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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.
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.
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).
We identified IL21R as a novel susceptibility gene for lupus.
Systemic lupus erythematosus (SLE or lupus) is a chronic systemic autoimmune disease that is associated with multiple organ involvement. The etiology and pathogenesis of lupus involves genetic, epigenetic, and environmental factors. Lupus is a heterogeneous disease with highly variable presentation. In 1982, the American College of Rheumatology (ACR) published 11 classification criteria (at least four of which must be met) used to aid in the classification of lupus. The one criterion common in nearly all lupus patients is the presence of anti-nuclear autoantibodies, considered a hallmark of the disease (1).
A large body of evidence supports the role of genetics in lupus. More than 100 possible genetic risk factors for lupus have been identified through case studies, linkage analyses of multiplex families, and candidate gene case-control studies (2). Epidemiological studies have shown familial clustering in lupus and increased risk for family members of someone with lupus to develop this disease (3, 4). There is, for example, more than a ten-fold increase in the concordance rate of lupus in monozygotic twins compared to dizygotic twins (5).
Interleukin (IL) 21 is a recently discovered member of the type I cytokine superfamily and is expressed primarily by activated CD4+ T cells and NK cells (6). Its receptor, IL21 receptor (IL21R), is expressed on a variety of cells including B cells, T cells, NK cells, and monocyte-derived dendritic cells (7-9). IL21 exerts a variety of effects on the immune system, and is important for B cell responsiveness, proliferation, plasma cell differentiation, and immunoglobulin production (10, 11). Further, IL21 is involved in Th17 cell differentiation (12) and modulates the function of both dendritic cells and NK cells (13).
IL21 overproduction has been reported in several murine lupus models including the BXSB-Yaa mouse (11). Excessive production of IL21 and a severe lupus-like autoimmune phenotype has been reported in the sanroque mouse strain, which has a mutation in a RING-type ubiquitin ligase protein family member (roquin) (14). In addition, an IL21R-Fc fusion protein significantly improves disease in MRL/lpr lupus-prone mice, suggesting that blocking IL21 might be a potential therapeutic approach in lupus patients (15).
In lupus patients, IL21R is underexpressed on total, naïve and memory B cells, and plasmablasts compared to controls, while there is no difference in the expression levels of IL21R in T cells (8). Decreased expression of IL21R is also associated with nephritis and a high titer of anti-dsDNA antibody in lupus patients (8). Lupus patients have higher concentrations of IL21 in the plasma, though plasma IL21 levels do not appear to correlate with lupus disease activity (6).
We recently reported that polymorphisms within IL21 are associated with lupus in two independent lupus cohorts (16). Herein, we evaluate genetic polymorphisms in IL21R in a large group of lupus patients and controls. We identify a genetic association between a single nucleotide polymorphism (SNP) within the IL21R gene and lupus in a European-derived cohort, which is confirmed in a second independent cohort of Hispanic lupus patients and controls.
Lupus patients and healthy controls were recruited and enrolled in the lupus genetics studies at the Oklahoma Medical Research Foundation and at collaborating sites in the US, UK, and Sweden. A total of 2,573 independent lupus patients and 3,075 healthy unrelated ethnically-matched controls were included in our European-derived cohort. A total of 657 independent lupus patients and 265 healthy unrelated ethnically-matched controls were included in our Hispanic cohort. All lupus patients met the 1997 ACR classification criteria for lupus. Clinical and serological data were acquired as previously described (17).
Genotyping was performed on DNA isolated from peripheral blood mononuclear cells, lymphoblastoid cell lines, or buccal cell swabs as previously described (18). We genotyped 17 SNPs located within the IL21R gene in two independent, ethnically divergent populations of lupus patients and controls. These SNPs were selected from the published SNP databases (http://www.ncbi.nlm.nih.gov/projects/SNP/) to cover the entire length of the IL21R gene. They were all validated SNPs, and were previously tested successfully using the Illumina genotyping platform that we utilized in this study. Genotyping was performed with the Illumina BeadStation 500GX instrument using Illumina Infinum II genotyping assays following manufacturer's recommendations.
Principal component analyses were used to examine sample homogeneity in both cohorts. To further exclude population substratification, we used the Genomic Control (GC) method, and calculated an inflation factor (λ) using 2218 “null” SNPs in the European-derived cohort and 2196 “null” SNPs in the Hispanic cohort.
This study was conducted utilizing a population-based case-control study design. Pearson's chi-square measures were calculated to determine whether allele frequencies were different between cases and controls. Uncorrected p values are reported with 95 percent confidence intervals. Permutation test was used to correct allele frequency differences for multiple testing, using Haploview 4.1 (19). Meta analysis was performed using the Cochran-Mantel-Haenszel test included in SAS ®. Logistic regression analysis was used to test for gene-gene interaction between the lupus-associated IL21 and IL21R SNPs.
We genotyped 17 SNPs located within the IL21R gene in two independent, ethnically divergent populations of lupus patients and controls. Our European-derived cohort included 2,573 independent lupus cases and 3,075 healthy controls while our Hispanic cohort included 657 independent lupus cases and 265 healthy controls. Principal component analyses were used to examine sample homogeneity in both cohorts. A total of 234 and 156 samples were identified as outliers in the European-derived and Hispanic cohorts, respectively, and were excluded from further analysis. Subsequently, GC analysis was performed and revealed no significant population substratification (λ= 1.13 and 1.17, in the European-derived and Hispanic cohorts, respectively). Fifteen of the 17 SNPs genotyped had a minor allele frequency of at least 1%, which we required for analysis. Genotyping success rate was ≥99.2%, and ≥97.8% in the European-derived and Hispanic cohorts, respectively.
We found a genetic association between rs3093301 (A/G) and lupus in both the European-derived and the Hispanic cohorts. The lupus-associated allele (A) had a frequency of 66.6% among European-derived lupus cases compared to 63.7% among controls (OR= 1.13, χ2 = 9.42, p=0.0022) (Table 1, Figure 1A). This association was confirmed in the Hispanic cohort, which had an associated allele frequency of 64.4% in lupus cases compared to 55.9% in controls (OR= 1.43, χ2 = 9.16, p=0.0025) (Table 2, Figure 1B). Using Cochran-Mantel-Haenszel test, the meta-analysis odds ratio for the associated allele in rs3093301 in lupus patients compared to controls was 1.16 (95%CI= 1.08-1.25, meta-analysis p value= 1.0×10-4) in the combined European-derived and Hispanic samples.
IL21R is located in proximity to the IL4R gene, another important candidate in autoimmunity. However, IL21R and IL4R are located in separate haplotype blocks in the European-derived samples (CEU) genotyped in the International HapMap Project (Figure 1). To further confirm that the genetic effect we describe in rs3093301 is not a surrogate for a genetic association in IL4R, we genotyped 5 SNPs within the IL4R gene in both our European-derived and Hispanic cohorts. None of the genotyped SNPs in IL4R (rs2107356, rs2234895, rs1805011, rs1805013, and rs2074570) showed an association with lupus in either cohort (p>0.05).
To determine whether the lupus-risk allele in rs3093301 is more frequently associated with any clinical or serological manifestation in lupus patients, we determined the frequency of the various lupus manifestations in female lupus patients homozygous for the risk allele (A/A) (n=197) compared to patients homozygous for the protective allele (G/G) (n=62) in the European-derived cohort. Variables tested included malar rash, discoid rash, photosensitivity, oral ulcers, arthritis, pericarditis, pleuritis, proteinuria, urine cellular casts, seizures, psychosis, hemolytic anemia, leucopenia, lymphopenia, thrombocytopenia, the presence of anti-dsDNA, anti-Sm, anti-nRNP, anti-Ro, anti-La, and anti-ribosomal P antibodies. We found that the A/A genotype in rs3093301 is associated with the presence of malar rash in European-derived female lupus patients compared to the G/G genotype (60.9% versus 35.5%, OR=2.83, CI = 1.56-5.13, χ2 = 12.31, p=0.00045). This association remains significant after correcting for multiple testing using Bonferroni correction (corrected p= 0.0095). It was previously reported that reduced expression of IL21R in peripheral blood B cells is associated with nephritis in lupus patients (8). We found no difference in the frequency of proteinuria or urine cellular casts between lupus patients with the A/A genotype compared to the G/G genotype in rs3093301 (p= 0.51 and 0.14, respectively).
We have previously reported a genetic association between IL21 and lupus. The SNP rs907715 within the IL21 gene was the most significantly associated SNP in our European-derived cohort (20). To test if there is gene-gene interaction between IL21 and IL21R upon lupus susceptibility, we performed logistic regression analysis to determine if the simultaneous presence of the risk alleles in the IL21 lupus-associated SNP (rs907715) and the IL21R lupus associated SNP (rs3093301) could explain the increased susceptibility to lupus. We find no evidence for a synergistic effect for the presence of the risk alleles in rs907715 and rs3093301 (interaction p=0.58), suggesting no genetic epistasis between the IL21 and IL21R loci upon lupus susceptibility.
IL21 is a pleiotropic cytokine that has multiple effects on a number of immunocompetent cells. It is critically involved in B cell activation, differentiation, and immunoglobulin production (6). We report on the genetic association between a SNP in IL21R (rs3093301) and lupus in two ethnically divergent lupus cohorts. This SNP is located on chromosome 16p11 in the first intron of IL21R just upstream of the translational start site (Figure 2).
European-derived lupus patients who are homozygous for the rs3093301 risk allele (A/A) have approximately 3 times the odds of suffering from malar rash compared to lupus patients homozygous for the protective IL21R allele (G/G). However, this finding should be considered preliminary and requires further replication in other independent cohorts of lupus patients. Plasma IL21 levels were elevated in a cohort of lupus patients, nonetheless, there was no correlation between IL21 levels and malar rash or any other symptom or sign of lupus in that cohort (unpublished data; reviewed in (6)).
IL21R is located on chromosome 16p11, which is close to 16p12, a region that has been previously linked to lupus susceptibility using linkage analysis studies in familial lupus (20). The 16p11 region also harbors the ITGAM gene which is a confirmed genetic association for lupus (21, 22). The ITGAM and IL21R genes are not in linkage disequilibrium. The IL21R gene is therefore a likely candidate gene contributing to the genetic linkage effect on chromosome 16p12 along with the ITGAM gene.
In summary, we report and confirm for the first time the genetic association between lupus and IL21R. Further studies are needed to identify the causal SNP for this association, and to determine the functional consequences of this polymorphism upon lupus susceptibility. These findings, along with our previous report on IL21 genetic association with lupus, indicate that the IL21/IL21R pathway is important in understanding the pathogenesis of this disease. However, there is no evidence for gene-gene interaction (epistasis) between the lupus associated SNPs in IL21 and IL21R upon the susceptibility to lupus.
We are thankful to Dr. Peter Gregersen for providing DNA control samples for our study.
This work was made possible by NIH Grant Number R03AI076729 from the National Institute of Allergy and Infectious Diseases, NIH Grant Number P20-RR015577 from the National Center for Research Resources, funding from the Arthritis National Research Foundation and the University of Oklahoma College of Medicine (AHS); NIH Grants Number AR42460, AI024717, AI31584, AR62277, AR048940, AR0490084, Kirkland Scholar award, Alliance for Lupus Research, and US Department of Veterans Affairs (JBH); NIH grant number P01-AR049084 (RPK); NIH grant number AR043274 (KLM); and NIH grant number AI063274 (PMG).