Genetics of Systemic Juvenile Idiopathic Arthritis Systemic juvenile idiopathic arthritis (sJIA) is an often severe, potentially life-threatening childhood inflammatory disease whose pathophysiology is poorly understood. To determine whether genetic variation within the major histocompatibility complex (MHC) locus on chromosome 6 influences sJIA susceptibility, we performed an association study of 982 children with sJIA and 8010 healthy control subjects from 9 countries. Using meta-analysis of directly observed and imputed single nucleotide polymorphism (SNP) genotypes and imputed classical human leukocyte antigen (HLA) types, we identified the MHC locus as a bona fide susceptibility locus with effects on sJIA risk that transcended geographically-defined strata. The strongest sJIA-associated SNP, rs151043342 (p = 2.8 x 10-17, OR 2.6), was part of a cluster of 482 sJIA-associated SNPs that spanned a 400 kb region and included the class II HLA region. Conditional analysis controlling for the effect of rs151043342 found that rs12722051 independently influenced sJIA risk (p = 1.0 x 10-5, OR 0.7). Meta-analysis of imputed classical HLA type associations in 6 study populations of Western European ancestry revealed that HLA-DRB1*11 and its defining amino acid residue, glutamate 58, were strongly associated with sJIA (p = 2.7 x 10-16, OR 2.3), as was the HLA-DRB1*11HLA-DQA1*05HLA-DQB1*03 haplotype (6.4 x 10-17, OR 2.3). By examining the MHC locus in the largest collection of sJIA patients assembled to date, this study solidifies the relationship between the class II HLA region and sJIA, implicating adaptive immune molecules in the pathogenesis of sJIA. A manuscript describing these findings has been submitted for publication. Genetics of Scleroderma in the African-American Population Scleroderma is an autoimmune disorder that is characterized by thickening and fibrosis of the skin, with systemic manifestations that can include renal involvement with accelerated hypertension, pulmonary involvement manifesting either pulmonary fibrosis or pulmonary arterial hypertension, gastrointestinal involvement with esophageal dysmotility, and cardiac involvement with various electrical conduction defects. Scleroderma remains one of the greatest challenges in clinical rheumatology, and although the advent of angiotensin converting enzyme inhibitors has had a major impact on scleroderma renal disease, treatment options for the involvement of other organs, particularly the lungs, are limited. Like many other autoimmune diseases, scleroderma does not usually present with a Mendelian pattern of inheritance, but the risk of developing scleroderma is greater for someone with an affected relative than for someone in the general population. The rationale for studying the genetics of scleroderma in the African-American population is that the disease appears to be more frequent and more severe among African-Americans than among the Caucasian population. It is therefore possible that genetic studies of scleroderma in African-Americans will uncover heretofore-unrecognized genes and pathways contributing to the pathogenesis of this illness and related fibrosing disorders. In addition, it may be possible to utilize admixture mapping techniques to identify risk alleles traceable to the African ancestry of these patients. This study has 4 phases: Phase 1, sample collection: We have established a large multicenter consortium denoted GRASP (Genome Research in African-American Scleroderma Patients) to recruit 1000 African-American scleroderma subjects. This consortium includes Johns Hopkins University, the University of California at San Francisco, the University of Texas at Houston, George Washington University, Northwestern University, Hospital for Special Surgery, Medical University of South Carolina, Tulane, Emory, University of Alabama at Birmingham, University of Pennsylvania, Stanford, Georgetown University, University of Michigan, University of Pittsburgh, Rutgers, University of Chicago, and New York University. Along with the DNA samples, detailed clinical information, autoantibody status, imaging results, demographic information, medication history, and disease progression are being captured in the clinical data collection form. To date GRASP has collected 969 African-American scleroderma samples, already the largest DNA collection from African-American scleroderma patients in existence. Dr Charles Rotimi of NHGRI has readily available DNA on 2000 African-American control samples. The sera from 1062 control samples was tested for anti-nuclear antibody by immunofluorescence and 1039 samples were found to be ANA negative at a titer of 1:80. 1000 controls have been selected from these 1039 controls for this study. Phase 2a, whole exome sequencing in African American scleroderma cases and controls: 400 African-American scleroderma patient DNAs along with 400 healthy control DNAs were submitted to the NIH Intramural Sequencing Center for whole exome sequencing (WES) to identify scleroderma-associated variants. The Nimblegen SeqCap EZ Exome+UTR kit was used for exome capture and the Illumina HiSeq2500 for sequencing. This capture kit targets 64 Mb of coding exons and miRNA regions, plus 32 Mb untranslated regions (UTRs). Novoalign was used to align the sequences and the NISC MPG pipeline was used to call the variants. We were able to obtain from Dr Rotimi an additional 80 WES from healthy African American controls using the same capture kit. We are currently combining these additional 80 controls with our 800 samples using the NISC MPG pipeline. We are also using the GATK pipeline for variant calling to complement the NISC MPG pipeline variant calls. Phase 2b, selection of rare variants from WES for custom genotyping array fabrication: we have identified 20,000 variants from the WES to be added to the Illumina Human OmniExpress Exome array. We selected exomic variants present in at least 2 individuals in the combined cohort of cases and controls, avoiding homozygous individuals, and removing variants already present on the genotyping arrays (Illumina HumanOmniExpressExome array, Illumina MEGA array). We selected loss of function variants (frameshift & non-frameshift insertion, deletion, substitution, splice, stop gain/loss single nucleotide variants, ncRNA splice) and non-synonymous single nucleotide variants predicted to be damaging. Phase 3, genotyping: Genotyping will be performed on 1000 African American scleroderma cases and controls, using two platforms. Illumina is fabricating a custom Human OmniExpress Exome array (about 1 million SNPs) to which 20,000 variants selected through WES will be added. In addition, we will genotype all cases and controls with the Illumina MEGA array, which contains about 1.6 million SNPs enriched for African-ancestry content. Phase 4, analysis: The current plan is to assemble the whole exome sequence data on 400 scleroderma cases and 480 controls and perform rare/low frequency variant analysis for loss of function and deleterious coding variants in FY2016. We also plan to complete our patient recruitment and meet our goal of 1000 African-American SSc patients in FY2016. During the FY2016 reporting period we hope to complete the genotyping using the two Illumina arrays and perform GWAS analysis including common variant association analysis, rare variant association analysis, and admixture mapping analysis. This comprehensive approach of testing both common and low frequency/rare variants (MAF < 5%) and also utilizing an admixture mapping strategy will help us identify scleroderma-associated loci.
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