During the current reporting period, we have continued to explore genetic and genomic factors that influence susceptibility to sJIA. We are conducting a genome-wide association study (GWAS) of sJIA in collaboration with investigators from the International Childhood Arthritis Genetics consortium (INCHARGE). INCHARGE includes investigators from major pediatric rheumatology centers in North America, South America, and Europe. We have generated single nucleotide polymorphism (SNP) genotype data for 988 children with sJIA and 431 healthy control subjects, and have combined these data with SNP genotypes, in silico, from 7579 additional healthy control subjects. The resulting dataset was divided into 9 strata by country of origin and strict quality control procedures were undertaken. Haplotype phasing, SNP imputation, and association testing of over 6.7 million SNPs were performed separately in each stratum, and the results were meta-analyzed. During this reporting period, this process was refined to utilize improved computational algorithms and the latest release of the 1000 Genomes project dataset. The major histocompatibility complex (MHC) locus was identified as the strongest sJIA susceptibility locus. Within the MHC locus, 482 sJIA-associated SNPs were mostly located in the class II human leukocyte antigen (HLA) gene cluster between NOTCH4 and HLA-DQB1. Within this region, there were two predominant peaks of association, the first in the BTNL2/HLA-DRA region (top SNP p = 2.8 times 10 to the negative 17, odds ratio = 2.6) and the second in the HLA-DRB1/HLA-DQA1 region (top SNP p = 1.9 times 10 to the negative 11, odds ratio = 2.2). Univariate regression controlling for the SNP most strongly associated with sJIA (near BTNL2/HLA-DRA) revealed an independent effect of variants near HLA-DRB1/HLA-DQA1 on sJIA risk (conditional p = 1 times 10 to the negative 5, odds ratio 0.7). Using HLA imputation, a statistical process that utilizes SNP genotype data to predict HLA alleles with high accuracy, we determined the classical HLA alleles in the 6 study populations of western European ancestry. Association meta-analysis of classical HLA alleles identified a strong association between sJIA and the class II HLA allele, HLA-DRB1*11 (p = 2.7 times 10 to the negative 16, odds ratio = 2.3). Furthermore, association meta-analysis of imputed HLA haplotypes revealed that the haplotype of HLA-DRB1*11/HLA-DQA1*05/HLA-DQB1*03 was also strongly associated with sJIA (p = 6.4 times 10 to the negative 17, odds ratio = 2.3). Overall, these data implicate the HLA-DR and HLA-DQ loci in the pathogenesis of sJIA. A manuscript describing the link between the MHC locus and sJIA is currently under review. The strong association of HLA-DRB1*11 with sJIA may indicate a role for the presentation of one or more specific antigens to helper T-cells in the pathophysiology of sJIA. Alternatively, the fact that the SNPs most strongly associated with sJIA do not alter protein structures raises the possibility that alteration of HLA-DR regulation or expression may underlie its role in sJIA. To address this question, we cross-referenced the list of sJIA-associated, non-coding, MHC region SNPs with publicly available regulatory and epigenetic data databases (IE. ENCODE and NIH Roadmap Epigenome Projects). This revealed a panel of 18 sJIA-associated SNPs that have been experimentally linked to the expression levels of a class II HLA gene, and for which strong evidence also supported an effect on transcription factor binding. Moreover, we found that sJIA-associated SNPs in the class II HLA locus were enriched within super-enhancers that were cell type-specific to B cells and monocytes (as reported by Hnisz et al., Cell 2013). Based on these findings, we are undertaking experiments to examine the effects of this group of sJIA-associated SNPs on transcription factor binding and gene expression in these cell types. Outside of the MHC locus, the sJIA GWAS has also identified 21 novel sJIA candidate susceptibility loci (p < 1 times 10 to the negative 5), the strongest of which was a susceptibility locus in a non-coding region of chromosome 1. As a secondary means of identifying sJIA susceptibility loci, we have used the panel of SNPs with at least modest association with sJIA (p < 1 times 10 to the negative 5) to perform gene-based association testing. This identified a list of over 100 genes that were associated with sJIA. We are now undertaking pathway and network-based analyses to determine whether specific biologic processes or pathways are enriched with sJIA-associated genes. In contrast to the GWAS of sJIA, which was undertaken to explore the role of common genetic variants in sJIA and identify susceptibility loci across the entire genome, we have initiated a targeted deep resequencing study of sJIA candidate genes to thoroughly interrogate genetic variation within a small set of genes and loci of interest. A key difference between these two studies is the ability of the resequencing study to identify rare or novel genetic variants that contribute to sJIA risk. The candidate genes and loci selected for resequencing were genes previously reported as sJIA-associated, genes known to cause monogenic periodic fever syndromes, genes known to cause hemophagocytic lymphohistiocytosis, and genes for which a modest association with sJIA (p < 1 times 10 to the negative 5) was identified in a preliminary analysis of the sJIA GWAS. In total, we have re-sequenced 100 sJIA candidate genes and loci in a collection of approximately 500 sJIA cases of Western European ancestry and 500 healthy control subjects of matched ancestry, and the analyses of these data are ongoing. In addition to sJIA, we have studied several other inflammatory phenotypes. In collaboration with the Milner group at NIAID, we have continued to investigate the genetic and molecular underpinnings of PLCG2-associated antibody deficiency and immune dysregulation (PLAID). We have examined a total of 36 subjects with PLAID or PLAID-like phenotypes, specifically evaporative cold urticaria and immune dysregulation. In many of these patients, causative mutations of PLCG2 were not identified, whereas the mutation-negative patients were phenotypically indistinguishable from those bearing PLCG2 mutations with respect to cold urticaria, cold-induced leukocyte activation, or the presence of neonatal-onset ulcerative and cutaneous lesions in cold-sensitive areas of the body. We have published these observations in JAMA Dermatology. In collaboration with investigators from NIAMS, NIAID, and several centers in Spain, we examined the relationship between food-related anaphylaxis and nonsteroidal anti-inflammatory drugs (NSAIDs). Lipid transport protein (LTP), an abundant plant protein, is a common food allergen in Mediterranean areas that causes a wide range of allergic reactions. 40% of cases of LTP-induced anaphylaxis (LTP-A) also require the presence of NSAIDs as a co-factor. To understand the relationship between LTP-A and NSAIDs, we compared subjects with LTP-A and NSAID-dependent LTP-A (NSAID-LTP-A) to healthy subjects. Whole transcriptome analysis identified altered expression patterns of genes regulating gastrointestinal epithelial renewal in both sets of patients. Changes indicative of inflammatory disease were also identified in the LTP-A group, including changes in B cell pathways, and increases in neutrophil activation markers and reactive oxygen species. In contrast, the NSAID-LTP-A group demonstrated reductions of interferon-gamma (IFNG) and IFNG-regulated genes, together with upregulated expression of adenosine receptor 3 and genes related to adenosine metabolism. These observations may explain the pathogenic divergence between the two processes. This work was published in the Journal of Allergy and Clinical Immunology.