Behçet disease (BD) is a genetically complex condition that is characterized by recurrent inflammatory attacks that affect the orogenital mucosa, eyes, and skin. Earlier genomewide association studies (GWAS) have linked the IL10 and IL23R-IL12RB2 loci to BD susceptibility [Remmers EF et al. Nat Genet 2010; Mizuki N et al. Nat Genet 2010]. Yohei Kirino, PhD, National Institutes of Health, Bethesda, Maryland, USA presented the findings of a GWAS analysis of imputed genotypes that are associated with BD.

According to Dr. Kirino, whole-genome imputation was used to identify additional BD susceptibility loci using 96 healthy Turkish controls who were genotyped on Illumina HumanOmni1M-Quad single-nucleotide polymorphism (SNP) chips as a reference. Imputation was conducted using MACH v1.0.15, providing 814,474 SNPs for analysis in 1215 BD cases and 1278 healthy controls. Sequenom iPLEX assays were used to validate the imputation results and to fine map the associated region. Two independent replication sets were genotyped for the most significant SNP.

A p-value cutoff of 1×10⁵ identified 114 non-HLA gene SNPs that were suggestive of association with BD. One imputed SNP, rs7616215 on chromosome 3, located approximately 38 kb from the 3′ UTR of the chemokine receptor-1 gene (CCR1) (OR, 0.71; p=1.9×10⁻⁸), exceeded genomewide significance (p<5×10⁻⁸). Fine mapping of the CCR1/CCR3 locus confirmed the imputation results for rs7616215 and identified 2 additional SNPs in strong linkage disequilibrium with rs7616215. They also exceeded genomewide significance.

The association of rs7616215 was replicated using additional Turkish and Japanese BD cases and controls in a meta-analysis of 2195 cases and 2187 controls (OR= 0.73; 95% CI, 0.66 to 0.81; p=1.8×10⁻¹⁰).

CCR1 belongs to the family of CC-motif chemokine receptors. It is expressed on neutrophils, monocytes, and T lymphocytes and binds several chemokine ligands, including CCL5/RANTES, CCL3/MIP-1α, and CCL4/MIP-1β. Serum levels of CCRI ligands, the chemokines MIP-α and RANTES, are increased in BD [Kim SK et al. Scan J Rhematol 2005; Ozer HT et al. Rheumatol Int 2005].

ENCODE data suggest that the CCRI variant could affect transcription of CCR1/CCR3 and that rs7616215 resides in a putative regulatory genomic domain. Analysis of CCR1 transcripts from the HapMap of European, Chinese, and Japanese subjects shows that the protective minor allele (C) correlates with significantly increased CCR1 expression (p<0.03). In addition, STAT4 expression is increased in cells with the BD risk allele. STAT4 is specifically activated by IL-12 and is a critical signaling mediator for the generation of IFN-γ-producing Th1 T cells.

In summary, imputation identified two novel loci, CCR1/CCR3 and STAT4, that were associated with BD. CCR1 expression and leukocyte chemotaxis are higher in individuals with the protective allele. STAT4 expression is higher in individuals with the risk allele. These results implicate leukocyte cell migration and Th1 T cells in the pathogenesis of BD.

To date, HLA-B51 has been the most strongly associated known genetic factor in BD [de Menthon M et al. Arthritis Rheum 2009]. This study's identification of CCR1 and STAT4 as novel gene loci in BD has potential implications in the regulation of inflammatory responses in the context of the disease. Its findings suggest novel therapeutic targets for BD.