During the previous reporting periods we established an international consortium for the purpose of identifying and obtaining DNA specimens from at least 1000 well-characterized cases of systemic onset JIA and 1000 ethnically-matched controls. This consortium includes investigators from several of the major pediatric rheumatology centers in North America, including the Childrens Hospital of Philadelphia, Cincinnati Childrens Hospital Medical Center, the Hospital for Sick Children in Toronto, Stanford University, and the University of Utah;from several of the major pediatric rheumatology centers in Europe, including Great Ormand Street Hospital in London, Hopital Necker-Enfants Malades in Paris, the Gaslini Institute in Genova, and the Wilhelmena Hospital in Utrecht;and other centers in Turkey, Argentina, Brazil, and Australia. In early May, 2010, we hosted a meeting of international collaborators at the NIH, in which there were discussions regarding recruitment strategies, appropriate informed consent for genome-wide association studies (GWAS), sample shipment, genotyping, and data analysis. Because of the difficulties for some centers to recruit appropriate ethnically matched controls, where necessary we planned to obtain in silico controls from existing online databases, using bioinformatic tools to ensure appropriate matching by ancestry. All genotyping has been performed in the Inflammatory Disease Section, using Illumina Human Omni 1M-Quad, v1.0 bead chips and an Illumina iScan Beadarray scanner. Altogether, we have generated single nucleotide polymorphism (SNP) genotypes on 988 children with SoJIA and 514 healthy control subjects. These data were combined with SNP genotypes, in silico, from 7370 additional healthy control subjects. After dividing the dataset into 9 strata by country of origin, we excluded samples and markers that failed to meet quality control standards. Haplotype phasing, SNP imputation, and association testing were performed independently in each stratum, and we then subjected the association results from greater than 1.6 million SNPs to fixed- and random-effects meta-analyses. A second round of more intensive imputation employing a more densely genotyped set of reference haplotypes was performed in each region with a minimal P value less than 10 to the negative seventh. Regions with association signals exceeding genomewide significance (P less than 1.7 time 10 to the negative eighth, correcting for multiple models) were further evaluated with logistic regression and conditional analysis. Genome wide meta-analysis of SoJIA identified disease associations with two regions, the major histocompatibility complex (MHC) locus and an intergenic region on chromosome 1, each of which were subjected to a second round of SNP imputation. In both regions, meta-analyses of the second round of imputation data identified significant associations. Meta-analyses of the MHC locus identified two strong association signals, the first centered around HLA-DRB1 (p = 1.6 times ten to the negative tenth, odds ratio = 1.5) and the second located between BTNL2 and HLA-DRA (P = 7.1 times 10 to the negative fifteenth, odds ratio = 2.2). Reciprocal univariate regression demonstrated that these two markers likely represent independent sources of SoJIA risk. On chromosome 1, we identified an SoJIA-associated cluster of 9 SNPs (P = 5.4 times 10 to the negative ninth, odds ratio = 2.0) that was nearest to LOC284661, encoding a long intergenic noncoding RNA. By cross referencing data from the ENCODE project with the 9 SoJIA-associated SNPs, we found evidence of transcriptional activity across the SoJIA-associated region and in close proximity to many of the SoJIA-associated SNPs. Furthermore, several of the SoJIA-associated SNPs were located within known histone marks or transcription factor binding sites. Overall, these data implicate the class II MHC molecule, HLA-DR, in the pathogenesis of SoJIA, and the intergenic location of the most strongly associated variants raises the possibility that an alteration of HLA-DR regulation or expression may underlie its role in SoJIA. The data implicating a long intergenic noncoding RNA is particularly intriguing, since these molecules may play an important role in coordinate regulation of multiple functionally related genes. We are currently preparing a manuscript reporting these findings. Since Dr. Michael Ombrello, the clinical fellow (Metzger Scholar) who has been conducting this project, recently took a position as an Assistant Clinical Investigator in NIAMS, the NHGRI Inflammatory Disease Section will be phasing out its involvement in this project as Dr. Ombrello builds his own independent research program. During the next year the IDS will continue to support Dr. Ombrellos genotyping efforts, and will assist him in developing followup functional studies, and in performing deep resequencing studies in search of rare disease-associated variants.
|Ombrello, Michael J; Remmers, Elaine F; Tachmazidou, Ioanna et al. (2015) HLA-DRB1*11 and variants of the MHC class II locus are strong risk factors for systemic juvenile idiopathic arthritis. Proc Natl Acad Sci U S A 112:15970-5|