRF5 rs10488631 was associated with increased IRF5, IFNα and IFN-inducible chemokine expression in CEU (pc=0.0005, 0.01 and 0.04, respectively). A haplotype containing these risk alleles of rs13242262, rs10488631 and rs2280714 was associated with increased IRF5, IFNα and IFN-inducible chemokine expression in CEU LCLs. In vitro studies showed specific activation of IFN-inducible genes in LCLs by IFNα.

Conclusions

SNPs of IRF5 in healthy individuals of a number of ethnic groups were associated with increased mRNA expression of IRF5. In European-derived individuals, an IRF5 haplotype was associated with increased IRF5, IFNα and IFN-inducible chemokine expression. Identifying individuals genetically predisposed to increased IFN-inducible gene and chemokine expression may allow early detection of risk for SLE.

Our understanding of the genetic basis of systemic lupus erythematosus (SLE) has been rapidly advanced using large-scale, case–control, candidate gene studies as well as genome-wide association studies during the past 3 years. These techniques have identified more than 30 robust genetic associations with SLE including genetic variants of HLA and Fcγ receptor genes, IRF5, STAT4, PTPN22, TNFAIP3, BLK, BANK1, TNFSF4 and ITGAM. Most SLE-associated gene products participate in key pathogenic pathways, including Toll-like receptor and type I interferon signaling pathways, immune regulation pathways and those that control the clearance of immune complexes. Disease-associated loci that have not yet been demonstrated to have important functions in the immune system might provide new clues to the underlying molecular mechanisms that contribute to the pathogenesis or progression of SLE. Of note, genetic risk factors that are shared between SLE and other immune-related diseases highlight common pathways in the pathophysiology of these diseases, and might provide innovative molecular targets for therapeutic interventions.

Systemic lupus erythematosus (SLE) is more common among women than men with a ratio of about 10 to 1. We undertook this study to describe familial male SLE within a large cohort of familial SLE. SLE families (two or more patients) were obtained from the Lupus Multiplex Registry and Repository. Genomic DNA and blood samples were obtained using standard methods. Autoantibodies were determined by multiple methods. Medical records were abstracted for SLE clinical data. Fluorescent in situ hybridization (FISH) was performed with X and Y centromere specific probes, and a probe specific for the toll-like receptor 7 gene on the X chromosome. Among 523 SLE families, we found five families in which all the SLE patients were male. FISH found no yaa gene equivalent in these families. SLE-unaffected primary female relatives from the five families with only-male SLE patients had a statistically increased rate of positive ANA compared to SLE-unaffected female relatives in other families. White men with SLE were 5 times more likely to have an offspring with SLE than were White women with SLE but there was no difference in this likelihood among Black men. These data suggest genetic susceptibility factors that act only in men.

Systemic Lupus Erythematosus (SLE) is a prototypic autoimmune disorder with a complex pathogenesis in which genetic, hormonal and environmental factors play a role. Rare mutations in the TREX1 gene, the major mammalian 3′-5′ exonuclease, have been reported in sporadic SLE cases. Some of these mutations have also been identified in a rare pediatric neurologic condition featuring an inflammatory encephalopathy known as Aicardi-Goutières syndrome (AGS). We sought to investigate the frequency of these mutations in a large multi-ancestral cohort of SLE cases and controls.

Methods

Forty single-nucleotide polymorphisms (SNPs), including both common and rare variants, across the TREX1 gene were evaluated in ∼8370 patients with SLE and ∼7490 control subjects. Stringent quality control procedures were applied and principal components and admixture proportions were calculated to identify outliers for removal from analysis. Population-based case-control association analyses were performed. P values, false discovery rate q values, and odds ratios with 95% confidence intervals were calculated.

Results

The estimated frequency of TREX1 mutations in our lupus cohort was 0.5%. Five heterozygous mutations were detected at the Y305C polymorphism in European lupus cases but none were observed in European controls. Five African cases incurred heterozygous mutations at the E266G polymorphism and, again, none were observed in the African controls. A rare homozygous R114H mutation was identified in one Asian SLE patient whereas all genotypes at this mutation in previous reports for SLE were heterozygous. Analysis of common TREX1 SNPs (MAF >10%) revealed a relatively common risk haplotype in European SLE patients with neurologic manifestations, especially seizures, with a frequency of 58% in lupus cases compared to 45% in normal controls (p=0.0008, OR=1.73, 95% CI=1.25-2.39). Finally, the presence or absence of specific autoantibodies in certain populations produced significant genetic associations. For example, a strong association with anti-nRNP was observed in the European cohort at a coding synonymous variant rs56203834 (p=2.99E-13, OR=5.2, 95% CI=3.18-8.56).

Conclusion

Our data confirm and expand previous reports and provide additional support for the involvement of TREX1 in lupus pathogenesis.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti–dsDNA autoantibody production, a SLE–related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti–dsDNA autoantibody positive (anti–dsDNA +, n = 811) and anti–dsDNA autoantibody negative (anti–dsDNA –, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti–dsDNA + SLE. Far fewer and weaker associations were observed for anti–dsDNA – SLE. For example, rs7574865 in STAT4 had an OR for anti–dsDNA + SLE of 1.77 (95% CI 1.57–1.99, p = 2.0E-20) compared to an OR for anti–dsDNA – SLE of 1.26 (95% CI 1.12–1.41, p = 2.4E-04), with pheterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti–dsDNA + SLE and were not associated with anti–dsDNA – SLE. In conclusion, we identified differential genetic associations with SLE based on anti–dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti–dsDNA – SLE.

Author Summary

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can involve virtually any organ system. SLE patients produce antibodies that bind to their own cells and proteins (autoantibodies) which can cause irreversible organ damage. One particular SLE–related autoantibody directed at double-stranded DNA (anti–dsDNA) is associated with kidney involvement and more severe disease. Previous genome-wide association studies (GWAS) in SLE have studied SLE itself, not particular SLE manifestations. Therefore, we conducted this GWAS of anti–dsDNA autoantibody production to identify genetic associations with this clinically important autoantibody. We found that many previously identified SLE–associated genes are more strongly associated with anti–dsDNA autoantibody production than SLE itself, and they may be more accurately described as autoantibody propensity genes. No strong genetic associations were observed for SLE patients who do not produce anti–dsDNA autoantibodies, suggesting that other factors may have more influence in developing this type of SLE. Further investigation of these autoantibody propensity genes may lead to greater insight into the causes of autoantibody production and organ damage in SLE.

Objective

To determine whether genetic substructure in European-derived populations is associated with specific manifestations of systemic lupus erythematosus (SLE), including mucocutaneous phenotypes, autoantibody production, and renal disease.

Methods

SLE patients of European descent (n=1754) from 8 case collections were genotyped for over 1,400 ancestry informative markers that define a north/south gradient of European substructure. Based on these genetic markers, we used the STRUCTURE program to characterize each SLE patient in terms of percent northern (vs. southern) European ancestry. Non-parametric methods, including tests of trend, were used to identify associations between northern European ancestry and specific SLE manifestations.

Results

In multivariate analyses, increasing levels of northern European ancestry were significantly associated with photosensitivity (ptrend=0.0021, OR for highest quartile of northern European ancestry compared to lowest quartile 1.64, 95% CI 1.13–2.35) and discoid rash (ptrend=0.014, ORhigh-low 1.93, 95% CI 0.98–3.83). In contrast, northern European ancestry was protective for anticardiolipin (ptrend=1.6 × 10−4, ORhigh-low 0.46, 95% CI 0.30–0.69) and anti-dsDNA (ptrend=0.017, ORhigh-low 0.67, 95% CI 0.46–0.96) autoantibody production.

Conclusions

This study demonstrates that specific SLE manifestations vary according to northern vs. southern European ancestry. Thus, genetic ancestry may contribute to the clinical heterogeneity and variation in disease outcomes among SLE patients of European descent. Moreover, these results suggest that genetic studies of SLE subphenotypes will need to carefully address issues of population substructure due to genetic ancestry.

Introduction

The aim of the study was to determine whether Olf1/EBF associated zinc finger protein (OAZ), a transcription factor encoded by a positional systemic lupus erythematosus (SLE) candidate gene, plays a functional role in the pathogenesis in SLE.

Methods

Gene expression levels in peripheral blood cells (PBLs) measured using quantitative real-time polymerase chain reaction (qPCR) were assessed for association with disease activity and the presence of specific autoantibodies. Peripheral blood mononuclear cells (PBMCs) were incubated with specific siRNAs for three days, then cells were harvested for measuring mRNA levels using qPCR, and supernatants for levels of total immunoglobulin (Ig)G and IgM as well as secreted cytokines, chemokine and antinuclear antibodies (ANA) using ELISA. Indirect immunofluorescence was also applied for ANA detection.

Results

OAZ gene expressions in PBLs from 40 ANA-positive SLE patients were significantly increased than those from 30 normal controls (P < 0.0001) and 18 patients with rheumatoid arthritis (P < 0.01). In SLE patients, OAZ transcripts were positively correlated with SLE disease activity index (SLEDAI) score (r = 0.72, P < 0.0001) and higher in those positive for anti-dsDNA or anti-Sm antibodies (both P < 0.05). Co-culturing with OAZ siRNAs reduced mRNA levels of OAZ by 74.6 ± 6.4% as compared to those co-cultured with non-targeting siRNA and OAZ silencing resulted in reduced total IgG, ANA, interferon (IFN)-γ, interleukin (IL)-10, IL-12 and IL-21, but elevated CCL2 levels in culture supernatants (P < 0.05). The declined ANA levels correlated with inhibited OAZ expression (r = 0.88, P = 0.05), reduced IL-21 levels (r = 0.99, P < 0.01), and elevated chemokine (C-C motif) ligand 2 levels (r = -0.98, P < 0.01). Expressions of ID1-3 were significantly down-regulated by 68.7%, 70.2% and 67.7% respectively after OAZ silence, while ID3 was also highly expressed in SLE PBLs (P < 0.0001) and associated with disease activity (r = 0.76, P < 0.0001) as well as anti-dsDNA or anti-Sm antibodies (both P < 0.05).

Conclusions

Elevated expression of OAZ transcripts in SLE PBLs were strongly correlated with disease activity. Suppression of OAZ expression inhibited downstream ID levels, and secretion of ANA and IL-21, implicating a role of OAZ pathway in the pathogenesis of SLE.

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

Objective

To test the hypothesis, utilizing 2 experimental mouse models, that plasmin is an important autoantigen that drives the production of certain IgG–anticardiolipin (aCL) antibodies in patients with the antiphospholipid syndrome.

Methods

BALB/cJ and MRL/MpJ mice were immunized with Freund’s complete adjuvant in the presence or absence of human plasmin. The mouse sera were analyzed for production of IgG-antiplasmin, IgG-aCL, and IgG–anti–β2-glycoprotein I (anti-β2GPI) antibodies. IgG monoclonal antibodies (mAb) were generated from the plasmin-immunized MRL/MpJ mice with high titers of aCL, and these 10 mAb were studied for their binding properties and functional activity in vitro.

Results

Plasmin-immunized BALB/cJ mice produced high titers of IgG-antiplasmin only, while plasmin-immunized MRL/MpJ mice produced high titers of IgG-antiplasmin, IgG-aCL, and IgG–anti-β2GPI. Both strains of mice immunized with the adjuvant alone did not develop IgG-antiplasmin or IgG-aCL. All 10 of the IgG mAb bound to human plasmin and cardiolipin, while 4 of 10 bound to β2GPI, 3 of 10 bound to thrombin, and 4 of 10 bound to the activated coagulation factor X (FXa). Functionally, 4 of the 10 IgG mAb inhibited plasmin activity, 1 of 10 hindered inactivation of thrombin by antithrombin III (AT), and 2 of 10 inhibited inactivation of FXa by AT.

Conclusion

Plasmin immunization leads to production of the IgG mAb antiplasmin, aCL, and anti-β2GPI in MRL/MpJ mice, but leads to production of only IgG-antiplasmin in BALB/cJ mice. IgG mAb generated from the plasmin-immunized MRL/MpJ mice bind to various antigens and exhibit procoagulant activity in vitro. These results suggest that plasmin may drive the potentially prothrombotic activities of aCL in genetically susceptible individuals.

Objective

To test the feasibility of applying a mimetic (specific for a patient-derived prothrombotic anticardiolipin antibody [aCL]) to study the homologous, disease-associated aCL in patients with antiphospholipid syndrome (APS).

Methods

We used the CL15 monoclonal aCL to screen 17 phage-display peptide libraries. Peptides (corresponding to recurrent peptide sequences) and their derivatives were synthesized and analyzed for binding to CL15 and for their abilities to inhibit CL15 from binding to cardiolipin. A peptide was chosen and used to study CL15-like IgG aCL in plasma samples from patients with APS, patients with systemic lupus erythematosus (SLE) but without APS, and normal healthy donors.

Results

Library screening with CL15 yielded 4 recurrent peptide sequences. Analyses of peptides showed that peptide CL154C reacted with antibody CL15 and inhibited binding of CL15 to cardiolipin, indicating that peptide CL154C may be a peptide mimetic for the CL15 aCL. Initial studies with plasma samples revealed that CL154C-reactive IgG was present (positivity defined as the mean + 3 SD optical density of the 25 normal controls) in 15 of 21 APS patients and 1 of 12 SLE patients.

Conclusion

These findings suggest that it is feasible to develop a specific enzyme-linked immunosorbent assay for each immunologically and functionally distinct disease-associated aCL. Additional testing of CL154C with a larger number of APS patients and SLE patients, as well as identification of peptide mimetics for each distinct aCL, will reveal the diagnostic potential of CL154C and other mimetics in identifying patients with aCL who are at risk of developing life-threatening thrombosis.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by various autoantibodies that recognize autoantigens displayed on the surface of cells undergoing apoptosis. The genetic contribution to SLE susceptibility has been widely recognized. We previously reported evidence for linkage to SLE of the human chromosome 1q41–q42 region and have now narrowed it from 15 to 5 cM in an extended sample using multipoint linkage analysis. Candidate genes within this region include (a) PARP, poly(ADP-ribose) polymerase, encoding a zinc-finger DNA-binding protein that is involved in DNA repair and apoptosis; (b) TGFB2, encoding a transforming growth factor that regulates cellular interactions and responses; and (c) HLX1, encoding a homeobox protein that may regulate T-cell development. Using a multiallelic, transmission-disequilibrium test (TDT), we found overall skewing of transmission of PARP alleles to affected offspring in 124 families (P = 0.00008), preferential transmission of a PARP allele to affected offspring (P = 0.0003), and lack of transmission to unaffected offspring (P = 0.004). Similar TDT analyses of TGFB2 and HLX1 polymorphisms yielded no evidence for association with SLE. These results suggest that PARP may be (or is close to) the susceptibility gene within the chromosome 1q41–q42 region linked to SLE.

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22–24 (LOD = 6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ∼1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [Pmeta = 5.20×10−14; odds ratio, 95% confidence interval = 0.82 (0.78–0.87)], and two missense variants, rs1990760 (Ala946Thr) [Pmeta = 3.08×10−7; 0.88 (0.84–0.93)] and rs10930046 (Arg460His) [Pdom = 1.16×10−8; 0.70 (0.62–0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis.

Author Summary

African-Americans (AA) are at increased risk of systemic lupus erythematosus (SLE), but the genetic basis of this risk increase is largely unknown. We used admixture mapping to localize disease-causing genetic variants that differ in frequency across populations. This approach is advantageous for localizing susceptibility genes in recently admixed populations like AA. Our genome-wide admixture scan identified seven admixture signals, and we followed the best signal at 2q22–24 with fine-mapping, imputation-based association analysis and experimental validation. We identified two independent coding variants and a non-coding variant within the IFIH1 gene associated with SLE. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis.

Objective

Several confirmed genetic susceptibility loci for lupus have been described. To date, no clear evidence for genetic epistasis is established in lupus. We test for gene-gene interactions in a number of known lupus susceptibility loci.

Methods

Eighteen SNPs tagging independent and confirmed lupus susceptibility loci were genotyped in a set of 4,248 lupus patients and 3,818 normal healthy controls of European descent. Epistasis was tested using a 2-step approach utilizing both parametric and non-parametric methods. The false discovery rate (FDR) method was used to correct for multiple testing.

Results

We detected and confirmed gene-gene interactions between the HLA region and CTLA4, IRF5, and ITGAM, and between PDCD1 and IL21 in lupus patients. The most significant interaction detected by parametric analysis was between rs3131379 in the HLA region and rs231775 in CTLA4 (Interaction odds ratio=1.19, z-score= 3.95, P= 7.8×10−5 (FDR≤0.05), PMDR= 5.9×10−45). Importantly, our data suggest that in lupus patients the presence of the HLA lupus-risk alleles in rs1270942 and rs3131379 increases the odds of also carrying the lupus-risk allele in IRF5 (rs2070197) by 17% and 16%, respectively (P= 0.0028 and 0.0047).

Conclusion

We provide evidence for gene-gene epistasis in systemic lupus erythematosus. These findings support a role for genetic interaction contributing to the complexity of lupus heritability.

Objective

Candidate gene and genome-wide association studies have identified several disease susceptibility loci in lupus patients. These studies have been largely performed in European-derived and Asian lupus patients. In this study, we examine if some of these same susceptibility loci increase lupus risk in African-American individuals.

Methods

Single nucleotide polymorphisms tagging 15 independent lupus susceptibility loci were genotyped in a set of 1,724 lupus patients and 2,024 normal healthy controls of African-American descent. The loci examined included: PTPN22, FCGR2A, TNFSF4, STAT4, CTLA4, PDCD1, PXK, BANK1, MSH5 (HLA region), CFB (HLA region), C8orf13-BLK region, MBL2, KIAA1542, ITGAM, and MECP2/IRAK1.

Results

We provide the first evidence for genetic association between lupus and five susceptibility loci in African-American patients (C8orf13-BLK, BANK1, TNFSF4, KIAA1542 andCTLA4; P values= 8.0 × 10−6, 1.9 × 10−5, 5.7 × 10−5, 0.00099, 0.0045, respectively). Further, we confirm the genetic association between lupus and five additional lupus susceptibility loci (ITGAM, MSH5, CFB, STAT4, and FCGR2A; P values= 7.5 × 10−11, 5.2 × 10−8, 8.7 × 10−7, 0.0058, and 0.0070, respectively), and provide evidence for a genome-wide significance for the association between ITGAM and MSH5 (HLA region) for the first time in African-American lupus patients.

Conclusion

These findings provide evidence for novel genetic susceptibility loci for lupus in African-Americans and demonstrate that the majority of lupus susceptibility loci examined confer lupus risk across multiple ethnicities.

Objective

Systemic lupus erythematosus (SLE; OMIM 152700) is a chronic autoimmune disease for which the aetiology includes genetic and environmental factors. ITGAM, integrin αΜ (complement component 3 receptor 3 subunit) encoding a ligand for intracellular adhesion molecule (ICAM) proteins, is an established SLE susceptibility locus. This study aimed to evaluate the independent and joint effects of genetic variations in the genes that encode ITGAM and ICAM.

Methods

The authors examined several markers in the ICAM1–ICAM4–ICAM5 locus on chromosome 19p13 and the single ITGAM polymorphism (rs1143679) using a large-scale case–control study of 17 481 unrelated participants from four ancestry populations. The single marker association and gene–gene interaction were analysed for each ancestry, and a meta-analysis across the four ancestries was performed.

Results

The A-allele of ICAM1–ICAM4–ICAM5 rs3093030, associated with elevated plasma levels of soluble ICAM1, and the A-allele of ITGAM rs1143679 showed the strongest association with increased SLE susceptibility in each of the ancestry populations and the trans-ancestry meta-analysis (ORmeta=1.16, 95% CI 1.11 to 1.22; p=4.88×10−10 and ORmeta=1.67, 95% CI 1.55 to 1.79; p=3.32×10−46, respectively). The effect of the ICAM single-nucleotide polymorphisms (SNPs) was independent of the effect of the ITGAM SNP rs1143679, and carriers of both ICAM rs3093030-AA and ITGAM rs1143679-AA had an OR of 4.08 compared with those with no risk allele in either SNP (95% CI 2.09 to 7.98; p=3.91×10−5).

Conclusion

These findings are the first to suggest that an ICAM–integrin-mediated pathway contributes to susceptibility to SLE.

Objective

Systemic lupus erythematosus is a clinically heterogeneous autoimmune disease. A number of genetic loci that increase lupus susceptibility have been established. This study examines if these genetic loci also contribute to the clinical heterogeneity in lupus.

Materials and methods

4001 European-derived, 1547 Hispanic, 1590 African-American and 1191 Asian lupus patients were genotyped for 16 confirmed lupus susceptibility loci. Ancestry informative markers were genotyped to calculate and adjust for admixture. The association between the risk allele in each locus was determined and compared in patients with and without the various clinical manifestations included in the ACR criteria.

Results

Renal disorder was significantly correlated with the lupus risk allele in ITGAM (p=5.0×10−6, OR 1.25, 95% CI 1.12 to 1.35) and in TNFSF4 (p=0.0013, OR 1.14, 95% CI 1.07 to 1.25). Other significant findings include the association between risk alleles in FCGR2A and malar rash (p=0.0031, OR 1.11, 95% CI 1.17 to 1.33), ITGAM and discoid rash (p=0.0020, OR 1.20, 95% CI 1.06 to 1.33), STAT4 and protection from oral ulcers (p=0.0027, OR 0.89, 95% CI 0.83 to 0.96) and IL21 and haematological disorder (p=0.0027, OR 1.13, 95% CI 1.04 to 1.22). All these associations are significant with a false discovery rate of <0.05 and pass the significance threshold using Bonferroni correction for multiple testing.

Conclusion

Significant associations were found between lupus clinical manifestations and the FCGR2A, ITGAM, STAT4, TNSF4 and IL21 genes. The findings suggest that genetic profiling might be a useful tool to predict disease manifestations in lupus patients in the future.

Objective

The overexpression of interferon (IFN)-inducible genes is a prominent feature of SLE, serves as a marker for active and more severe disease, and is also observed in other autoimmune and inflammatory conditions. The genetic variations responsible for sustained activation of IFN responsive genes are unknown.

Methods

We systematically evaluated association of SLE with a total of 1,754 IFN-pathway related genes, including IFN-inducible genes known to be differentially expressed in SLE patients and their direct regulators. We performed a three-stage design where two cohorts (total n=939 SLE cases, 3,398 controls) were analyzed independently and jointly for association with SLE, and the results were adjusted for the number of comparisons.

Results

A total of 16,137 SNPs passed all quality control filters of which 316 demonstrated replicated association with SLE in both cohorts. Nine variants were further genotyped for confirmation in an average of 1,316 independent SLE cases and 3,215 independent controls. Association with SLE was confirmed for several genes, including the transmembrane receptor CD44 (rs507230, P = 3.98×10−12), cytokine pleiotrophin (PTN) (rs919581, P = 5.38×10−04), the heat-shock DNAJA1 (rs10971259, P = 6.31×10−03), and the nuclear import protein karyopherin alpha 1 (KPNA1) (rs6810306, P = 4.91×10−02).

Conclusion

This study expands the number of candidate genes associated with SLE and highlights the potential of pathway-based approaches for gene discovery. Identification of the causal alleles will help elucidate the molecular mechanisms responsible for activation of the IFN system in SLE.

Objective

Previous genome wide association study conducted in a population of European ancestry identified rs4963128, a KIAA1542 SNP 23kb telomeric to IRF7, in strong association with SLE. This study was undertaken to investigate whether genetic polymorphism within IRF7 is a risk factor for the development of SLE.

Methods

We genotyped one KIAA1542 SNP rs4963128 and one IRF7 SNP rs1131665 (Q412R) in an Asian population (cases vs. controls: 1302 vs.1479) to assess their association with SLE using custom-designed Beadstation Infinium II platform (Illumina). Subsequently, rs1131665 was further genotyped in independent panels of Chinese (528 vs.527), European American (EA) (446 vs.461) and African American (AA) (159 vs.115) by Taqman genotyping assay to seek confirmation of association in various ethnic groups. Luciferase reporter assay was used to assess the effect of Q412R polymorphism on the activation of IRF7.

Results

Consistent association of rs1131665 (Q412R) with SLE was identified in Asian, EA and AA populations (case vs. control: 2435 vs. 2582; Pmeta = 6.18×10−6, OR = 1.42[1.22–1.65]). Expression of IRF7 412Q risk allele resulted in a 2-fold increase in ISRE transcriptional activity compared with expression of IRF7 412R (P = 0.0003), suggesting IRF7 412Q confers elevated IRF7 activity and may therefore affect downstream IFN pathway.

Conclusion

We showed that the major allele of a nonsynonymous SNP rs1131665 (412Q) in IRF7 confers elevated IRF7 activation and predisposes to the development of SLE in multiple ethnic groups. This result provides direct genetic evidence supporting IRF7 may be a risk gene for human SLE.

In spite of the well-known clustering of multiple autoimmune disorders in families, analyses of specific shared genes and polymorphisms between systemic lupus erythematosus (SLE) and other autoimmune diseases (ADs) have been limited. Therefore, we comprehensively tested autoimmune variants for association with SLE, aiming to identify pleiotropic genetic associations between these diseases. We compiled a list of 446 non–Major Histocompatibility Complex (MHC) variants identified in genome-wide association studies (GWAS) of populations of European ancestry across 17 ADs. We then tested these variants in our combined Caucasian SLE cohorts of 1,500 cases and 5,706 controls. We tested a subset of these polymorphisms in an independent Caucasian replication cohort of 2,085 SLE cases and 2,854 controls, allowing the computation of a meta-analysis between all cohorts. We have uncovered novel shared SLE loci that passed multiple comparisons adjustment, including the VTCN1 (rs12046117, P = 2.02×10−06) region. We observed that the loci shared among the most ADs include IL23R, OLIG3/TNFAIP3, and IL2RA. Given the lack of a universal autoimmune risk locus outside of the MHC and variable specificities for different diseases, our data suggests partial pleiotropy among ADs. Hierarchical clustering of ADs suggested that the most genetically related ADs appear to be type 1 diabetes with rheumatoid arthritis and Crohn's disease with ulcerative colitis. These findings support a relatively distinct genetic susceptibility for SLE. For many of the shared GWAS autoimmune loci, we found no evidence for association with SLE, including IL23R. Also, several established SLE loci are apparently not associated with other ADs, including the ITGAM-ITGAX and TNFSF4 regions. This study represents the most comprehensive evaluation of shared autoimmune loci to date, supports a relatively distinct non–MHC genetic susceptibility for SLE, provides further evidence for previously and newly identified shared genes in SLE, and highlights the value of studies of potentially pleiotropic genes in autoimmune diseases.

Author Summary

It is well known that multiple autoimmune disorders cluster in families. However, all of the genetic variants that explain this clustering have not been discovered, and the specific genetic variants shared between systemic lupus erythematosus (SLE) and other autoimmune diseases (ADs) are not known. In order to better understand the genetic factors that explain this predisposition to autoimmunity, we performed a comprehensive evaluation of shared autoimmune genetic variants. First we considered results from 17 ADs and compiled a list with 446 significant genetic variants from these studies. We identified some genetic variants extensively shared between ADs, as well as the ADs that share the most variants. The genetic overlap between SLE and other ADs was modest. Next we tested how important all the 446 genetic variants were in our collection with a minimum of 1,500 SLE patients. Among the most significant variants in SLE, the majority had already been identified in previous studies, but we also discovered variants in two important immune genes. In summary, our data identified diseases with common genetic risk factors and novel SLE effects, and this supports a relatively distinct genetic susceptibility for SLE. This study helps delineate the genetic architecture of ADs.

Objective

We previously reported association of co-occurrence of HLA-DRB1 shared epitope (SE) and RANKL SNPs with younger age of RA onset in 182 rheumatoid factor positive (RF) European American (EA) early RA patients. Here, we fine-mapped the 48 kb RANKL region in the extended 210 EA RF-positive early RA cohort, sought replication of RA-associated SNPs in additional 501 EA and 298 African-Americans (AA) RA cohorts, and explored functional consequences of RA-associated SNPs.

Methods

SNP genotyping was conducted using pyrosequencing or TaqMan PCR assays. Associations of rs7984870 with RANKL expression in plasma, PBMC and isolated T cells were quantified using ELISA and RT-PCR. Site-directed mutagenesis of rs7984870 within the 2kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay.

Results

A single promoter SNP rs7984870 was consistently significantly associated with earlier age of RA onset in 3 independent seropositive (RF or anti-cyclic citrullinated peptide antibody positive) RA cohorts but not in seronegative RA patients. The risk C allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive RA patients, significantly elevated RANKL mRNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to transcription factor SOX5.

Conclusion

The RANKL promoter allele that increased transcriptional levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to younger RA onset in seropositive EA and/or AA individuals.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic predisposition, characterized by an upregulated type I interferon pathway. MicroRNAs are important regulators of immune homeostasis, and aberrant microRNA expression has been demonstrated in patients with autoimmune diseases. We recently identified miR-146a as a negative regulator of the interferon pathway and linked the abnormal activation of this pathway to the underexpression of miR-146a in SLE patients. To explore why the expression of miR-146a is reduced in SLE patients, we conducted short parallel sequencing of potentially regulatory regions of miR-146a and identified a novel genetic variant (rs57095329) in the promoter region exhibiting evidence for association with SLE that was replicated independently in 7,182 Asians (Pmeta = 2.74×10−8, odds ratio = 1.29 [1.18–1.40]). The risk-associated G allele was linked to reduced expression of miR-146a in the peripheral blood leukocytes of the controls. Combined functional assays showed that the risk-associated G allele reduced the protein-binding affinity and activity of the promoter compared with those of the promoter containing the protective A allele. Transcription factor Ets-1, encoded by the lupus-susceptibility gene ETS1, identified in recent genome-wide association studies, binds near this variant. The manipulation of Ets-1 levels strongly affected miR-146a promoter activity in vitro; and the knockdown of Ets-1, mimicking its reduced expression in SLE, directly impaired the induction of miR-146a. We also observed additive effects of the risk alleles of miR-146a and ETS1. Our data identified and confirmed an association between a functional promoter variant of miR-146a and SLE. This risk allele had decreased binding to transcription factor Ets-1, contributing to reduced levels of miR-146a in SLE patients.

Author Summary

Genome-wide association studies have identified quite a number of susceptibility loci associated with complex diseases such as systemic lupus erythematosus (SLE). However, for most of them, the intrinsic link between genetic variation and disease mechanism is not fully understood. SLE is characterized by a significantly upregulated type I interferon (IFN) pathway, and we have previously reported that underexpression of a microRNA, miR-146a, contributes to alterations in the type I IFN pathway in lupus patients. Here we identified a novel genetic variant in the promoter region of miR-146a that is directly related to reduced expression of miR-146a and is associated with SLE susceptibility. The risk allele of this variant confers weaker binding affinity for Ets-1, which is a transcription factor encoded by a lupus susceptibility gene found in recent GWAS. These findings suggest that reduced expression of Ets-1 and its reduced binding affinity to the miR-146a promoter both may contribute to low levels of this microRNA in SLE patients, which may contribute to the upregulated type I IFN pathway in these patients. To our knowledge, this is also the first piece of evidence showing association between a genetic variant in a promoter region of a miRNA gene and a human disease.

Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. Recent studies have greatly expanded the number of established SLE risk alleles, but the distribution of multiple risk alleles in cases versus controls and their relationship to subphenotypes have not been studied. We studied 22 SLE susceptibility polymorphisms with previous genome-wide evidence of association (p<5×10−8) in 1919 SLE cases from 9 independent Caucasian SLE case series and 4813 independent controls. The mean number of risk alleles in cases was 15.1 (SD 3.1) while the mean in controls was 13.1 (SD 2.8), with trend p = 4×10−128. We defined a genetic risk score (GRS) for SLE as the number of risk alleles with each weighted by the SLE risk odds ratio (OR). The OR for high-low GRS tertiles, adjusted for intra-European ancestry, sex, and parent study, was 4.4 (95% CI 3.8–5.1). We studied associations of individual SNPs and the GRS with clinical manifestations for the cases: age at diagnosis, the 11 American College of Rheumatology classification criteria, and double-stranded DNA antibody (anti-dsDNA) production. Six subphenotypes were significantly associated with the GRS, most notably anti-dsDNA (ORhigh-low = 2.36, p = 9e−9), the immunologic criterion (ORhigh-low = 2.23, p = 3e−7), and age at diagnosis (ORhigh-low = 1.45, p = 0.0060). Finally, we developed a subphenotype-specific GRS (sub-GRS) for each phenotype with more power to detect cumulative genetic associations. The sub-GRS was more strongly associated than any single SNP effect for 5 subphenotypes (the above plus hematologic disorder and oral ulcers), while single loci are more significantly associated with renal disease (HLA-DRB1, OR = 1.37, 95% CI 1.14–1.64) and arthritis (ITGAM, OR = 0.72, 95% CI 0.59–0.88). We did not observe significant associations for other subphenotypes, for individual loci or the sub-GRS. Thus our analysis categorizes SLE subphenotypes into three groups: those having cumulative, single, and no known genetic association with respect to the currently established SLE risk loci.

Author Summary

Systemic lupus erythematosus is a chronic disabling autoimmune disease, most commonly striking women in their thirties or forties. It can cause a wide variety of clinical manifestations, including kidney disease, arthritis, and skin disorders. Prognosis varies greatly depending on these clinical features, with kidney disease and related characteristics leading to greater morbidity and mortality. It is also complex genetically; while lupus runs in families, genes increase one's risk for lupus but do not fully determine the outcome. The interactions of multiple genes and/or interactions between genes and environmental factors may cause lupus, but the causes and disease pathways of this very heterogeneous disease are not well understood. By examining relationships between the presence of multiple lupus risk genes, lupus susceptibility, and clinical manifestations, we hope to better understand how lupus is triggered and by what biological pathways it progresses. We show in this work that certain clinical manifestations of lupus are highly associated with cumulative genetic variations, i.e. multiple risk alleles, while others are associated with a single variation or none at all.