Imputation of BD SNP data To discover additional BD susceptibility loci, we carried out whole-genome imputation using as a reference 96 Turkish healthy controls genotyped on Illumina HumanOmni1M-Quad SNP chips. Imputation was conducted using MACH v1.0.15 providing 779,465 SNPs for analysis in 1,209 Turkish BD cases and 1,278 healthy controls from our initial collection. Sequenom iPLEX assays were used to validate the imputation results and to fine map the associated region. Two independent replication sets comprising 838 Turkish cases and 630 controls, as well as 612 Japanese cases and 740 controls (if the SNP was polymorphic in the Japanese collection), were genotyped for the most significant SNPs. By Cochran-Mantel-Haenszel (CMH) meta-analysis, we identified associations at CCR1 (rs7616215, P = 4.30 times 10 to the negative thirteenth, OR = 0.72), encoding a chemokine receptor expressed on myeloid cells;KLRC4 (rs2617170, P = 1.34 times 10 to the negative ninth, OR = 0.78), encoding a receptor found on natural killer (NK) cells and gamma-delta T-cells;and STAT4 (rs7574070, P = 1.29 times 10 to the negative ninth, OR = 1.27), encoding a transcription factor expressed in T-lymphocytes. The protective CCR1 C-allele was associated with higher mRNA expression and increased leukocyte chemotaxis. The STAT4 risk allele was also associated with increased mRNA expression. It differs from rs7574865, a SNP associated with rheumatoid arthritis, systemic lupus erythematosus, and several other autoimmune diseases, although both reside in the third intron of STAT4. It is also noteworthy that we found evidence for epistasis between KLRC4, which encodes an NK cell receptor, and the disease-associated HLA-B*51/MICA haplotype, which encodes an NK receptor ligand. Dr. Yohei Kirino presented this work in a plenary lecture at the annual meeting of the American College of Rheumatology in November, 2011, and we currently have a manuscript under revision that describes our findings. Clinical Subset Analyses and Gene-Gene Interactions In the subset of BD patients with uveitis, we found that two coding SNPs in ERAP1 conferred risk for BD under a recessive model. By CMH meta-analysis of the 435 case BD uveitis discovery collection and 1,278 controls, and a 370 case uveitis replication group with 630 Turkish controls, the overall P value for the R725Q coding variant was 4.73 times 10 to the negative eleventh, with an OR of 4.56. Moreover, a CMH meta-analysis of this SNP in the GWAS and replication collections (i.e., including both uveitis and non-uveitis BD patients) found significant association of the homozygous R725Q genotype with BD susceptibility (P = 4.35 times 10 to the negative eighth, OR = 3.08). ERAP1 is an endoplasmic reticulum-expressed amino peptidase, which functions to trim peptides and load them onto MHC Class I. Of note, the R725Q ERAP1 variant confers protection from ankylosing spondylitis and psoriasis, but only in patients who are HLA-B*27 positive in AS (Evans et al., Nature Genet 2011) or HLA-C*06 positive in psoriasis (Strange et al., Nature Genet 2010). Similarly, by logistic regression we found that ERAP1 R725Q preferentially conferred risk for BD in HLA-B*51 positive individuals (P = 0.0009). These data suggest that the disease-associated peptidase variant contributes to BD susceptibility through an interaction, perhaps mediated by specific peptides produced by the variant peptidase, with the HLA-B*51 protein, and suggest a mechanistic explanation for the long-observed but poorly understood HLA-B*51 association with BD. Our findings add substantially to an emerging body of data delineating common pathogenetic mechanisms for BD, ankylosing spondylitis, and psoriasis. All 3 are inflammatory disorders affecting the skin, eyes, and joints, all 3 have important HLA Class I associations, variants of the IL-23 receptor are associated with susceptibility in all 3, and now there is convincing evidence in all 3 diseases for epistasis between Class I MHC and ERAP1. Copy Number Variation Analysis SNP intensity data from our previous BD Illumina 370,000 SNP GWAS was used for the analysis. We evaluated homozygous deleted regions in a genome-wide fashion, thus applying a recessive model for copy number variation association. The detected common CNV was validated with real-time PCR and a break-point assay. Meta-analysis across various inflammatory diseases was performed. We identified a haplotype tagged by a 3.2 kb deletion polymorphism within the first intron of the LEPREL1 gene that is associated with protection against BD in multiple populations (combined cases = 2056, controls = 2120, OR = 0.63, P = 7.89 times 10 to the negative sixth). The LEPREL1 homozygous deletion was also found to be protective against various inflammatory diseases (cases = 9082, controls = 9243, OR = 0.85, P = 3.4 times 10 to the negative fourth). LEPREL1, which encodes prolyl 3-hydroxylase 2 (P3H2), was found to be expressed primarily in plasmacytoid/lymphocytoid dendritic cells (DCs) and lipopolysaccharide induced its expression in a murine DC line. Allelic expression analysis in cells heterozygous for the deletion showed that LEPREL1 mRNA is lower from the allele with the deletion. Individuals with the homozygous deletion produced lower levels of TNF-alpha in PBMC stimulated with innate-immune agonists. A manuscript reporting these findings is in preparation. Deep Resequencing of Targeted Genes To determine whether rare and low frequency variants are associated with BD, we have performed deep re-sequencing of two GWAS-identified genes (IL23R and IL10) and 11 genes known to have roles in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD ASC, NOD2, and TLR4) in 766 Japanese and 768 Turkish samples (equal numbers of cases and controls, sequenced by a pooling strategy). Non-synonymous variants identified by deep exonic re-sequencing were validated and evaluated by individual genotyping of 4,955 samples, including the discovery collection. Applying the C-alpha test for the statistical analysis of each gene, we found a non-random distribution of rare and low-frequency variants in cases and controls that implicated IL23R, MEFV, TLR4, and NOD2 in BD susceptibility. Associations of variants in IL23R in both populations and TLR4 in the Turkish population were corroborated by the adaptive sum test and step-up approaches. Carriage of the MEFV-M694V mutation, known to cause FMF, conferred BD risk in Turkish samples (OR = 2.65, P = 1.79 times 10 to the negative twelfth). These findings implicate innate immune and bacterial sensing mechanisms in BD pathogenesis. We are currently analyzing rare and low-frequency variants in the genes identified in the latest round of GWAS (CCR1, STAT4, KLRK1, KLRC1-4, and ERAP1). Because a previous Turkish familial BD linkage study showed the strongest peak in the KLRK1 complex, we also included those familial BD cases in the analysis for the KLRK1 and KLRC1-4 NK receptor complex. During the upcoming reporting period, our efforts will be directed towards: (1) completing and publishing four manuscripts reporting the findings described above;(2) following up on functional analyses of genetic variants identified by GWAS;(3) further delineating the role of HLA and interacting genes in BD;(4) additional targeted high-density genotyping with the Illumina Immunochip;and (5) developing therapeutic strategies that target the immunologic pathways identified by these genetic studies.

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Takeuchi, Masaki; Ombrello, Michael J; Kirino, Yohei et al. (2016) A single endoplasmic reticulum aminopeptidase-1 protein allotype is a strong risk factor for Behçet's disease in HLA-B*51 carriers. Ann Rheum Dis 75:2208-2211
Zhou, Qing; Wang, Hongying; Schwartz, Daniella M et al. (2016) Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease. Nat Genet 48:67-73
Takeuchi, Masaki; Kastner, Daniel L; Remmers, Elaine F (2015) The immunogenetics of Behçet's disease: A comprehensive review. J Autoimmun 64:137-48
Ombrello, Michael J; Kastner, Daniel L; Remmers, Elaine F (2015) Endoplasmic reticulum-associated amino-peptidase 1 and rheumatic disease: genetics. Curr Opin Rheumatol 27:349-56
Bakir-Gungor, Burcu; Remmers, Elaine F; Meguro, Akira et al. (2015) Reply to Stoimenis et al. Eur J Hum Genet 23:1280
Ombrello, Michael J; Sikora, Keith A; Kastner, Daniel L (2014) Genetics, genomics, and their relevance to pathology and therapy. Best Pract Res Clin Rheumatol 28:175-89
Ombrello, Michael J; Kirino, Yohei; de Bakker, Paul I W et al. (2014) Behçet disease-associated MHC class I residues implicate antigen binding and regulation of cell-mediated cytotoxicity. Proc Natl Acad Sci U S A 111:8867-72
Bakir-Gungor, Burcu; Remmers, Elaine F; Meguro, Akira et al. (2014) Identification of possible pathogenic pathways in Behcet's disease using genome-wide association study data from two different populations. Eur J Hum Genet :
Kirino, Yohei; Zhou, Qing; Ishigatsubo, Yoshiaki et al. (2013) Targeted resequencing implicates the familial Mediterranean fever gene MEFV and the toll-like receptor 4 gene TLR4 in Behcet disease. Proc Natl Acad Sci U S A 110:8134-9
Kirino, Yohei; Bertsias, George; Ishigatsubo, Yoshiaki et al. (2013) Genome-wide association analysis identifies new susceptibility loci for Behçet's disease and epistasis between HLA-B*51 and ERAP1. Nat Genet 45:202-7

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