During the current reporting period we have focused on 3 major projects: 1) Genetics of Scleroderma in the African-American Population Through a collaborative group denoted GRASP (Genome Research in African American Scleroderma Patients), we have secured the largest collection of African-American (AA) SSc patients ever assembled. GRASP consists of 23 centers outside of the NIH. With help from the Scleroderma Research Foundation, we have collected DNA samples from over 1100 AA SSc patients, and we have identified over 1000 antinuclear antibody (ANA) negative AA controls provided by Charles Rotimi. Phenotypic characteristics of the GRASP cohort were published during the previous reporting period. Consistent with the underlying assumptions of this study, GRASP patients exhibited more severe phenotypes than a control European ancestry (EA) cohort. 1008 AA SSc patients and 1008 AA SSc controls have been genotyped on 2 Illumina GWAS arrays. We have completed whole-exome sequencing (WES) on 400 AA SSc patients and 482 AA controls. Variants identified in the discovery phase by GWAS and WES are now undergoing sequencing in a replication study of 600 AA SSc patients and 360 AA controls. During the current reporting period we completed a study of imputed classical HLA types among 662 AA SSc patients and 946 AA controls, compared with 723 EA SSc patients and 5347 EA controls obtained from dbGaP. In the AA population, the African ancestry-predominant HLA-DRB1*08:04 and HLA-DRB1*11:02 alleles were associated with overall SSc risk and HLA-DRB1*08:04 was strongly associated with the severe anti-fibrillarin (AFA) antibody subset of SSc (OR=7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, the HLA-DPB1*13:01 and DRB1*07:01 alleles were more strongly associated with anti-topoisomerase (ATA) and anti-centromere antibody positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance of HLA in defining autoantibody subtypes. The association of the HLA-DPB1*13-01 allele with the ATA+ subset of SSc in both AA and EA patients demonstrated a trans-ancestry effect. A direct correlation between SSc prevalence and HLA-DPB1*13:01 frequency in multiple populations was observed (r=0.98, P=3.2e-6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA-alpha/beta allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase I, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link between HLA alleles, autoantibodies, and environmental triggers in the pathogenesis of SSc. A manuscript describing these findings has been submitted. 2) Genetic and Immunologic Studies of PFAPA Recent GWAS data from our own laboratory and others indicate that there are susceptibility loci in common between Behcet's disease and recurrent aphthous ulcers. These include IL10, STAT4, CCR1-CCR3, IL12A, RIPK2, NOD2, IRF8, and CEBP. We screened three cohorts of patients with PFAPA (in total 265 patients) for single nucleotide polymorphisms (SNPs) previously associated with Behcet's disease and/or recurrent aphthous stomatitis. A variant upstream of IL12A (rs17753641) was consistently the most significant variant in all three cohorts and in the meta-analysis with an OR of 2.07 (p=3.8e-10). Four additional variants near IL10, STAT4, CCR1-3, and IL23R-IL12RB2 were also significantly associated with PFAPA in the meta-analysis. IL12A encodes the p35 subunit of both the pro-inflammatory cytokine IL-12 and the anti-inflammatory cytokine IL-35. We found that monocytes from individuals from the NIH Blood Bank who were heterozygous or homozygous for the risk allele secreted significantly more IL-12 into the supernatant than those of individuals without the risk allele suggesting that this risk allele leads to heightened monocyte and CD4+ T cell activation. Overall, these data support the concept that PFAPA is on a disease spectrum with recurrent aphthous ulcers and Behcet's disease. We also aimed to identify HLA risk alleles for PFAPA in European-American and Turkish cohorts. We found that a class II HLA haplotype consisting of HLA-DQB1*0603, HLA-DRB1*1301, and DQA1*0103 was significantly associated with PFAPA (ORmeta for HLA-DQB1*0603 is 2.09, corrected p=0.0002). We also found that HLA-B*1501 was significantly associated with PFAPA in the meta-analysis with an OR of 1.97 and corrected p value of 0.004. HLA-B*1501 has previously been associated with both Behcets disease and recurrent aphthous stomatitis. We find that the classical MHC associations contribute more to the disease than in recurrent aphthous ulcers but less so than in Behcet's disease. This suggests that presence of particular HLA subtypes may play a role in determining the ultimate phenotype in patients with risk alleles. A manuscript describing these findings is currently in preparation. 3) Trisomy 8-Associated Autoinflammatory Disease During the current reporting period we studied 11 patients with trisomy 8 mosaicism, ranging in age from 5 to 37 years. Eight-two percent had recurrent fever, 82 percent had oral ulcerations, and 72 percent had severe oral ulcerations larger than 1 cm or lasting more than one week. Five patients reported genital, esophageal, or colonic ulcers. Nine patients had cognitive or motor delay. Two patients had symptoms of a bleeding diathesis with subdural hematoma, petechiae, and/or menorrhagia with normal platelet counts; the platelets of these patients had fewer dense granules by electron microscopy. Patients had elevated peripheral absolute monocyte count (average 970 cells/microliter), but none had chronic myelomonocytic leukemia, acute myeloid leukemia, or myelodysplasia. Whole blood gene expression revealed upregulation of IL-1-, TLR- and NF-kappaB-related genes. CD14+ monocytes had a significantly higher percentage of cells with trisomy 8 compared to CD3+ T cells and CD19+ B cells (chromosome 8 copy number was 2.8 in CD14+ cells vs. 2.5 in CD19+ p=0.01 and 2.3 in CD3+ cells p =0.001). Four out of 5 patients had partial improvement on daily colchicine, 3 out of 6 reported improvement with intermittent or daily anakinra, 2 out of 3 had improvement on TNF inhibitors (etanercept or infliximab), and one patient improved following hematopoietic stem cell transplant. A manuscript describing these findings is currently in preparation.

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9
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2019
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National Human Genome Research Institute
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Manthiram, Kalpana; Correa, Hernan; Boyd, Kelli et al. (2018) Unique histologic features of tonsils from patients with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome. Clin Rheumatol 37:1309-1317
Gourh, Pravitt; Remmers, Elaine F; Boyden, Steven E et al. (2018) Brief Report: Whole-Exome Sequencing to Identify Rare Variants and Gene Networks That Increase Susceptibility to Scleroderma in African Americans. Arthritis Rheumatol 70:1654-1660
Richards, Robert I; Robertson, Sarah A; Kastner, Daniel L (2018) Neurodegenerative diseases have genetic hallmarks of autoinflammatory disease. Hum Mol Genet 27:R108-R118
Arthur, Victoria L; Shuldiner, Emily; Remmers, Elaine F et al. (2018) IL1RN Variation Influences Both Disease Susceptibility and Response to Recombinant Human Interleukin-1 Receptor Antagonist Therapy in Systemic Juvenile Idiopathic Arthritis. Arthritis Rheumatol 70:1319-1330
Morgan, Nadia D; Shah, Ami A; Mayes, Maureen D et al. (2017) Clinical and serological features of systemic sclerosis in a multicenter African American cohort: Analysis of the genome research in African American scleroderma patients clinical database. Medicine (Baltimore) 96:e8980
Manthiram, Kalpana; Li, Suzanne C; Hausmann, Jonathan S et al. (2017) Physicians' perspectives on the diagnosis and management of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome. Rheumatol Int 37:883-889
Manthiram, Kalpana; Lapidus, Sivia; Edwards, Kathryn (2017) Unraveling the pathogenesis of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis through genetic, immunologic, and microbiologic discoveries: an update. Curr Opin Rheumatol 29:493-499
Ombrello, Michael J; Arthur, Victoria L; Remmers, Elaine F et al. (2017) Genetic architecture distinguishes systemic juvenile idiopathic arthritis from other forms of juvenile idiopathic arthritis: clinical and therapeutic implications. Ann Rheum Dis 76:906-913
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
Ombrello, Michael J; Sikora, Keith A; Kastner, Daniel L (2014) Genetics, genomics, and their relevance to pathology and therapy. Best Pract Res Clin Rheumatol 28:175-89

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