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.

We previously reported that the G allele of rs3853839 at 3′untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10−10, odds ratio (OR) (95%CI) = 1.27 (1.17–1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10−11, OR = 1.24 [1.18–1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3′UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R2 = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3′UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10−19, OR = 1.25 [1.20–1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.

Author Summary

Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease contributed to by excessive innate immune activation involving toll-like receptors (TLRs, particularly TLR7/8/9) and type I interferon (IFN) signaling pathways. TLR7 responds against RNA–containing nuclear antigens and activates IFN-α pathway, playing a pivotal role in the development of SLE. While a genomic duplication of Tlr7 promotes lupus-like disease in the Y-linked autoimmune accelerator (Yaa) murine model, the lack of common copy number variations at TLR7 in humans led us to identify a functional single nucleotide polymorphism (SNP), rs3853839 at 3′ UTR of the TLR7 gene, associated with SLE susceptibility in Eastern Asians. In this study, we fine-mapped the TLR7-TLR8 region and confirmed rs3853839 exhibiting the strongest association with SLE in European Americans, African Americans, and Amerindian/Hispanics. Individuals carrying the risk G allele of rs3853839 exhibited increased TLR7 expression at the both mRNA and protein level and decreased transcript degradation. MicroRNA-3148 (miR-3148) downregulated the expression of non-risk allele (C) containing transcripts preferentially, suggesting a likely mechanism for increased TLR7 levels in risk-allele carriers. This trans-ancestral mapping provides evidence for the global association with SLE risk at rs3853839, which resides in a microRNA–gene regulatory site affecting TLR7 expression.

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

Genetic association of the IL2/IL21 region at 4q27 has been previously reported in lupus and a number of autoimmune and inflammatory diseases. Herein, using a very large cohort of lupus patients and controls, we localize this genetic effect to the IL21 gene.

Methods

We genotyped 45 tag SNPs across the IL2/IL21 locus in two large independent lupus sample sets. We studied a European-derived set consisting of 4,248 lupus patients and 3,818 healthy controls, and an African-American set of 1,569 patients and 1,893 healthy controls. Imputation in 3,004 WTCCC additional control individuals was also performed. Genetic association between the genotyped markers was determined, and pair-wise conditional analysis was performed to localize the independent genetic effect in the IL2/IL21 locus in lupus.

Results

We established and confirmed the genetic association between IL2/IL21 and lupus. Using conditional analysis and trans-ethnic mapping, we localized the genetic effect in this locus to two SNPs in high linkage disequilibrium; rs907715 located within IL21 (OR=1.16 (1.10–1.22), P= 2.17 ×10−8), and rs6835457 located in the 3’-UTR flanking region of IL21 (OR= 1.11 (1.05–1.17), P= 9.35×10−5).

Conclusion

We have established the genetic association between lupus and IL2/IL21 with a genome-wide level of significance. Further, we localized this genetic association within the IL2/IL21 linkage disequilibrium block to IL21. If other autoimmune IL2/IL21 genetic associations are similarly localized, then the IL21 risk alleles would be predicted to operate in a fundamental mechanism that influences the course of a number of autoimmune disease processes.

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, Pmeta = 6.6×10−8, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, Pmeta = 2.9×10−7, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ∼146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (Pmeta = 3.2×10−7, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (Pmeta = 3.5×10−4, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.

Author Summary

Systemic lupus erythematosus (SLE) is a complex autoimmune disease, associated with increased complement activation. Previous studies have provided evidence for the presence of SLE susceptibility gene(s) in the chromosome 1q31-32 locus. Within 1q32, genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) may contribute to the development of SLE, because genetic variants of these genes impair complement regulation and predispose to various human diseases. In this study, we tested association of genetic variants in the region containing CFH and CFHRs with SLE. We identified genetic variants predisposing to SLE in European American, African American, and Asian populations, which might be attributed to the deletion of CFHR3 and CFHR1 genes but not previously identified disease-associated exonic variants of CFH. This study provides the first evidence for consistent association between CFH/CFHRs and SLE across multi-ancestral SLE datasets, providing new insights into the role of complement regulators in the pathogenesis of SLE.

Objective

Interleukin (IL) 21 is a member of the type I cytokine superfamily that exerts a variety of effects on the immune system including B cell activation, plasma cell differentiation, and immunoglobulin production. The expression of IL21R is reduced in B cells from lupus patients, while IL21 serum levels are increased in both lupus patients and some lupus-murine models. We recently reported that polymorphisms within the IL21 gene are associated with increased susceptibility to lupus. Herein, we examined the genetic association between SNPs within IL21R and lupus.

Methods

We genotyped 17 SNPs in the IL21R gene in two large cohorts of lupus patients and ethnically-matched healthy controls. Genotyping was performed with the Illumina BeadStation 500GX instrument using Illumina Infinum II genotyping assays.

Results

We identified and confirmed the association between rs3093301 within the IL21R gene and lupus in two independent European-derived and Hispanic cohorts (meta analysis odds ratio= 1.16, 95% CI= 1.08-1.25, meta analysis p=1.0×10-4).

Conclusion

We identified IL21R as a novel susceptibility gene for lupus.

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs. 32.6% in controls, P = 0.016, OR = 0.90 [0.82-0.98]). Two of these SNPs are in exon 10, directly 5′ of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs as well as a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.