Genome-wide association analysis identifies new susceptibility loci for Behçet's disease and epistasis between HLA-B*51 and ERAP1

Abstract

Individuals with Behçet's disease suffer from episodic inflammation often affecting the orogenital mucosa, skin and eyes. To discover new susceptibility loci for Behçet's disease, we performed a genome-wide association study (GWAS) of 779,465 SNPs with imputed genotypes in 1,209 Turkish individuals with Behçet's disease and 1,278 controls. We identified new associations at CCR1, STAT4 and KLRC4. Additionally, two SNPs in ERAP1, encoding ERAP1 p.Asp575Asn and p.Arg725Gln alterations, recessively conferred disease risk. These findings were replicated in 1,468 independent Turkish and/or 1,352 Japanese samples (combined meta-analysis P < 2 × 10(-9)). We also found evidence for interaction between HLA-B*51 and ERAP1 (P = 9 × 10(-4)). The CCR1 and STAT4 variants were associated with gene expression differences. Three risk loci shared with ankylosing spondylitis and psoriasis (the MHC class I region, ERAP1 and IL23R and the MHC class I-ERAP1 interaction), as well as two loci shared with inflammatory bowel disease (IL23R and IL10) implicate shared pathogenic pathways in the spondyloarthritides and Behçet's disease.

Figure 1. Manhattan plot of imputed SNPs

The −log₁₀P values for association of 779,465 autosomal imputed SNPs by basic allelic test in 1,209 Turkish BD cases and 1,278 controls. The results were segregated by chromosome and are shown by genomic position. The red horizontal line indicates genome-wide significance, p=5 × 10⁻⁸.

Figure 2. Regional association plots

Regional association plots (-log₁₀ P values) and LD structures of the disease-associated regions surrounding (a) CCR1-CCR3, (b) STAT4, (c) KLRK1-KLRC1, and (d) ERAP1-ERAP2. The associations in (a-c) are for basic allelic tests, whereas the associations in (d) are for a recessive model test. Data are from the discovery collection with the genome-wide analysis genotypes (blue), imputed genotypes (green) and the fine-mapping genotypes (red). Orange diamonds show the meta-analyses results described in Table 1. Red horizontal lines in (a-c) indicate genome-wide significance (p=5 × 10⁻⁸) and in (d) indicates genome-wide significance accounting for three genetic models (p=1.67 × 10⁻⁸). SNP rs7574865 in (b) is a STAT4 intronic SNP associated with multiple autoimmune diseases. The LD structures of the same regions are shown in the upper panels, with red filled squares linking pairs of markers that indicate the strength of LD by intensity of fill: D′ = 1 (intense red) to D′ = 0 (no fill).

Figure 3. Epistasis between HLA-B*51 and ERAP1 rs17482078 (ERAP1 p.Arg725Gln) coding variant in BD

Epistasis was analyzed in the combined Turkish GWAS and replication samples. HLA-B*51+ indicates individuals either heterozygous or homozygous for HLA-B*51. In the replication samples, the surrogate marker rs2848713 (r²=0.68 with HLA-B*51 in Turkish GWAS) was used to predict HLA-B*51 positivity. Odds ratios for BD were determined comparing the frequency of the two marker genotypes in cases versus controls. The odds ratios shown are relative to the lowest disease-risk genotype group (HLA-B*51 negative and ERAP1 rs17482078 CC). Error bars represent the 95% confidence intervals. In an analysis restricted to the HLA-B*51 positive individuals, the ERAP1 TT genotype had an odds ratio of 3.78 (95% CI: 1.94-7.35), whereas in the HLA-B*51 negative individuals, the TT genotype odds ratio was 1.48 (95% CI: 0.78-2.80).

Figure 4. Association of CCR1 and STAT4 variants with gene expression levels and function in human cells

(a) Mean CCR1 mRNA expression in human primary monocytes according to rs7616215 genotype (data from Zeller et al). Error bars represent the standard error of the mean. (b) CCR1 mRNA expression in human primary monocytes from our study according to rs7616215 genotype. (c) Chemotaxis of human primary monocytes against a gradient of MIP1-α according to rs7616215 genotype. “Migration index” indicates number of migrated cells in the presence of MIP1-α divided by the number of migrated cells in the absence of MIP1-α from the same sample. (d) Correlation between monocyte CCR1 expression and chemotactic activity. Comparison between CCR1 mRNA level and chemotaxis migration index were determined in monocytes isolated from the same blood sample. Spearman correlation test was performed. (e) STAT4 mRNA expression in Human Variation Panel samples (GEO GSE24277). rs7572482 is a surrogate marker for rs7574070 (r²=1, D′=1 in HapMap CEU samples). (f) STAT4 mRNA expression in HapMap JPT and CHB samples according to rs7574070 genotype (GEO GSE6536). In panels b, c, e, and f, the horizontal bars indicate median values and the p-values shown are from the Kruskal-Wallis rank sum test.