During the current reporting period we have focused on 3 major projects: 1) Genetics of Systemic Juvenile Idiopathic Arthritis In the previous reporting period, we focused on the MHC, and published a paper in the PNAS demonstrating that, in 6 study populations of Western European ancestry, HLA-DRB1*11 and its defining amino acid, glutamate 58, were strongly associated with sJIA. In the current reporting period, we followed up with a paper published in the Annals of the Rheumatic Diseases, reporting the results of a genome-wide association study (GWAS) of 770 children with sJIA collected in nine countries by the International Childhood Arthritis Consortium. Single nucleotide polymorphisms were tested for association with sJIA. Weighted genetic risk scores were used to compare the genetic architecture of sJIA with other JIA subtypes. The major histocompatibility complex locus and a noncoding locus on chromosome 1 each showed association with sJIA exceeding the threshold for genome-wide significance, while 23 other novel loci were suggestive of association with sJIA. Using a combination of genetic and statistical approaches, we found no evidence of shared genetic architecture between sJIA and other common JIA subtypes. The lack of shared genetic risk factors between sJIA and other JIA subtypes supports the hypothesis that sJIA is a unique disease process and argues for a different classification framework. Research to improve sJIA therapy should target its unique genetics and specific pathophysiological pathways. 2) Genetics of Scleroderma in the African-American Population Scleroderma is a chronic multisystem disease that is clinically characterized by progressive fibrosis of the skin and internal organs, vasculopathy, and autoimmunity. It is the cause of significant morbidity and mortality, and the treatment options are not nearly as effective as those available for other autoimmune diseases. Scleroderma is both more common and more severe in the African-American population. Hence, a study of scleroderma in African Americans has the potential both to identify previously unrecognized multi-ethnic scleroderma susceptibility loci, and perhaps to identify population-specific genes that could have a major impact in this underserved population with more severe disease. Through a collaborative group denoted GRASP (Genome Research in African American Scleroderma Patients), we have secured the largest collection of African American scleroderma patients ever assembled. GRASP consists of 19 centers outside of the NIH: Johns Hopkins, Georgetown, George Washington, University of Pennsylvania, University of Pittsburgh, Rutgers, New York University, Hospital for Special Surgery, Medical University of South Carolina, Emory, Miami, University of Alabama at Birmingham, Tulane, Northwestern, University of Chicago, Michigan, University of Texas at Houston, University of California San Francisco, and Stanford. With help from the Scleroderma Research Foundation, in the first phase of the project we have collected DNA samples from 1052 African American scleroderma patients, and we have identified 1039 antinuclear antibody (ANA) negative African-American controls provided by Charles Rotimi. During the current reporting period, we have completed three separate studies that will be reported as podium presentations at the 2017 American College of Rheumatology meeting. The first of these three studies compared the phenotypic manifestations of scleroderma in 1009 subjects in the GRASP cohort to that reported in the European League Against Rheumatism Scleroderma Trials and Research (EUSTAR) cohort. 945 (94 percent) patients met the 2013 ACR/EULAR classification criteria for scleroderma, with the remaining 64 (6 percent) meeting the 1980 ACR or CREST criteria. The majority (57 percent) had the more diffuse subtype and a young age at symptom onset, in marked contrast to the EUSTAR cohort. 1 in 10 patients had a severe Medsger cardiac score of 4. Pulmonary fibrosis evident on computed tomography of the chest was present in 43 percent of patients, and was significantly associated with anti-topoisomerase I positivity. 38 percent of patients with CT evidence of pulmonary fibrosis had a severe restrictive ventilator defect. 1 in 6 GRASP patients required oxygen therapy, compared to 3 percent in the EUSTAR cohort. A significant association was noted between longer disease duration and higher odds of pulmonary hypertension. The prevalence of potentially fatal scleroderma renal crisis was 3.5 times higher than that reported in the EUSTAR cohort. In the second study, we used the Illumina MEGA array to conduct the first genome-wide association study (GWAS) in African-American scleroderma. 1.7 million variants were genotyped and then imputed using Beagle software and 1000 Genomes as the reference population. Admixture and principal component analysis (PCA) confirmed that the patients and controls were well-matched, with a lambdaGC of 1.03. Association analysis was performed on the imputed data in 934 patients and 946 controls. The MHC region demonstrated the strongest association after accounting for the top 10 PCAs. rs35915063 was the top variant in the HLA-DQB1 gene (P=2.2 x 10e-17, OR=1.96). Stepwise conditional regression analysis using an additive model revealed the DQA gene variant rs9271620 and the DPB1 variant rs2071354 as the next 2 independent risk variants. The rs59063398 variant showed the strongest non-MHC association with P=2.2 x 10e-8, OR=0.47). This variant is part of an evolutionarily conserved halpoblock in the IFT43 gene region that is African ancestry specific. This gene region includes the promoter for TGFB3, suggesting potential alteration of TGFB3 promoter activity and hence regulation of TGFB3 expression. In the third study, we used MHC region genotypes from the MEGA array to impute classical HLA types and analyzed their associations with scleroderma in the GRASP cohort. We extracted the genotypes of 25,256 markers from 20 Mb encompassing the greater MHC region and used the Michigan Imputation Server with 1000G Phase 3 v5 reference and Eagle phasing algorithm to obtain input data for the HLA*IMP:03 web server. The most significantly scleroderma-associated HLA type was a predominantly African allele, HLA-DRB1*08:04, OR=2.95. Regression analysis conditioning on the disease-associated alleles identified another African DRB1 allele, *11:02, as well as HLA-DPB1*13:01, and HLA-DRB4*01:01 as independent contributors to disease risk, but no association was found for MHC class I alleles. 34.6 percent of GRASP cases carry either DRB1*08:04 or *11:02 compared with 16.3 percent of controls. Analysis of 246 anti-topoisomerase antibody positive cases revealed a strong disease risk conferred by HLA-DPB1*13:01 (OR=4.1). 30.5 percent of anti-topoisomerase Ab cases carry HLA-DPB1*13:01, compared with 9.7 percent of controls. 3) Genetics of PFAPA We are also studying the genetic and immunologic underpinnings of PFAPA syndrome, the most common periodic fever syndrome in children. We are searching for rare, causative variants by whole exome sequencing of familial cases. Our results suggest that PFAPA is a polygenic disease, and we are now assessing allele frequencies of candidate genes involved in other complex ulcerative autoinflammatory diseases in a cohort of PFAPA patients. Tonsillectomy is curative in many patients with PFAPA; therefore, we are characterizing lymphoid and myeloid cell populations in the tonsils of patients with PFAPA with immunophenotyping, comparisons of gene expression, and functional studies. In addition, we have observed that patients with trisomy 8 mosaicism and chromosome 8 duplications have a propensity to autoinflammatory disease with recurrent fever and severe mucocutaneous ulcers; studies to identify causal genes are underway.
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