High-Resolution Analysis of the HLA Locus in BD Susceptibility The HLA protein, HLA-B*51, encoded by HLA-B in the MHC, is the strongest known genetic risk factor for BD. Associations between BD and other factors within the MHC have been reported also, although strong regional linkage disequilibrium complicates their confident disentanglement from HLA-B*51. In the current study, we examined a combination of directly obtained and imputed MHC-region SNPs, directly obtained HLA-B locus types, and imputed classical HLA types with their corresponding polymorphic amino acid residues for association with BD in 1190 cases and 1257 controls. SNP mapping with logistic regression of the MHC identified the HLA-B/MICA region and the region between HLA-F and HLA-A as independently associated with BD (P <1.7 x 10 to the negative 8th). HLA-B*51, -A*03, -B*15, -B*27, -B*49, -B*57, and A*26 each contributed separately to BD risk. We directly examined rs116799036, a MICA-associated noncoding SNP upstream of HLA-B that was recently suggested to underlie the association of HLA-B*51 with BD, but we were unable to replicate that finding in our collection. Instead, we mapped the BD association to seven MHC class I (MHC-I) amino acid residues, including anchor residues that critically define the selection and binding of peptides to MHC-I molecules, residues known to influence MHC-I-killer immunoglobulin-like receptor interactions, and a residue located in the signal peptide of HLA-B. The locations of these variants collectively implicate MHC-I peptide binding in the pathophysiology of BD. Furthermore, several lines of evidence suggest a role for altered regulation of cellular cytotoxicity in BD pathogenesis. High-Density Genotyping of Immune-Related Genes We have genotyped 1900 Turkish BD patients and 1799 controls with the Immunochip, a custom array with 196,524 markers in 186 loci selected from analysis of 12 autoimmune diseases. After quality control, we performed association tests. For novel loci with association test P <5x10 to the negative sixth, additional SNPs in the region were imputed using the 1000 Genomes reference. For replication, we imputed the genotype data of 608 Japanese cases and 737 controls from a previous GWAS. The Cochran-Mantel-Haenszel test was performed for meta-analysis. Thresholds for statistical significance were 5 x 10 to the negative 8th and 1.67 x 10 to the negative 8th in the basic allele test analysis and the three model (additive, dominant, recessive) genotypic analysis, respectively. The basic allele association test identified 4 novel loci, IL1A-IL1B, SHCIP1-IL12A, IRF8, and PTPN1, which exceeded genome-wide significance. In addition, the FUT2 locus showed genome-wide significance in the dominant model genotypic analysis. Imputation data provided an additional novel locus with genome-wide significance, EGR2. Meta-analysis of the Turkish and Japanese population data revealed novel loci, RIPK2 and LACC1. These results greatly expand the list of genes with common variants that influence BD susceptibility and implicate variants influencing both innate and adaptive immunity in disease pathogenesis. Analysis of Killer Immunoglobulin-like Receptor (KIR) Genes in BD Susceptibility HLA-B*51 (B*51) is a ligand for a pair of allelic KIR receptors present on cytotoxic cells, KIR3DL1, which inhibits their cytotoxicity, and KIR3DS1, which activates their cytotoxic activity. KIRs are inherited on evolutionarily conserved haplotypes in which KIR3DL1 and KIR3DS1 are mutually exclusive. We therefore tested the hypothesis that KIR-regulated cytotoxic mechanisms contribute to BD by testing for association between KIR3DL1/KIR3DS1 alleles and BD in Turkish individuals. Turkish BD patients (n = 1900) and controls (n = 1779) were genotyped for the KIR3DL1 and KIR3DS1 alleles with two sequence-specific PCR assays. Genotypes of 6994 SNPs from the HLA region were determined with the Immunochip (Illumina) and used to impute the individuals HLA types using SNP2HLA and a reference panel of 5225 European individuals. A chi squared test for association was used to evaluate the contribution of KIR3DL1/KIR3DS1 to BD. A P-value less than 0.05 was considered significant. Classical HLA types were determined by imputation in all samples and types with posterior probability greater than 0.9 were included in analyses. KIR3DL1 and KIR3DS1 genotypes were determined for 1799 of the cases and 1710 controls. In these subjects, the presence of the cytotoxic-activating KIR3DS1 allele did not differ significantly between cases and controls (42.7% vs. 41.0%, P = 0.29). Furthermore, the activating allele did not appear to interact with HLA-B alleles. It was present at similar frequencies in B*51-positive cases and controls (44.3% vs. 43.0%, P = 0.63), in Bw4-positive cases and controls (43.0% vs. 41.1%, P = 0.31), and in cases and controls bearing the Bw4 motif with isoleucine at position 80 (43.7% vs. 41.4%, P = 0.32). Similarly, no disease association was found for the inhibitory KIR3DL1 allele in all the samples or in any of the HLA-B subsets. In conclusion, we found no association of BD with the presence of the KIR3D activating (KIR3DS1) or inhibitory (KIR3DL1) receptors, which together regulate cytotoxic activity by binding a subset of HLA class I molecules, including the BD-associated HLA-B*51. Due to the complexity of this locus (i.e., sequence variation, copy number variation), lack of association between BD and the presence/absence of KIR3DS1 or KIR3DL1 does not exclude a role for KIRs in the pathogenesis of BD, and thus further studies of KIR3DL1/KIR3DS1 types and copy number variants, as well as of other KIRs, are warranted.
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