ObjectiveSystemic lupus erythematosus (SLE), an autoimmune disorder, has been associated with nearly 100 susceptibility loci. Nevertheless, these loci only partially explain SLE heritability and their putative causal variants are rarely prioritised, which make challenging to elucidate disease biology. To detect new SLE loci and causal variants, we performed the largest genome-wide meta-analysis for SLE in East Asian populations.MethodsWe newly genotyped 10 029 SLE cases and 180 167 controls and subsequently meta-analysed them jointly with 3348 SLE cases and 14 826 controls from published studies in East Asians. We further applied a Bayesian statistical approach to localise the putative causal variants for SLE associations.ResultsWe identified 113 genetic regions including 46 novel loci at genome-wide significance (p<5×10−8). Conditional analysis detected 233 association signals within these loci, which suggest widespread allelic heterogeneity. We detected genome-wide associations at six new missense variants. Bayesian statistical fine-mapping analysis prioritised the putative causal variants to a small set of variants (95% credible set size ≤10) for 28 association signals. We identified 110 putative causal variants with posterior probabilities ≥0.1 for 57 SLE loci, among which we prioritised 10 most likely putative causal variants (posterior probability ≥0.8). Linkage disequilibrium score regression detected genetic correlations for SLE with albumin/globulin ratio (rg=−0.242) and non-albumin protein (rg=0.238).ConclusionThis study reiterates the power of large-scale genome-wide meta-analysis for novel genetic discovery. These findings shed light on genetic and biological understandings of SLE.
BackgroundThat Epstein–Barr virus (EBV) infection is associated with systemic lupus erythematosus (SLE) is established. The challenge is to explain mechanistic roles EBV has in SLE pathogenesis. Previous studies identify four examples of autoantibody cross-reactions between SLE autoantigens and Epstein–Barr nuclear antigen 1 (EBNA1). For two of these examples, the earliest detected autoantibody specifically cross-reacts with EBNA1; thereby, defined EBNA1 epitopes induce a robust autoantibody response in animals. These results suggest that the autoantibodies initiating the process leading to SLE may emerge from the anti-EBNA1 heteroimmune response. If this hypothesis is true, then anti-EBNA1 responses would be more frequent in EBV-infected SLE patients than in EBV-infected controls. We tested this prediction.MethodsWe evaluated published East Asian data by selecting those with a positive anti-viral capsid antigen (VCA) antibody immunoglobulin G (IgG) test and determining whether anti-EBNA1 was more common among the EBV-infected SLE cases than among matched EBV-infected controls with conditional logistic regression analysis.ResultsAll the qualifying SLE patients (100%) in this dataset were EBV-infected compared to age- and sex-matched controls (92.2%) [odds ratio (OR) = 28.6, 95% CI 6.4–∞, p = 8.83 × 10-8], confirming the known close association of EBV infection with SLE. Furthermore, virtually all the SLE cases have both anti-VCA IgG and anti-EBNA1 IgG antibodies [124 of 125 (99.2%)], which are more frequently present than in age- and sex-matched EBV-infected controls [232 of 250 (93.2%)] (OR = 9.7, 95% CI 1.5–414, p = 0.0078) for an 89.7% SLE attributable risk from anti-EBNA1, which is in addition to the 100% SLE risk attributable to EBV infection in these data.ConclusionsThe association of EBV infection with SLE is reconfirmed. The prediction that anti-EBNA1 is more frequent in these SLE cases than in EBV-infected controls is true, consistent with the hypothesis that anti-EBNA1 contributes to SLE. This second EBV-dependent risk factor is consistent with a molecular mimicry model for the generation of SLE, starting with EBV infection, progressing to anti-EBNA1 response; then molecular mimicry leads to anti-EBNA1 antibodies cross-reacting with an SLE autoantigen, causing autoantibody epitope spreading, and culminating in clinical SLE. These results support the anti-EBNA1 heteroimmune response being a foundation from which pathogenic SLE autoimmunity emerges.
ALL, the most common pediatric cancer, originates from progenitor B-cells in ~85% of cases. IKAROS Family Zinc Finger 1 (IKZF1) is an important locus for ALL. Our goal was to identify the functional variants, thereby nominating a potential causal mechanism for ALL. We evaluated all 27 genetic polymorphisms in disequilibrium at r2>0.8 with the tag, the most highly associated risk variant at IKZF1 locus. They map to intron 7, 3′ UTR and 3′ flanking sequence of IKZF1. Fidgetin-like protein 1 (FIGNL1) is 45,524 bases from the tag variant and is nominated by eQTL and DNA looping data along with IKZF1 as possible targets. Epstein-Barr virus (EBV) transformed GM12878 cells were transduced with lentiviral vectors for stable expression of fusion proteins dead (d) Cas9-VP64 and activator MS2-p65-HSF1 (synergistic activation mediator (SAM) system). We designed 27 single guide (sg) RNAs, one for each of the 27 variants and evaluated expression perturbation in cells transfected with pools and individual sgRNA. Expression of FIGNL1, but not IKZF1, was convincingly altered in this system. The reduction of FIGNL1 expression by ~50% was isolated to two neighboring variants separated by 1 kb. The risk allele at these variants increases FIGNL1 expression in wild type cells, however in EBV transformed B-cells FIGNL1 expression is decreased. Other data show that the EBV encoded DNA binding co-factor EBNA3C binds at or very near this locus in EBV transformed B cells. Our results nominate two closely located SNPs in the last intron of IKZF1 as potentially causal variants, controlling the expression of FIGNL1, which ordinarily participates in homologous recombination during DNA double-strand break repair, maintenance of genomic stability and prevention of cancer.
Our recent study has demonstrated induction of regulatory B cells (Bregs) by bacterial DNA in MRL/lpr mice leading to attenuation of SLE. This suggests a regulatory role for the gut bacteria in SLE development. However, the mechanism by which bacterial DNA induces Breg cell differentiation remains to be elucidated. Bacterial DNA contains unmethylated CpG motifs which are recognized by the innate immune molecule, toll-like receptor 9 (TLR9). We therefore hypothesize that gut bacteria-derived DNA induces Bregs in a TLR9-dependent manner, which in turn protects against lupus initiation. To test our hypothesis, TLR9 global knockout (Tlr9 -/-) MRL/lpr mice and their heterozygous (Tlr9 +/-) littermates were randomized into 2 groups per genotype. Beginning at 4 weeks of age, one group in each genotype received 80 mg E. coli double-stranded DNA (dsDNA) once a week by oral gavage for 4 consecutive weeks, whereas the other group received phosphate buffered saline (PBS) as control. Urine samples were collected before euthanasia whereas blood, spleen, lymph nodes, and kidneys were collected and processed following euthanasia at 15 weeks of age. In vitro culture treatment with bacterial DNA was used to investigate a TLR9-dependent, B cell-intrinsic or -extrinsic mechanism in Breg cell induction. Our data showed a trend in decrease of proteinuria, as well as sizes of spleen and lymph nodes, with bacterial DNA treatment in Tlr9 +/mice. Interestingly, serum levels of anti-dsDNA IgM, which has been shown to be protective against lupus, were significantly increased in bacterial DNA-treated Tlr9 +/mice, although the pathogenic anti-dsDNA IgG remained unchanged. Additionally, flow cytometry analysis of splenocytes showed an increasing trend in the percentage of interleukin-10 positive (IL-10 + ) Bregs in bacterial DNA-treated Tlr9 +/mice and a concomitant decrease in double negative (DN, or CD4 -CD8 -) T cells. In the kidney, bacterial DNA treatment also decreased the percentage of CD45 + cells in Tlr9 +/mice. Importantly, all bacterial DNA-induced changes observed in Tlr9 +/-MRL/lpr mice, including those of proteinuria, organ weight, anti-dsDNA IgM, splenic Bregs, splenic DN T cells and renal CD45 + cells, were abrogated in Tlr9 -/-MRL/lpr mice. Bacterial DNA treatment of cocultures of B and non-B cells from Tlr9 +/+ and Tlr9 -/-MRL/lpr mice revealed a TLR9-dependent, but B cell-extrinsic, induction of Bregs mediated by IL-6 produced most likely from myeloid cells. Altogether, our study suggests possible attenuation of SLE by gut bacteria-derived DNA through a B cell-extrinsic, TLR9-dependent mechanism that involves the induction of IL-10 + Bregs and secretion of potentially protective anti-dsDNA IgM.
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