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.
Systemic lupus erythematosus (SLE, [MIM 152700]) is a clinically heterogeneous autoimmune disease with a strong genetic component, characterized by inflammation, dysregulation of type-1 interferon responses and autoantibodies directed towards nuclear components. SLE overwhelmingly targets women, and its incidence and clinical course differ dramatically between ethnic populations. In particular, SLE occurs with at least 3–5 times higher prevalence and more severe complications in African-Americans (AA) compared to Americans with European ancestry (EA) [1]. However, the genetic basis of this increased risk is largely unknown. The recently “admixed” AA population is likely to provide critical information necessary to identify chromosomal regions that harbor variants associated with SLE and provide insights about allele frequency differences among distinct ancestral populations (i. e. , European and African). Admixture mapping (AM) has proven to be a powerful method to leverage ancestry information to identify chromosomal segments linked to disease [2]–[9]. For instance, AM has helped identify the risk gene MYH9 in idiopathic focal segmental glomerulosclerosis in AA [10], and risk alleles in several genes associated with breast [11] and prostate cancer [12]. In addition to the greater lupus incidence, studying AA populations offers a second advantage. Africans have the smallest haplotype blocks of all human populations: African average population recombination distance is 6 kb, while it is 22 kb in Europeans and Asians [13], [14]. This 3-fold smaller haplotype size gives rise to correspondingly tighter genomic associations in admixed populations such as AA, making causal mutations easier to decipher. Although several genes for SLE susceptibility have been found through candidate gene analysis and genome wide association scans (GWAS), none or very few causal mutations have been identified in each gene. In this study we employed AM in AA to identify admixture signals, and performed a follow-up association study on AA and EA to further identify and localize variants associated with SLE. We experimentally validated predicted variants with biochemistry, cell culture experiments and sequencing of patient-isolated samples. We showed distinct functions of two coding SNPs including changes in gene expression. Through electrophoretic mobility shift assays (EMSAs), protein identification and in vitro protein binding assays, we determined that the intronic SNP disrupts function of a transcriptional enhancer of the IFIH1 locus. Taken together, these results explain the effects of three independent causal mutations on SLE, and begin to elucidate the disparity in disease prevalence between different human populations.
Since case-only analysis has greater statistical power than case-control, we first performed a case-only admixture scan [3], [6], [8] on 1032 AA SLE cases (Figure 1, Table S1). Individual admixture estimates and genome scans for admixture mapping were analyzed using STRUCTURE [15] and ANCESTRYMAP [7] and later verified with ADMIXMAP [6]. As expected, a two-ancestral population model (African and European) best explained the population structure of these samples. By applying the ANCESTRYMAP software, we identified seven potential admixture signals that exceeded our predefined LOD threshold of 2 (Figure 2A, Table S2). Specifically, we identified a genomewide significant association [7] of SLE risk with European ancestry at 2q22–q24 (highest LOD = 6. 28 was achieved between rs6733811 and rs4129786) using a weighted prior risk model; the strongest association at the same locus was observed at a fixed prior risk of 1. 5, which represents a 1. 5-fold increased risk of SLE due to one European ancestral allele at this locus. To evaluate how the prior risk model could influence the ANCESTRYMAP results, we also applied a uniform prior risk model and found consistent genomewide evidence for association at 2q22–q24 (LOD = 5. 86). We also reassessed the strength of the admixture signal at 2q22–24 using alternate markers (LODodd = 3. 65, LODeven = 4. 32), and computer simulation (P = 0. 02). We also validated case-only admixture signals with a case-control admixture scan with 800 ancestry informative markers (AIMs), using 1726 controls from the Dallas Heart Study (DHS) (Table S2). We next repeated the admixture scan using ADMIXMAP, which uses a classical (non-Bayesian) hypothesis test (i. e. , score tests for allelic associations with the trait, conditional on individual admixture and other covariates). All the admixture signals identified by the case-only design using ANCESTRYMAP were strongly validated by ADMIXMAP (Table S2). The strongest peak was identified at 2q22–24 through ADMIXMAP (P = 2. 99×10−8, Table S2). Both AM programs found that the strongest effect was on the AIM rs6733811 (P = 2. 99×10−8, LOD = 5. 65). Two weaker signals were also found on chromosome 2 (LOD = 3. 61, P = 4. 82×10−3; and LOD = 3. 52, P = 1. 64×10−4), as well as on chromosomes 7 (LOD = 3. 26, P = 6. 49×10−6), 9 (LOD = 3. 43, P = 1. 41×10−5), 14 (LOD = 2. 44, P = 3. 84×10−5) and 19 (LOD = 3. 20, P = 1. 15×10−5) (Table S2). To identify SLE-susceptibility gene (s) within 2q22–24, we performed a follow-up case-control (CC) association study in two ethnically diverse groups: CCAA (1525 cases, 1810 controls) and CCEA (3968 cases, 3542 controls) (Figure 1, Table S1). Individual ancestry was estimated using 216 highly informative AIMs. Case-control association tests were performed using 284 SNPs from 20 plausible candidate genes spanning ∼21 megabases of 2q22–q24 (95% CI (142. 4–163. 6), Table S3). Forty-two SNPs from 10 genes (IFIH1, CACNB4, ACVR1C, KCNH7, NEB, STAM2, ZEB2, NMI, ARHGAP15, and ACVR2A) showed significant association for the allelic test (Puncorrected<0. 05) in CCAA, whereas 23 SNPs (in IFIH1, CACNB4, NEB, ARHGAP15, and TNFA1P6) showed significant association in CCEA (Table S4). The strongest associations occurred at IFIH1 (interferon-induced helicase 1; PAA = 3. 52×10−5, PEA = 8. 82×10−5) and CACNB4 (voltage-gated calcium channel, beta subunit; PAA = 9. 07×10−5, PEA = 2. 61×10−2), which are separated by 10. 2 Mb. Among the 22 SNPs tested within IFIH1,13 were significantly (P<0. 05) associated in AA and 4 in EA. Among 23 CACNB4 SNPs, 12 were significant in AA and 2 in EA. Considering the number of associated SNPs, level of replication and involvement in autoimmune phenotypes [16]–[20], we considered IFIH1 as the strongest candidate to explain the admixture-mapping signal. Of the 13 SNPs significantly associated in AA, a preliminary imputation-based association analysis and comparing linkage disequilibrium (LD) determined that 11 were sufficient to tag the 13 associated SNPs (Table S6). To increase the statistical power to detect variants associated with SLE, we genotyped these 11 IFIH1 SNPs in 949 healthy AA controls from the DHS, along with additional out-of-study controls (Figure 1, Table S1). Using single SNP analysis (allelic and genotypic models), followed by conditional analysis and LD analysis across two populations, we detected three SNPs with potentially independent SLE association (Table 1, Figure 2B and 2E). Based on an allelic model, intronic variant rs13023380 [PAA = 4. 33×10−5, PEA = 9. 52×10−11; Pmeta = 5. 20×10−14; OR = 0. 82 (0. 78–0. 87) ], and a missense (Ala946Thr) variant rs1990760 [PAA = 2. 02×10−4, PEA = 1. 22×10−4; Pmeta = 3. 08×10−7; OR = 0. 88 (0. 84–0. 93) ], were associated with SLE in both AA and EA. Another non-synonymous missense variant (Arg460His), rs10930046, was initially associated only with SLE in AA (PAA = 1. 81×10−7; OR = 0. 80 (0. 73–0. 87) ), where the best fit genetic model was identified as dominant (Pdom = 1. 16×10−8, OR = 0. 70 (0. 62–0. 79) ) (Table 1, Table S5). This SNP is rare in EA, having a minor allele frequency (MAF) of only 1. 3% in controls and 1. 6% in cases (PEA = 0. 086) (Table 1). However, after conditioning on the other two associated SNPs (rs13023380 and rs1990760), the rs10930046 became marginally significant in EA (PEA = 0. 017; OR = 1. 2). These SNPs remained significant in both AA and EA after adjusting for ancestry (Table 1). Strikingly, the ancestral alleles at these three SNPs (all ‘G’) are the minor alleles in at least one population: all three ancestral alleles are the minor alleles in EA; the ancestral rs10930046 allele is minor in AA as well. We analytically estimated the joint population attributable risk (PAR) [21] using these three SNPs (rs13023380, rs10930046 and rs1990760) for AA (18. 1%) and EA (14. 7%). Most of the increased PAR (%) in AA was attributable to rs1090046 (12. 5% PAR), whereas for EA very little was attributable to this SNP (0. 3% PAR), likely due to the extremely low MAF. For AA, we also sought to determine how much of the European ancestry risk ratio (λ = 1. 5, estimated by ANCESTRYMAP) was attributable to the three SNPs at 2q22–24. Using the estimated ORs in AAs and the SNP allele frequencies of the two ancestral populations (YRI, the Yoruba people of West Africa, was used as an African ancestral population; CEPH, Utah residents with Northern and Western European heritage, was used as an European ancestral population (Table S8), we estimated the locus-specific ancestry risk ratio (λ; see Methods) for each SNP (λrs1990760 = 1. 12, λrs10930046 = 1. 15, λrs13023380 = 1. 12). Assuming that each SNP contributes to the ancestry risk ratio independently, about 80% of the increased risk due to one copy of the European ancestry alleles estimated from ANCESTRYMAP (∼1. 5) can be explained by the three SNPs at 2q22–24, reinforcing our conclusion from admixture mapping that local European ancestry increases the disease risk at 2q22–24. We also repeated our admixture mapping by stratifying the cases by three genotype (‘AA’, ‘AG’ and ‘GG’) at the most differentiated (FstCEPH-YRI = 0. 38) SNP, rs10930046 (NAA = 279, NAG = 323, NGG = 114). Even with the small samples, we found a dramatically increased risk of European ancestry at rs10930046 (LOD = 10. 78) in the homozygous ‘AA’ compared to the other genotypes, where ancestry association is insignificant (LOD for ‘GG’ = −6. 46 and ‘AG’ = −3. 38). To identify additional SLE-associated variants, we performed an imputation-based association analysis in and around IFIH1 using MACH [22] with reference data from AA (207 controls) and EA (594 controls) using genotyping data from the ImmunoChip (Figure 2B, 2E and Tables S6, S7). Using stringent predefined criteria for imputation, there were 61 additional SNPs for AA, but only 1 for EA later used for conditional analysis. Inefficiency of EA imputation was mainly due to presence of many low frequency (<1%) alleles and strikingly different LD structure (Figure 2D and 2G, Table S7). A pair-wise logistic regression analysis conditioned on each SNP revealed that the three previously identified SNPs were each independently associated with SLE. While in AA, rs13023380, rs10930046 and rs1990760 accounted for the entire association spanning the whole gene (Figure 2B, 2C), in EA, rs13023380 and rs10930046 were independently associated with SLE and accounted for the association (Figure 2E, 2F). Finally, comparing LD (r2) between these three SNPs across nine datasets from seven ethnic populations, we concluded that these three SNPs are also physically independent (Figure S2). Interestingly, using D′ we found that these SNPs are on the same haplotype in EA and AA, but most likely they are not in the ancestral populations (Figure S2). In order to discover the ancestral origin of the risk (‘A’ in each of the 3 SNPs) and protective (‘G’ in each case) alleles for these three SNPs, we estimated local ancestry around the SNPs, then compared (by allele frequency and fixation index) AA individuals whose both haplotypes were European (AAEUR, N = 129) or African (AAAFR, N = 2124), and to individuals from HapMap populations CEPH and YRI (Table S8). Risk allele frequencies derived from the haplotypes were similar between AAEUR and CEPH, and between AAAFR and YRI. Alignment of the human genome with other genomes strongly suggests that the protective alleles (‘G’) are ancestral, and that the risk (‘A’) alleles are derived. For the two coding SNPs, the ‘G’ allele of rs1990760 (and the resulting alanine amino acid) is ∼100% conserved across 34 mammalian genomes (Table S9); the ‘G’ allele of rs10930046 (and the resulting arginine amino acid) is ∼100% conserved across 50 vertebrate genomes (Table S10). Introns are typically less conserved than protein-coding sequence, and accordingly the intronic sequence surrounding the rs13023380 SNP is only strongly conserved in primates; the base corresponding to rs13023380 is ‘G’ in each case (Figure S6). In AA, only the rs10930046 risk allele is major; interestingly, all three ‘A’ risk, derived alleles are the major alleles in EA and the rs10930046 risk allele is almost fixed (Table 1). This suggests a strong selective pressure against the SLE-protective alleles in humans [23], which is not manifest in other animal species. Given the strong association of these three SNPs in IFIH1 with SLE, we evaluated their effect on the function of the IFIH1 gene. IFIH1 has been implicated in binding with dsRNA complexes generated as replication intermediates during RNA viral infections, leading to inflammation and apoptosis [24], [25]. The full length IFIH1 protein contains 1025aa in the following domains: caspase recruitment (CARD) (aa115–200), helicase ATP-binding (aa305–493), helicase C-terminal (aa743–826) and RIG-I regulatory (aa901–1022) (Figure 5A). Deletion of the ATP-binding domain, which includes rs10930046, induces apoptosis in melanoma cells [26]. The RIG-I regulatory domain, which includes rs1990760, recognizes dsRNA, upon which the helicase domains are activated [27]. Apoptosis has been associated with SLE pathogenesis in humans and mice [28]. Furthermore, Ingenuity Pathway Analysis (IPA) indicates that IFIH1 interacts with several genes involved in apoptosis and inflammation (Figure S3). To assess the effects of coding variants in apoptosis and inflammation, we mutagenized IFIH1 cDNA cloned in a mammalian expression vector with a poly-cistronic (IRES) GFP marker at the C-terminus. We over-expressed IFIH1 in a K562 leukemia cell line and measured cell death for each risk SNP, comparing with the ancestral protective allele. The rs10930046 risk allele ‘A’ significantly increased apoptosis over the protective allele ‘G’ (14. 6% average increase at each time point between 44 and 92 hours, P = 0. 014) (Figure 3A). In contrast, the risk allele ‘A’ of rs1990760 had little impact on apoptosis (P = 1. 0), as expected since it is located in the RIG-1 regulatory domain, which is not involved in apoptosis. To assess the effect of these polymorphisms on expression of downstream genes, additional transfected K562 cells were sorted for GFP+ cells by FACS and total RNAs were isolated from these cells. These were subjected to RT-qPCR of 11 genes related to apoptosis, inflammation or viral response: NFκ-B1, NFκ-B2, RELA, CASP8, CASP9, TNFα, MAPK8, MAVS, IFNA, IFIT1 and MX1. Gene expression analysis showed that over-expression of the ‘A’ allele of rs10930046 significantly increased expression of NFκ-B1 (>2. 8-fold, P = 2. 1×10−2), CASP8 (>1. 8-fold, P = 4. 5×10−4), CASP9 (>3. 5-fold, P = 7. 2×10−6) and MAVS (>2-fold, P = 9. 6×10−3) compared to the ‘G’ allele (Figure 3B, 3D, 3E, 3H) but did not affect expression of NFκ-B2 (P = 0. 14), TNFα (P = 0. 7) or MAPK8 (P = 0. 9) (Figure 3C, 3F, 3G). While the ‘A’ allele of rs1990760 had no significant effect on expression of NFκ-B1 (P = 0. 14) or CASP9 (P = 0. 08), it showed a significant decrease of TNFα (>5-fold, P = 1. 8×10−7), NFκ-B2 (>3-fold, P = 1. 3×10−3) and CASP8 (>1. 5-fold, P = 1. 6×10−6) expression (Figure 3B, 3C, 3D, 3E, 3F). The rs1990760 risk allele also significantly increased expression of MAPK8 (>2-fold, P = 6. 2×10−3) and MAVS (>1. 6-fold, P = 8. 5×10−3) (Figure 3G, 3H). Strikingly, interferon alpha (IFNA) expression was reduced for both risk alleles (rs10930046, >2-fold, P = 4. 2×10−3; rs1990760,2-fold, P = 9. 1×10−3) (Figure 3I). Reduced IFNA expression in SLE patients had been predicted for the risk allele of rs1990760 [29]. Our results not only confirmed this but also showed that expression of the risk allele of rs10930046 similarly reduced IFNA expression (Figure 3I). Similarly, expression of IFIT1 was also reduced (rs10930046, >0. 75-fold, P = 8. 7×10−3; rs1990760, >3. 5-fold, P = 9. 1×10−7); and MX1 expression was decreased for rs10930046 (>2. 5-fold, P = 2. 1×10−3) but was increased for rs1990760 (>1. 5-fold, P = 3. 3×10−4) (Figure 3J, 3K). Following induction of transfected cells with Type-1 interferon IFN beta (IFNB), IFIT1 and MX1 showed strong up-regulation (Figure 3L, 3M) by both SNPs (IFIT1: rs10930046, >2-fold, P = 2. 0×10−2; rs1990760, >2-fold, P = 2. 7×10−2; MX1: rs10930046, >1. 3-fold, P = 3. 1×10−2; rs1990760, >2. 2-fold, P = 4. 7×10−5). RELA expression did not change significantly (for rs10930046, P = 0. 61 and for rs1990760, P = 0. 77) for either risk allele (not shown). In our expression analysis, significant up-regulation of CASP8, CASP9 and NFκ-B1 (and unchanged NFκ-B2 and TNFα levels) by the rs10930046 risk allele would be expected to dramatically increase apoptosis, as observed. For rs1990760, levels of these five pro-apoptotic factors are dramatically lowered, consistent with absence of an apoptosis phenotype. MAVS (mitochondrial antiviral-signaling protein) expression was increased for both risk alleles. MAVS is an antiviral protein in the host defense system whose virus-triggered cleavage is necessary to attenuate apoptosis [30], [31]. However, without viral attack MAVS induces apoptosis through caspase and NFKB activation [30]. In our case, it could promote apoptosis, particularly for the risk allele of rs10930046. In terms of inflammation, the expression data shows some interesting effects. For rs10930046, neither TNFα nor MAPK8 changed, but for rs1990760, TNFα decreased while MAPK8 increased leading to inflammation signaling through non-apoptotic pathways. We next examined known transcriptional networks in the context of our expression data. At the root, IFIH1 and type-1 interferons constitute a positive-feedback loop (Figure 3P). We verified this in our cellular model: indeed, in control cells, IFIH1 expression increased several hundred-fold upon IFNB treatment (Figure 3N), and in IFIH1 over-expressing cells, IFNA expression increased (Figure 3O). Taken together, our results support the predicted IFIH1-Type1 interferon feedback loop through IRF7 [32] and MAVS [33]. IFNA and TNFα are known to drive IFIT1 expression [34], and the IFIH1 SNP-driven decrease in IFIT1 may be mediated through decreased IFNA and/or TNFα. MX1 (interferon-induced GTP-binding protein) is also driven by IFNA and TNFα [35]. Surprisingly, although both IFNA and TNFα decreased in the presence of rs1990760 ‘A’, MX1 was significantly up-regulated. This result is similar to a recent paper [29], which showed that when SLE patients' cells were induced with IFNA, the rs1990760 ‘A’ risk allele displayed higher levels of MX1 than ‘G’ allele patients, even though these patients had lower circulating IFNA levels. Our data suggest that IFIH1 risk alleles at these two coding SNPs down-regulate IFNA expression (either through reduced expression or activity of IFIH1) and, in turn, IFNA down-regulates interferon regulatory antiviral genes, potentially conferring viral susceptibility. It is also possible that the rs1990760 risk variant in IFIH1 may increase sensitivity of cells to IFNA pathway activation and subsequent IFN-induced gene transcription [36]. The intronic variant rs13023380 could influence IFIH1 function either by producing a functional miRNA or by altering the binding efficiency to one or more nuclear regulatory proteins. Through bioinformatic analyses (miRBase: http: //www. mirbase. org/) we confirmed that no reported or predicted miRNA-producing or binding sites were present in these sequences. To address whether rs13023380 alters nuclear protein-DNA interaction, we performed EMSAs on nuclear protein extracts from K562 and JURKAT cell lines, using 150-bp PCR products amplified from genomic DNA of ‘AA’ and ‘GG’ homozygous patients. Both PCR products containing the ‘A’ risk sequences or ancestral ‘G’ sequences bound to nuclear protein extract, but DNA containing ‘A’ sequences consistently showed ∼2-fold reduced binding efficiency to a protein complex compared to ‘G’ sequences (Figure 4A, Figure S4F). To identify any DNA-bound proteins, we performed mass spectrometric sequencing (MALDI-TOF) on the protein/DNA complexes isolated using two separate methods: 2D electrophoresis and protein pull-down. In 2D electrophoresis, the visible DNA-bound protein complex in EMSA was excised from a native PAGE gel (Figure S4A–S4C) and sequenced directly, which identified lupus autoantigen Ku70/80 (XRCC5/6), nucleolin (NCL) and HSP90AA1/AB1 as the major constituents of the DNA–protein band (Table S11). Using the second method, we performed EMSA with biotinylated PCR products and pulled down the DNA-bound proteins using immobilized streptavidin-coated agarose beads. Subsequent fractionation by SDS-PAGE (Figure S4D), and sequencing of two distinct visible protein bands (not present in the control pull-down product), confirmed NCL and HSP90AB1 (Table S11). We did not identify Ku70/80 in the streptavidin method, possibly because these two proteins were washed off or were present in insufficient quantities to detect and sequence. However, when we performed “super-shift” assays with antibodies to these proteins, surprisingly, anti-NCL and anti-Ku70/80 antibodies released EMSA-bound DNA instead of super-shifting the complex (Figure S4E). It is possible that the antibodies either induce conformational changes in their targets to release DNA or compete with target proteins for DNA binding. Autoantibodies against NCL and Ku70/Ku80 are characteristic features of SLE [37], [38] and release of free DNA from EMSA-bound DNA in vitro implies that autoantibodies in vivo could impair the function of these proteins by disrupting the binding of bound proteins from target DNA, including the rs13023380 locus. In light of the observed competition of added antibodies to protein-DNA binding, we determined whether purified recombinant proteins of NCL and Ku70/80 bound to these DNAs. Both recombinant proteins, purified from insect cells, produced identical gel shifts as the nuclear extract (Figure 4B, 4C), but again the risk ‘A’ allele bound to the recombinant proteins with ∼2-fold decreased efficiency relative to the protective ‘G’ allele. These results prompted us to enquire whether DNA sequence containing rs13023380 and its surroundings could act as a transcriptional regulatory element (TRE, e. g. enhancer/silencer) in vivo, and if the risk allele has any effect on transcription. The same sequences used for EMSA were cloned before a minimal TKmin promoter and a luciferase reporter gene, and luminescence assays were performed. Both sequences increased reporter gene activity over the core vector, suggesting that the rs13023380 locus contains a transcriptional enhancer. The risk allele-carrying sequences showed almost a 2-fold reduction (Figure 4D) in luciferase activity compared to those with the ancestral allele. Taken together, these results suggest that the rs13023380 locus recruits transcriptional activity of IFIH1 through binding of Ku70/80, NCL and HSP90AA1/AB1 (and potentially more proteins), and that the risk allele at this base position interferes with this enhancer activity, potentially decreasing IFIH1 transcript levels. The absolute conservation of Ala946 (rs1990760, Ala946Thr) in all sequenced mammalian genomes, with diverse codons, strongly suggests selection at the amino acid level (Table S9). We performed molecular modeling of IFIH1-Thr946, based on the protein structure of the IFIH1 C-terminal domain (PDB 2RQB), and the full-length structure of the homologous enzyme RIG-I, bound to dsRNA (PDB 3TMI). Ala946 is placed directly at the mouth of the helicase active site; in RIG-I this region makes contact with the helicase “cap”, which mediates dsRNA entry and processing [27]. Mutation of alanine to the bulker threonine side-chain (Figure 4E, 4F) may alter the sterics and/or dynamics of this protein region, leading to loss-of-function. Similarly, Arg460 (rs10930046, Arg460His) is conserved in all vertebrate genomes sequenced, with diverse codons, again implying amino acid-level selection (Table S10). Comparison with RIG-I (PDB 3TMI) suggests that in the ancestral protein, Arg460 may form hydrogen bonds with the 419–433 loop, most likely with the strictly conserved acidic side-chains of Glu425 and Glu428, and the conserved Gln433 (Figure 4E, 4G). Intriguingly, the crystal structure of the human IFIH1 ATP-binding (DECH) domain (PDB 3B6E) incorporates the pervasive rs10930046 risk mutation. In this structure the His460 side-chain does not make favorable contacts with the 419–433 loop and much of this loop is poorly structured. Loss of stabilizing interactions of Arg460 might lead to weakened structural integrity of the helicase ATP-binding domain (the 3B6E domain is internally shifted ∼1. 5 Å relative to the RIG-I structure; Figure 4G), and subsequently with the helicase C-terminal and RIG-I regulatory domains. DsRNA binding, which occurs at a site proximal to the rs10930046 mutation (Figure 5A), leads to RIG-I dimerization [39]. The disruptive nature of the rs10930046 risk allele on overall protein structural integrity apparently decreases dimerization, as the 3B6E structure was determined as a monomer (all related structures are dimers). Indeed it is likely that the rs10930046 risk allele structure “poisons” an ancestral binding partner, leading to a dominant negative phenotype, consistent with the genetically dominant model, especially in AA. The intronic rs13023380 risk allele has no effect on the protein-coding sequence of IFIH1. The region directly surrounding rs13023380 is rich in strongly conserved C/G bases (Figure S6A). Given the binding of the locus to NCL and other nuclear regulatory proteins, we hypothesized that the site might play a role in mRNA processing. Modeling of the region around rs13023380 predicts a highly structured pre-mRNA, with strongly favorable folding free energies (CentroidFold, ncRNA. org) (Figure S6B). In the ancestral pre-mRNA, the rs13023380 base is part of a highly structured 7-mer RNA stem with a 7-base loop (Figure S6B). In the risk allele pre-mRNA, mutation of the conserved rs13023380 base disrupts RNA stem formation, and likely perturbs structure and stability of the loop (Figure S6C, S6D), which might disrupt the binding of RNA-binding proteins (such as NCL [40]), impairing pre-mRNA trafficking and processing.
Our whole genome admixture scan identified 7 admixture peaks associated with SLE in AA, with the strongest at 2q22–24, containing the IFIH1 gene. Three SNPs (two coding: rs1990760 and rs10930046, and one intronic: rs13023380) accounted for the increased risk. IFIH1 has been associated with Type 1 diabetes (T1D) [41], IgA deficiency [18], Graves' disease [17], and suggestively linked to SLE [20], [42]. The role of IFIH1 in apoptosis and inflammation makes it potentially critical for SLE progression. Moreover, allele frequency differences in associated and non-associated SNPs (high FST values), together with the differences in the number of rare variants between EA and AA, imply a strong positive selection in EA (intriguingly, for the SLE-risk alleles at all three positions), as previously suggested [23]. In AA, local European ancestry at these loci correlates with increased risk. Variant rs1990760 has been recently reported to affect expression of viral resistance genes IFIT1 and MX1 in SLE patients [29]. The risk allele of rs1990760 positively correlated with interferon-induced gene expression in SLE patients who were positive for anti-dsDNA antibodies [29]. Another report on rs1990760 suggested that the risk allele correlated with increased expression of IFIH1 in T1D patients [32]. The rs10930046 risk allele has been implicated in psoriasis susceptibility [43]. Here we have systematically examined the effects of the two coding SNPs on immune cell biology, and demonstrated that the rs10930046 risk allele dramatically increases apoptosis, and that both significantly perturb inflammatory gene profiles. The intronic risk allele disrupts a transcriptional enhancer that recruits nucleolin, lupus autoantigen Ku70/80 and HSP90, potentially decreasing IFIH1 transcript levels. Combined with molecular modeling, our results strongly suggest that these effects are due to several specific amino acid and nucleotide substitutions, rather than to indirect effects due to LD with other SNPs. SLE is commonly identified with an up-regulation of the interferon pathway [44]. Intriguingly, our results suggest that the two non-synonymous IFIH1 mutations down-regulate interferon signaling. However, recent findings demonstrated that SLE patients with anti-DNA antibodies have lower serum IFNA levels [29], and this dose-dependent decrease suggests that there exists a sub-population of SLE patients with lower serum IFNA levels with increased IFN sensitivity [36]. Heterogeneity is also observed in clinical TNFα levels; rs1990760 would seem a likely candidate to be associated with low TNFα levels in this patient sub-population [45]. The intronic SNP (rs13023380) discovered in this study has not been previously implicated in SLE or any other medical condition. The transcriptional enhancer uncovered in this genomic region, and the risk allele' s disruption of its activity, opens up new avenues for investigation. Nucleolin, in addition to contributing to RNA polymerase 1 function [46], is known to be a principal component of the B-cell transcription factor complex LR1 [47], which binds the Ig heavy chain switch region and functions in Ig recombination. Disruption of nucleolin binding to the rs13023380 risk allele may dysregulate polymerase binding, IFIH1 transcription, autoantibody production and interaction. The region surrounding rs13023380 is rich in highly conserved C/G bases, which are preferentially recognized by NCL and Ku70/80 [48], [49]. In addition to perturbing transcription at the locus, molecular modeling implicates the base substitution in destabilizing non-spliced mRNA, further altering proper regulation of expression levels. Nucleolin, as a matrix-binding protein [50], could also provide a scaffold for matrix and DNA during immunoglobulin hyper-recombination. Thus in SLE patients, a positive feedback loop potentially exists where genetic susceptibility creates a biochemical imbalance, dysregulating NCL, which may then promote antibody hypermutation and autoantibody production, further destabilizing the cellular network. Similarly, Ku70/80 facilitates DNA repair and promotes transcription initiation by complexing with RNA polymerase 1. Disruption of Ku70/80 binding to the rs13023380 locus would be expected to have similar consequences for autoantibody production and interaction. Additionally, Ku70/80 mediates the predominant pathway of non-homologous end joining (NHEJ) during immunoglobulin class switch recombination (CSR) [51], [52]. Our discovery that antibodies directed against NCL and Ku70/80 promote release of dsDNA by nuclear proteins suggests that in SLE, hallmark autoantibodies against these two proteins may alter their activity. Thus in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates NCL, Ku70/80, or other nucleic acid regulatory proteins binding to the rs13023380 locus (and other DNA sequences). Follow-up studies could systematically explore the effect of both antibodies and SNP-induced protein mutations on the DNA-binding and transcriptional properties of a number of gene products implicated in SLE and other diseases. From an evolutionary point of view, evidence suggests that IFIH1 is under strong positive selection, especially in EA. The derived allele of rs10930046 (risk in AA) is highly differentiated in ethnically diverse populations and allele frequency increases from AA (60%) to EA (>98%), and may be acting as a protective allele (for some condition other than SLE). This indicates that at some time point, the risk allele may have offered competitive advantage to individuals by increasing apoptosis in defense to new threats of infection encountered during migrations to the New World from Africa [53]. This selection is evident in an observed gradient of geographical distribution (Figure S5) of the allele frequency [53]. Therefore, the different haplotype block structure between the two groups derived from African and European homozygotes is expected. To evaluate the epidemiological significance of IFIH1 polymorphism in the genetic and ethnic background of SLE in AA and EA populations, we estimated the joint PAR. The joint PAR from the three SNPs for AA and EA are 18. 1% and 14. 7%, respectively. Most of the increased PAR in AA was attributable to rs1090046 (12. 5%). Interestingly, the admixture peak at 2q22–24 is associated with increased local European ancestry, suggesting that European ancestry at this locus confers a higher risk of SLE compared to African ancestry. Indeed, we observed the strongest locus-specific LOD score at 2q22–24 using a fixed prior risk of 1. 5, meaning that carrying one European ancestry allele confers 1. 5 fold increased risk of SLE relative to having no European ancestry alleles. Furthermore, we addressed the question whether the locus-specific ancestry risk ratio calculated from the estimated OR of the three SNPs in AA and their allele frequencies in ancestral populations accounts for the European ancestry risk ratio of ∼1. 5 estimated from the admixture scan. The total increased ancestry risk (45%) due to these three SNPs (λrs1990760 = 1. 12, λrs10930046 = 1. 12, λrs13023380 = 1. 16) were close to the increased risk 50% estimated from our admixture mapping (λ = 1. 5). Therefore, the locus-specific ancestry risk ratio corroborates the ancestry risk ratio estimated from the admixture scan. In summary, to our knowledge this is the first study to use a whole-genome admixture mapping design to identify SLE susceptibility loci, confirm case-control association analysis in AA and EA, and identify novel variants within IFIH1 associated with SLE susceptibility. We report three independently associated IFIH1 variants with significant ethnic variation, providing a possible basis for differences in SLE risk between ethnically diverse populations. In addition, we show allele-specific differential cellular signaling and predict an in vivo role of Ku70/80 and NCL autoantibodies that could impair function of IFIH1 by disrupting DNA binding. Therefore, these results clearly establish IFIH1 as an SLE susceptibility gene and provide mechanisms for the IFIH1 variants in SLE etiopathogenesis.
We designed our study in four stages: In Stage 1, we performed a case-only admixture scan on 1032 AA SLE cases (Figure 1, Table S1) followed by exploring the largest admixture peak at 2q22–24 with a more focused candidate gene analysis in both AA and EA populations (Stage 2), including out-of-study controls from dbGaP for AA (2675) and EA (6208) to boost statistical power. In Stage 3, an imputation-based analysis was performed to fine-map our selected gene using SNPs from ImmunoChip arrays [54]. In Stage 4, we experimentally tested the biochemical function of associated SNPs in cellular models, in vitro protein experiments and molecular modeling. For our admixture mapping (AM), we used 1032 African-American (AA) SLE cases and 1726 AA controls (Table S1). Individuals were recruited by the coordinating institutions: Lupus Family Repository and Registry (LFRR) at the Oklahoma Medical Research Foundation (OMRF, 540 AA), and the University of Alabama at Birmingham (UAB) (492 AA) through the PROFILE study group. Cases fulfilled at least 4 of 11 criteria from the American College of Rheumatology (ACR) [55], [56] based on medical record review. In the follow-up case control study (CC), we used both AA (CCAA: 1525 cases and 1810 controls,) and European-Americans (EA) (CCEA: 3968 cases and 3542 controls). There was an overlap of 737 cases between AM and CC. We increased the sample size using out of study controls from publicly available datasets in dbGaP [57] (dbGaP see Web Resources), including 942 AA and 2267 EA controls from the Study of Addiction (SAGE); 784 AA and 1449 EA controls from Health ABC (HABC); 2492 EA controls from the Wellcome Trust Consortium (WTCCC see Web Resources); 949 AA controls from the Dallas Heart Study (DHS see Web Resources) provided by Dr. Helen H. Hobbs. AA and EA samples were collected by the coordinating institutions: LFRR, BIOLUPUS, Medical University of South Carolina, the PROFILE study group, the Oklahoma Lupus cohort, the Feinstein Institute of Medical Research and ODRCC. All individuals were de-identified prior to being genotyped. Within each stage of this experiment, all cases and controls were independent. This study was approved by the Institutional Review Boards of the OMRF or the ethical committees at the institutions where subjects were recruited. Rigorous quality control (QC) was applied to all data used in this study. Subjects were excluded from analysis if they had <95% genotyping success or were population-stratification outliers. Using ancestral informative markers (AIMs), we performed principal components analysis (PCA) using EIGENSOFT [58] (EIGENSOFT see Web Resources) and STRUCTURE [59] (STRUCTURE see Web Resources) to identify outliers, hidden population structure and estimate individual ancestry proportions (European). Relatedness between individuals was calculated using PLINK [60] (PLINK see Web Resources) and GCTA [61]. All related and duplicate individuals (r>0. 25) were removed. SNPs were removed for >10% missing genotyping, being out of Hardy-Weinberg equilibrium (HWE, P<0. 001 in controls) or for poor clustering. SNPs were also removed for minor allele frequency (MAF) <1%. We used AIMs which passed QC, had a minimum intermarker distance >1 Megabase and were not in linkage disequilibrium (LD) in the ancestral populations. In addition to these QC measures, imputed SNPs were included in the analysis only if Rsq >0. 90. This ensures the all high quality imputed SNPs were included in the analysis. Genomic DNA samples in AM from SLE patients were genotyped at OMRF using Affymetrix MALD 3K panel, including 2154 SNPs that passed QC. Fourteen duplicate individuals were removed. DHS samples were genotyped by Perlegen Sciences including 800 AIMs and 10 SNPs within 2q22–24. Samples in the follow-up CC study were genotyped on a custom Illumina iSelect platform. SAGE, HABC and WTCCC genotypes were merged with our data for further QC. PCA was performed for all samples using 401 SNPs to detect PCA outliers from SAGE, HABC and WTCCC. We used 163 AIMs to compare our samples with SAGE, and 755 AIMs to compare our samples with DHS (Figure S1). After QC, individuals were removed from analysis if they were related or duplicate (57 AA and 138 EA), had a missing genotype call rate >5% (19 AA and 361 EA), or were within 2 standard deviations of the mean first eigenvector (30 AA and 51 EA) (Figure S1). Since we used several thousand out of study controls from different publicly available sources [dbGaP (SAGE, HABC) and WTCCC], we had to take very strict QC. Some from the out of study control samples were identified as outliers by PCA; these individuals were mainly the AA samples with >75% European ancestry, and EA samples with <90% European ancestry. All SNPs that passed QC were used in the analysis. After QC our analysis included 1525 AA SLE cases and 1810 AA healthy controls. We genotyped 347 highly informative AIMs to detect hidden population structure and correct for spurious associations. To assess robustness of these results we analyzed 3968 EA cases and 3542 EA. In order to follow the admixture signal within 2q22–24, we genotyped 284 SNPs from 20 candidate genes. The peak and 95% CI of the 2q22–24 region spanned about 21 MB. We used logistic regression in PLINK, with individual admixture estimates as a covariate to identify and remove individual outliers, and to correct for admixture and population stratification in our data analysis. We increased our sample sets using out-of-study controls for a total of 1525 cases and 4485 controls in AA, and 3968 cases and 9750 controls in EA. The URLs for the data presented here are as follows: PLINK, http: //pngu. mgh. harvard. edu/~purcell/plink STRUCTURE, http: //pritch. bsd. uchicago. edu/structure. html EIGENSOFT, http: //genepath. med. harvard. edu/~reich/Software. htm ANCESTRYMAP, http: //genepath. med. harvard. edu/~reich/Software. htm ADMIXMAP, http: //homepages. ed. ac. uk/pmckeigu/admixmap MACH, http: //www. sph. umich. edu/csg/abecasis/MACH/index. html HAPMAP, http: //www. hapmap. org dbGaP, http: //www. ncbi. nlm. nih. gov/gap Wellcome Trust Consortium, http: //www. wtccc. org. uk DHS, http: //clinicaltrials. gov/ct2/show/NCT00344903 Ingenuity Pathway Analysis, http: //www. ingenuity. com Capital Bioscience, MD: http: //www. capitalbiosciences. com/ Becton Dickinson Bioscience: http: //www. bd. com/ Stratagene: www. stratagene. com/ Invitrogen, USA: www. invitrogen. com Totallabquant: http: //www. totallab. com UCSC Genome Browser: http: //genome. ucsc. edu NCBI sequencing Trace Archive: http: //blast. ncbi. nlm. nih. gov ClustalW: http: //www. clustal. org/clustal2/ Protein Data Bank: http: //www. rcsb. org PyMOL: http: //www. pymol. org/ CentroidFold: http: //www. ncrna. org LMBCR: www. ouhsc. edu/lmbcr miRBase: http: //www. mirbase. org/
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.
molecular cell biology
gene expression
genetics
molecular genetics
biology
population genetics
genetics of disease
genetics and genomics
Admixture Mapping in Lupus Identifies Multiple Functional Variants within IFIH1 Associated with Apoptosis, Inflammation, and Autoantibody Production