A substantial genetic contribution to systemic lupus erythematosus (SLE) risk is conferred by major histocompatibility complex (MHC) gene (s) on chromosome 6p21. Previous studies in SLE have lacked statistical power and genetic resolution to fully define MHC influences. We characterized 1,610 Caucasian SLE cases and 1,470 parents for 1,974 MHC SNPs, the highly polymorphic HLA-DRB1 locus, and a panel of ancestry informative markers. Single-marker analyses revealed strong signals for SNPs within several MHC regions, as well as with HLA-DRB1 (global p = 9. 99×10−16). The most strongly associated DRB1 alleles were: *0301 (odds ratio, OR = 2. 21, p = 2. 53×10−12), *1401 (OR = 0. 50, p = 0. 0002), and *1501 (OR = 1. 39, p = 0. 0032). The MHC region SNP demonstrating the strongest evidence of association with SLE was rs3117103, with OR = 2. 44 and p = 2. 80×10−13. Conditional haplotype and stepwise logistic regression analyses identified strong evidence for association between SLE and the extended class I, class I, class III, class II, and the extended class II MHC regions. Sequential removal of SLE–associated DRB1 haplotypes revealed independent effects due to variation within OR2H2 (extended class I, rs362521, p = 0. 006), CREBL1 (class III, rs8283, p = 0. 01), and DQB2 (class II, rs7769979, p = 0. 003, and rs10947345, p = 0. 0004). Further, conditional haplotype analyses demonstrated that variation within MICB (class I, rs3828903, p = 0. 006) also contributes to SLE risk independent of HLA-DRB1*0301. Our results for the first time delineate with high resolution several MHC regions with independent contributions to SLE risk. We provide a list of candidate variants based on biologic and functional considerations that may be causally related to SLE risk and warrant further investigation.
Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease characterized by autoantibody production and involvement of multiple organ systems. Although the etiology of SLE remains unknown, several lines of evidence underscore the importance of genetic factors, including a high sibling risk ratio (λs = 8–29), familial clustering, where approximately 10 to 12% of SLE patients have an affected first-degree relative, and higher concordance rates in monozygotic twins (24–69%) relative to dizygotic twins and non-twin siblings (2–9%) [1], [2], [3]. Similar to many other autoimmune diseases, genes within the major histocompatibility complex (MHC) on the short arm of chromosome 6p21. 3 exhibit strong association with the risk for SLE. The MHC is a gene-dense region of the human genome spanning approximately 4. 5 Mb and known to encode more than 180 expressed genes [4], [5]. Forty percent of the expressed loci have functions related to immune activation and response. The class III region, with more than 55 expressed loci, is the most dense subregion of the MHC and the entire human genome [6]. Historically, interest in the MHC region for SLE has focused on the highly polymorphic HLA class I and II genes that encode membrane glycoproteins that present peptides for recognition by T lymphocytes, as well as genes within the HLA class III region, particularly the tumor necrosis factor and complement component C4 gene loci. Indeed, inherited deficiency of complement genes, particularly C4A (null) alleles, has long been recognized as strong, albeit rare, genetic risk factors for SLE [7]. Work by Graham and colleagues [8], [9] examining ∼50 microsatellite markers across the MHC region highlighted the importance of HLA class II haplotypes involving the HLA-DRB1 and –DQB1 loci, particularly those corresponding to serologic types HLA-DR2 and DR3 (DRB1*1501-DQB1*0602 and DRB1*0301-DQB1*0201, respectively). More recently, genome wide association scans in SLE also underscore the importance of the MHC [10], [11], [12]. However, strong linkage disequilibrium (LD) between particular alleles within the MHC has interfered with disease variant identification and previous studies have not been able to distinguish between associated MHC variants. Long-range, extended or ‘ancestral’ haplotypes, sometimes spanning greater than 2 Mb, have been observed [8], [13]. Recent studies in European-derived populations have examined the distribution of LD across the MHC and have suggested that SNPs can help dissect causal variation within this region [14], [15]. A recent analysis of 314 UK SLE families examining 68 SNPs across the HLA class II and III regions and HLA-DRB1 suggested two distinct association signals centered on DRB1*0301 and the T allele of rs419788 in intron 6 of the class III region gene SKIV2L [16]. Of interest, the class III signal appears to exclude the TNF -308 promoter polymorphism, which has been a focus of prior work [17]. Herein we extend this work by characterizing a much larger collection of SLE cases (and parents) for 1,974 MHC genetic markers plus the HLA-DRB1 locus. We also characterized SLE cases for a set of ancestry informative markers (AIMs) to identify population outliers and assess the possible impact of substructure within the European population on our genetic association results. Further, we utilized several analytic strategies to determine whether multiple distinct alleles or haplotypes contribute to SLE risk.
Table 1 summarizes characteristics of the 1,610 Caucasian SLE cases and available parents (n = 1,470) from the complete trio families included in this study. Clinical characteristics of the SLE cases are consistent with those reported for Caucasian patients, generally [18]. Table 2 summarizes association results for HLA-DRB1 alleles in SLE cases and non-transmitted control alleles from parents of the trio families [19]. Due to the highly polymorphic nature of this locus and prior evidence suggesting that multiple HLA-DRB1 alleles influence SLE risk, we employed a relative predispositional effects (RPE) analysis [20] (see Methods). Results indicated that three specific HLA-DRB1 alleles were strongly associated with SLE risk: DRB1*0301 (odds ratio, OR = 2. 21, p = 2. 53×10−12), *1401 (OR = 0. 50, p = 0. 0002) and *1501 (OR = 1. 39, p = 0. 0032). The DRB1*0801 allele, which has been shown in previous work to be associated with SLE risk [8] was not significantly associated with SLE in the current RPE analysis, after accounting for testing of multiple DRB1 alleles (full results shown in Table S1). Figure 1 displays single marker association results for the 1,974 MHC SNPs (out of 2,360) passing quality control filters (see Methods) in SLE cases compared to controls (additional results are shown in Table S2). We observed strong association signals across a broad region encompassing the HLA class I, III and II regions, with weaker evidence of association in the extended class I and II regions. The strongest evidence of association was observed at the multiallelic DRB1 locus (global p = 9. 99×10−16 and ORs as shown in Table 2). The MHC region SNP demonstrating the strongest evidence of association with SLE was rs3117103 (bp 32,457,535), with OR = 2. 44 and p = 2. 80×10−13. This SNP is flanking C6orf10 at a distance of 9. 8 kb. Given the extensive LD across this region, we utilized conditional analyses to identify association signals that were not due to rs3117103. Similarly, we repeated these analyses conditioning instead on the HLA-DRB1 locus, including the DRB1*0301, *1401 and *1501 alleles. The rs number and map position for all SNPs associated with SLE using the conditional haplotype method (CHM) (total = 171 SNPs with p<0. 01) are provided in Table S3. Following these conditional analyses, several analytic approaches were utilized to further define genetic variants with the most compelling evidence of association with SLE risk, as shown in Figure 2. Figure 3 displays initial association results for the MHC region variants (n = 11, including HLA-DRB1) that were shown to be associated with SLE risk based on conditional haplotype and stepwise logistic regression analyses, with p<0. 001 (See Figure 2 and Methods). In addition to the HLA-DRB1 locus, there were several variants with evidence of association with SLE, including one variant in the extended class II region, five variants in HLA class II (including HLA-DRB1), one variant in HLA class III, two in HLA class I, and two in the extended class I region. Figure 4 shows an LD plot representing the correlation (r2) among HLA-DRB1 and the aforementioned 10 associated SNPs that met our significance threshold of p<0. 001 based on conditional analyses (see Methods and Table S4). There was very little evidence of LD among these 10 SNPs and HLA-DRB1 with the exception of SNPs within the MICB (r2 = 0. 58), C6orf10 (r2 = 0. 53) and HLA-DQB2 loci (r2 = 0. 35) and strong LD between HLA-DRB1 and the C6orf10 locus (r2 = 0. 77). To further evaluate the evidence supporting association of these 10 variants with SLE we performed transmission disequilibrium testing [21], [22] among 650 complete trio families. These analyses were consistent with the prior results, including strongest evidence of association for rs3117103 (OR = 2. 44, p = 1. 58×10−14). Additional details are shown in Table S5. We also evaluated these 10 SNPs among a more homogeneous subset of cases (and non-transmitted controls) estimated by AIMs to have ≥90% Northern European ancestry. These results also supported our main analyses, with the strongest evidence of association for rs3117103 (OR = 2. 49, p = 1. 62×10−13) (see Table S5 for additional details). Similarly, results for the top four DRB1 risk alleles did not change when analyses were repeated among the Northern European subset of cases and controls (data not shown). A distinguishing feature of the MHC region is the extensive, long-range LD, which has been observed particularly among European-derived populations [15] and is haplotype specific [23]. Thus, to further assess the independence of the 11 variants (including DRB1) and determine whether one or more extended haplotypes are strongly associated with SLE risk, we estimated haplotypes involving these variants and evaluated the evidence of association with SLE (see Methods). When case and control chromosomes were considered together, a total of 171 unique haplotypes were observed. When MHC haplotypes were compared between SLE cases and controls, evidence of association was revealed for two common haplotypes. The haplotype with the strongest evidence of association with SLE contains the DRB1*0301 allele (15% transmitted vs. 6% non-transmitted haplotypes, OR = 2. 63, p = 8. 32×10−15), which represents ∼70% of the DRB1*0301 haplotypes. The other associated haplotype contains the *1501 allele (9% transmitted vs. 6% non-transmitted haplotypes, OR = 1. 54, p = 0. 0025) (data not shown). Finally, we considered our data both a) excluding and b) conditioning on haplotypes containing the SLE-associated DRB1 alleles. More specifically, we compared MHC SNP allele frequencies among cases vs. controls after sequentially removing haplotypes containing *0301, *1501, and then *1401 (see Table 3 and Table S6). After removing haplotypes containing these alleles, we observed no evidence for independent association of rs3117103 with SLE (p>0. 30; see Table 3 and Table S6). However, four MHC region variants remained significantly associated with SLE, including rs362521 (OR2H2, extended class I region; p = 0. 0060), rs8283 (CREBL1, class III; p = 0. 011), rs7769979 and rs10947345 (DQB2, class II; p = 0. 0030 and 0. 0004, respectively). Of interest, two HLA class II region SNPs at the DQB2 locus remained associated with SLE even in the absence of DRB1 risk alleles. This is also the first study to support a role in SLE for variants within the extended MHC class I region, near OR2H2. Next, the CHM was used to investigate associations with the top 10 MHC region SNPs conditioned on haplotypes containing the SLE-associated HLA-DRB1 alleles (see Table 4 and Table S6). When conditioning on DRB1*0301 haplotypes, we observed significant association with SLE (p = 0. 0059) for a SNP at the MICB locus (HLA class I). In contrast, conditioning on DRB1*1501 or DRB1*1401 revealed no evidence for other MHC effects on SLE risk, however limited power was present for conditional analyses involving DRB1*1401 given the infrequency of that allele in this dataset. Finally, logistic regression analyses involving the aforementioned five SNPs and HLA-DRB1 revealed evidence of independent association with SLE for all variants (p-values = 0. 003 to <10−6) with the exception of one of the DQB2 SNPs (rs7769979) (data not shown). In summary, we have characterized a large set of SLE cases and parents of European ancestry for the highly polymorphic HLA-DRB1 locus and ∼2,000 SNPs across 4. 9 Mb of the MHC. We have employed methods to ensure that our main results are not due to confounding by population admixture or major substructure within the European population. Our results support the existence of multiple, independent association signals across this region as opposed to a single primary association signal (with other effects explained by LD). Independent associations within extended class I and both class II and III regions were present, even after accounting for HLA-DRB1 effects. However, we cannot exclude the possibility that multiple associated variants within MHC haplotypes function together to influence SLE risk through co-regulation or other mechanisms. An important limitation of the current analysis is the lack of comprehensive coverage in the MHC region. For example, the MHC Panel Set of markers was not designed to directly assess rare variants or multi-allelic markers, such as insertion-deletion or copy number variants. In addition, we have not characterized these families for other classical HLA genes (besides HLA-DRB1), and in fact our results implicate a role for other HLA class II region genes. Thus, we cannot determine which, if any, of the associated variants we have identified represent underlying causal variants, versus proxies for causal variants as a result of LD. In order to begin to address this important question we identified SNPs and other variants tagged by the independently associated SNPs and summarized their known function, as well as what is known about these variants themselves (see Table S7). One of the novel loci highlighted in the current study is OR2H2, within the extended class I region, which encodes olfactory receptor family 2, subfamily H, number 2. The olfactory receptor proteins are G-protein-coupled receptors that share structure with many neurotransmitter and hormone receptors [24]. To our knowledge, this gene has not been implicated in risk of SLE or other human autoimmune diseases. Our top SNP in this region tags three additional SNPs in a single locus, gamma-aminobutyric acid (GABA) B receptor, 1 (GABBR1, see Table S7), which is also a neurotransmitter receptor [25]. Of relevance to this finding, recent chromosome 6 sequencing efforts have now fully characterized the extended MHC in humans [26], spanning a total of 7. 6 Mb and comprised of five subregions that include the original ‘classical’ MHC [27]. Of the 421 loci in this region, a total of 252 (60%) are classified as expressed genes. Although our study included 305 SNPs within the extended class I MHC and 247 within the extended class II MHC, more work is needed to interrogate the entire extended MHC region. Our top SNP within the class III region was located in the 3′ untranslated region of CREBL1, which encodes c-AMP responsive element binding protein-like 1 [28]. Of interest, we did not identify additional variants tagged by our top SNP in this region (see Table S7). Unfortunately, our MHC marker set did not include SNPs within or near the C4 locus, which has been previously associated with SLE. In particular, copy number variation at this locus has been strongly implicated in SLE risk. However, the two markers flanking the C4 locus did not reveal evidence for association with SLE in the current study (rs389512 and rs1009382, p>0. 15). Given the density of genes within the class III region and previous work suggesting a contribution of class III variants to SLE risk, further study of this region is clearly warranted, including direct assessment of C4 variation. We also demonstrated association of SLE with a variant near the 5′ end of the MHC class I chain-related B (MICB) gene, which encodes a heavily glycosylated protein that serves as a ligand for the NKG2D type 2 receptor [29]. Binding of this ligand activates the cytolytic response of natural killer cells. This finding is also of interest given previous evidence of association of the closely related MICA gene to SLE [30], although results have been contradictory [31]. The MICB gene has not been previously implicated in SLE risk. All other variants tagged by our top SNP in this region are within or very near the MICB locus (see Table S7). Lastly, two HLA class II region SNPs at the DQB2 locus remained associated with SLE even in the absence of DRB1 risk alleles, and these two SNPs tag variants of at least four additional class II loci (see Table S7). However, our study lacked power to detect additional rare DRB1 associations (beyond *0301, *1501 and *1401), thus it is possible that additional DRB1 effects explain, at least in part, the DQB2 association observed in the current study. We also sought to determine how our results compare to those reported by Fernando, et al. , based on their analysis of 68 SNPs in the HLA class II and III regions, plus HLA-DRB1, in 314 UK SLE families [16]. Two distinct association signals centered on DRB1*0301 and the T allele of rs419788 in intron 6 of the class III region gene SKIV2L were observed. Five SNPs within this gene were included in our marker panel, including their top SNP, rs419788. Four of the five SNPs were associated with SLE with p<0. 01 in our single marker analyses, however, only two of these SNPs (but not rs419788) met the significance threshold (p<0. 01) for our CHM analyses. Neither of these two SNPs were among the group of ten SNPs selected for further study based on conditional haplotype based analyses or stepwise logistic regression. We have not examined other, non-European populations in this study and this represents a very important focus for future work given the increased burden of SLE among those groups. Further, comparison of MHC association results across major ethnic groups, where LD patterns are substantially different, or the analysis of specific disease subtypes may improve our ability to localize causal variants within this region.
Written consent was obtained from all study participants and ethical approval for this study was obtained from the University of California, San Francisco Committee on Human Research. SLE trio families and cases were derived from three independent case series, including the UCSF Lupus Genetics Project collection, the Lupus Genetics Studies and Lupus Family Registry and Repository (http: //lupus. omrf. org/) at the Oklahoma Medical Research Foundation (OMRF) and the University of Minnesota SLE collection. SLE cases from trio families (n = 735) were a subset of the total set of SLE cases (n = 1,610), as shown in Table 1. Details of recruitment and enrollment procedures have been reported previously [32], [33], [34]. All subjects were self-reported Caucasian and all SLE cases met the American College of Rheumatology classification criteria for SLE [35], [36]. Non-transmitted HLA-DRB1 and MHC SNP alleles (described below) from SLE trio family parents were used as ‘controls’ for all comparisons with SLE cases, as previously described [19]. DNA was extracted from whole blood using the Gentra Puregene Whole Blood Kit. DNA was obtained from buccal cells and extracted using the Gentra Puregene Mouse Tail Kit. A total of 50 ng of buccal cell or 25 ng of blood DNA (when necessary) was whole genome amplified (WGA) using the Sigma WGA2 Kit prior to genotyping. WGA samples were column purified using the Sigma Genelute PCR Clean-up Kit. DNA was also extracted from DNA Genotek Oragene saliva sample collection kits according to the manufacturer' s protocol. Of the total 3,080 individual DNA samples used for genotyping in this study, 69% were derived from whole blood, 25% from buccal cells, and 6% from saliva; 72% of all samples (including both blood and buccal) were WGA prior to genotyping.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and involvement of multiple organ systems. Although the cause of SLE remains unknown, several lines of evidence underscore the importance of genetic factors. As is true for most autoimmune diseases, a substantial genetic contribution to disease risk is conferred by major histocompatibility complex (MHC) gene (s) on chromosome 6. This region of the genome contains a large number of genes that participate in the immune response. However, the full contribution of this genomic region to SLE risk has not yet been defined. In the current study we characterize a large number of SLE patients and family members for approximately 2,000 MHC region variants to identify the specific genes that influence disease risk. Our results, for the first time, implicate four different MHC regions in SLE risk. We provide a list of candidate variants based on biologic and functional considerations that may be causally related to SLE risk and warrant further investigation.
rheumatology/connective tissue disease
genetics and genomics/genetics of the immune system
genetics and genomics/complex traits
rheumatology/systemic lupus erythematosos
rheumatology/autoimmunity, autoimmune, and inflammatory diseases
immunology/genetics of the immune system
immunology/autoimmunity
High-Density SNP Screening of the Major Histocompatibility Complex in Systemic Lupus Erythematosus Demonstrates Strong Evidence for Independent Susceptibility Regions