Genome-Wide Association Study of African and European Americans Implicates Multiple Shared and Ethnic Specific Loci in Sarcoidosis Susceptibility

<div><p>Sarcoidosis is a systemic inflammatory disease characterized by the formation of granulomas in affected organs. Genome-wide association studies (GWASs) of this disease have been conducted only in European population. We present the first sarcoidosis GWAS in African Americans (AAs, 818 cases and 1,088 related controls) followed by replication in independent sets of AAs (455 cases and 557 controls) and European Americans (EAs, 442 cases and 2,284 controls). We evaluated >6 million SNPs either genotyped using the Illumina Omni1-Quad array or imputed from the 1000 Genomes Project data. We identified a novel sarcoidosis-associated locus, <em>NOTCH4</em>, that reached genome-wide significance in the combined AA samples (rs715299, <em>P</em>_AA-meta = 6.51×10⁻¹⁰) and demonstrated the independence of this locus from others in the MHC region in the same sample. We replicated previous European GWAS associations within <em>HLA-DRA, HLA-DRB5, HLA-DRB1</em>, <em>BTNL2,</em> and <em>ANXA11</em> in both our AA and EA datasets. We also confirmed significant associations to the previously reported <em>HLA-C</em> and <em>HLA-B</em> regions in the EA but not AA samples. We further identified suggestive associations with several other genes previously reported in lung or inflammatory diseases.</p> </div

Sample summary before and after quality control (QC).

a<p>Taken from the Illumina YRI-ASW iControlDB;</p>b<p>175 Caucasian healthy controls from the Illumina iControlDB, 1047 controls from the dbGaP GENEVA Melanoma study, and 1986 controls from the dbGAP CIDR: NGRC Parkinson’s Disease Study.</p

Replication of previously reported SNPs associated with sarcoidosis [9], [11], [25], [27].

1<p>Major/minor allele of AAs as the reference;</p>2<p>Minor allele frequency;</p>3<p>The odds ratio (OR) was calculated with respect to the minor allele of AAs.</p

Regions of association meeting genome-wide significance and their most significant SNPs grouped by sample.

1<p>Major/minor allele of AAs as the reference;</p>2<p>Minor allele frequency;</p>3<p>The odds ratio (OR) was calculated with respect to the minor allele of AAs.</p>a<p>Previously reported sarcoidosis loci meeting genome-wide significance in the AA discovery set.</p>b<p>Potentially novel region meeting genome-wide significance after the meta-analysis of AA datasets.</p>c<p>Previously reported sarcoidosis loci meeting genome-wide significance in the EA dataset.</p><p>Note that stepwise conditional analysis results to identify independent signals within the MHC region can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043907#pone.0043907.s006" target="_blank">Tables S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043907#pone.0043907.s007" target="_blank">S4</a>.</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

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

SLE-associated <i>rs2205960</i> predicted to be part of a decameric motif for NF-κB p65 (RELA).

<p><i>A</i>. Degeneracy within the core 10-base motif is illustrated at all positions apart from position 7 which is non-degenerate by the stacked letters at each position. The relative height of each letter is proportional to its over-enrichment in the motif. A dashed line is boxed around <i>rs2205960-T</i>, this SLE-associated allele is predicted to form the 8^th nucleotide in the motif. Predictions were made using the non-degenerate set of matrix profiles in the Jaspar Core database. <i>B</i>. Altering the <i>rs2205960</i> allele from <i>-T</i> to <i>-G</i> decreases the binding affinity for NF-κB p65 by over 10%. <i>C</i>. Binding of NF-κB at <i>rs2205960</i>, suggested by genome-wide ChIP-seq ENCODE data. Profiles were generated for lymphoblatoid cell lines and stored in the UCSC genome database.</p

Single marker association results for East Asian (As), European (Eur) and Hispanic (Hisp) SLE-control cohorts.

<p>Variants in bold are imputed using 1000 Genomes phase 1 (v3).</p

LD plots at <i>TNFSF4</i> locus in four populations.

<p>This section of chromosome 1q25.1 encompasses the <i>TNFSF4</i> gene and upstream region as defined by custom algorithm in Haploview 4.2. The measure of LD was used to depict 57 SNPs common to all cohorts, post QC and 1000 genomes imputation. The pair-wise correlations between <i>TNFSF4</i> markers is illustrated in these plots by the correlation coefficient R²(where r² = 0 = no correlation, white; 02<1, gradations of grey; R² = 1 = complete correlation, black). The <i>TNFSF4</i> gene is positioned above the plots relative to haplotype blocks (black triangles) and grey ticks indicate SNP locations to scale.</p