The SLE-risk G allele of rs3853839 confers elevated TLR7 expression through slower mRNA degradation.

<p>(A) Association of rs3853839 genotypes with <i>TLR7/8</i> transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter <i>in vitro</i>. <i>TLR7</i> 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in <i>TLR7</i> transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in <i>TLR7</i> transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. ^*<i>P</i><0.05. (E) Summary of the G/C allele ratio in <i>TLR7</i> transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; <i>P</i> = 0.04; paired <i>t</i> test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.</p

Association of rs3853839 with SLE in multiple ancestries.

*<p>AS: Previously published data in population of Eastern Asian descent (5).</p><p>Abbreviation: AS, Eastern Asian; AA, African American; EA, European American; HS, Amerindian/Hispanics; OR, odds ratio; CI, confidence interval.</p

Binding assay for rs13023380 and molecular model of <i>IFIH1</i>.

<p>(A) EMSA was performed using nuclear protein extracts from K562 cells (A) with 141-bp PCR products including either the protective (‘G’) or risk (‘A’) sequence at rs13023380. Both ‘G’ and ‘A’ allele-containing PCR products bound to a protein complex in the nuclear extracts. However, the ‘A’ allele bound with at least 2-fold reduced efficiency compared to the ‘G’ allele-carrying PCR product, as measured by the intensity of the shifted band relative to the free DNA band in the same lane. As a nonspecific (NS) DNA control, a 140-bp DNA sequence not present in the genome was created by PCR amplification of bisulfite-modified genomic DNA. (B, C) EMSA for purified recombinant Nucleolin and Ku70/80 protein with PCR products carrying the ‘G’ or ‘A’ allele of rs13023380. In both the cases, the ‘G’ allele binds both of these proteins with increased efficiency. +signs are used to denote the increasing amount of protein added in the reaction. Numbers below EMSA pictures denote the ratio between the intensities of protein bound DNA to the free DNA. (D) Luciferase activities of intronic DNA sequences carrying ancestral ‘G’ or risk allele ‘A’. The protective allele has approximately 2-fold higher promoter activity (luciferase units) than risk allele ‘A’ carrying sequences. <i>Tkmin</i>-only vector, MCS-vector with multiple cloning sites, 380G-protective allele, 380A-risk allele. (E) Crystal structure of RIG-I in complex with dsRNA (from PDB 3TMI) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003222#pgen.1003222-Jiang1" target="_blank">[27]</a>. Side-chains are shown in red for the positions corresponding to the two coding SNPs in <i>IFIH1</i>. Both mutations are in close proximity to the dsRNA-binding pocket. (F) Close-up of the side-chain of Ala946, modeled from 3TMI. The side-chain makes close contact with the opposing helicase “cap” domain; together these two domains regulate dsRNA entry and processing. Threonine is shown in transparent colors. (G) Superimposition of the RIG-I ATP-binding domain (PDB 4A2W) in blue, and the human <i>IFIH1</i> ATP-binding domain (PDB 3B6E) in green. The <i>IFIH1</i> structure contains the histidine side-chain resulting from the rs10930046 risk allele. Large portions of the <i>IFIH1</i> structure are absent in the 3B6E model, and the two helices are shifted by 1.5 Å. In the ancestral protein, Arg460 likely interacts with the Leu421 main-chain oxygen, as well as the negative helix dipole and the side-chains of Gln433, and Glu425 and 428 (not present in 3B6E).</p

Allelic associations of SNPs in the <i>TLR7-TLR8</i> region with SLE.

<p>(A) The genomic structure of the <i>TLR7-TLR8</i> region and the location of all studied SNPs are indicated. (B) Association signals (−log₁₀<i>P</i>) are plotted against the position of each SNP (based on GRch37/hg19) in European Americans (EA), African Americans (AA), and Hispanics (HS). Genotyped and imputed SNPs are depicted with circles and triangles, respectively. The diamond identifies the <i>TLR7</i> 3′UTR SNP rs3853839. SNPs are highlighted using different colors according to their LD strength (r²) with rs3853839. (C) A trans-ancestral meta-analysis is conducted on 40 genotyped SNPs (circles) and 14 imputed SNPs (triangles) that are shared by the three ancestries (SNPs listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003336#pgen.1003336.s006" target="_blank">Table S1</a>) using fixed and random model, respectively. The dashed line represents the significance level of 5×10⁻⁸.</p

The SLE-risk G allele of rs3853839 displays reduced transcript modulation by miR-3148.

<p>(A) TargetScan's predicted miR-3148-binding site in <i>TLR7</i> 3′UTR. The C allele, rather than G allele of rs3853839 corresponds to the second base of this seed region. (B) Inverse correlation of miR-3148 and <i>TLR7</i> transcript levels in PBMCs from 16 patients with SLE (solid circles) and 21 controls (open diamonds). (C) HEK-293 cells were cotransfected with empty reporter vector (EV), luciferase constructs driven by <i>TLR7</i> 3′UTR segment containing either C or G allele of rs3853839 and increasing concentrations (1, 6, 12, and 48 nM) of miR-3148 or nontarget control (NC) mimics. Luciferase activity was determined 24 hours after transfection. Normalized luciferase activity is the Renilla/Firefly ratio of miR-3148-treated reporter vector compared with the same NC-treated reporter vector. Data show the mean ± SEM and are representative of cumulative data from three independent experiments. <i>P</i> = 0.0003 over all miR-3148-treated C-allele vector groups, and not significant over all miR-3148-treated G-allele or empty vector groups (ANOVA test). <i>P*</i> = 0.02, <i>P**</i><0.0001 (Student's <i>t</i> test) for the comparison of indicated groups.</p

Study design.

<p>Our study design had four stages (sample sizes for cases/controls are in parentheses). In Stage 1, we performed admixture mapping of African Americans (AA) in a case-only analysis with 1032 cases and in a case-control analysis with 1726 controls. In Stage 2, the major admixture mapping signal at 2q22–24 was followed by a candidate gene analysis using case-control association in CC_AA (1525/1810) and European Americans (CC_EA) (3968/3542), with 737 cases used for stages 1 and 2. In order to focus on our best candidate locus (<i>IFIH1</i>), we used out-of-study controls to increase control sample sizes to 4485 for AA and 9750 for EA. In Stage 3, we performed imputation based analysis on AA (1525/4485) and EA (3968/9750) to confirm our candidate gene analysis. In Stage 4, we performed functional analyses for the three confirmed SNPs. For the coding SNPs rs10930046 and rs1990760, we used an apoptosis assay to assess possible changes in protein function, and a gene expression assay to evaluate the effects of these SNPs on expression of genes related to apoptosis, inflammation and viral response. For the intronic variant rs13023380, we used EMSA to investigate whether the variant affected binding of the local DNA sequence to nuclear proteins.</p

Admixture mapping and conditional analysis.

<p>(A) A whole-genome admixture scan on AA SLE cases identified 7 admixture signals that achieved the predefined LOD score >2 (red dashed line). (B) We performed an imputation-based association analysis of <i>IFIH1</i>, which was identified as the most promising candidate gene by a case-control study on 20 candidate genes in the largest peak (2q22–24), followed by 4-SNP haplotype conditional analysis (C). Filled dots indicate the −log₁₀ P values for the association to SLE, and color coding represents inter-marker correlation (r²) between the strongest associated SNP, rs10930046 (“purple diamond”), and the individual SNPs, as shown in the color bar. (C) After conditioning the 4-marker haplotypes for the three markers rs1990760–rs10930046–rs13023380, all individual SNP associations are explained as shown. (D) We analyzed the LD between SNPs on the ImmunoChip, and these LD values were used as a reference panel for imputation in AA. Darker color denotes higher correlation between markers (r²). The LD pattern showed high correlation between markers, making it possible to increase SNP density by imputation. The three independently associated SNPs identified in (B) are denoted by arrows. (E) We performed an imputation based case-control association analysis in EA. Filled dots indicate the −log₁₀ P values for each SNP, and color coding represents the inter-marker correlation (r²) between each individual SNP and the strongest associated SNP, rs13023380 (“purple diamond”), as shown in the color bar. (F) We then performed a two SNP haplotype analysis followed by a three marker haplotype analysis conditioned on the two independent variants rs10930046 and rs13023380. (G) LD analysis of SNPs on the ImmunoChip reference panel showed low inter-marker correlation, which largely precluded imputation based association. Darker color indicates greater r². Arrows indicate the position of the independent SNPs.</p

Clinical criteria sub-analysis results for European-Americans and Hispanics.

<p>F_P = frequency for SLE cases positive for sub-criteria, F_N = frequency for SLE cases negative for sub-criteria, F_U = frequency for controls.</p

SNPs of the <i>IL10</i> gene cluster associated with SLE in European Americans.

<p>(A) Association of 19 genotyped SNPs with SLE in EA (red), AA (yellow), AS (blue) and HS (green). Allelic <i>P</i> value (−log₁₀<i>P</i>) of each SNP was plotted against its genomic position. (B) Association of 19 genotyped and an additional 109 imputed SNPs with SLE in EA. Genotyped and imputed SNPs were indicated as circles and triangles, respectively. Based on its pairwised LD strength with rs3024505, each SNP was highlighted as red (r²>0.9) or grey (r²<0.9). (C) Genomic structure of the <i>IL10</i> gene cluster and the location of each SNP. (D) Haplotypic analysis in EA. Haplotypes were constructed using four SLE-associated SNPs shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003870#pgen-1003870-g001" target="_blank">Figure 1B</a> (rs3024505, rs3024495, rs3024493 and rs3122605), three previously reported SLE-associated SNPs (rs1800872, rs1800871 and rs1800896 in the promoter of <i>IL10</i>) and rs1518111 (the T allele associated with Bechet's disease). Risk alleles of four SLE-associated SNPs shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003870#pgen-1003870-g001" target="_blank">Figure 1B</a> were bolded and italicized.</p

Dose-dependent association of rs3122605 risk G-allele with elevated levels of <i>IL10</i> mRNA and protein.

<p><i>IL10</i> mRNA (A) and protein levels (B) were measured in PBMCs and plasma from EA SLE patients and healthy controls, respectively. Each symbol represents an individual and horizontal lines indicate mean ± SEM values.</p