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ObjectiveRelapsing polychondritis (RP) is a systemic inflammatory disease of unknown etiology. The study objective was to examine the contribution of rare genetic variations in RP.MethodsWe performed a case-control exome-wide rare variant association analysis including 66 unrelated European American RP cases and 2923 healthy controls. Gene-level collapsing analysis was performed using Firth’s logistics regression. Pathway analysis was performed on an exploratory basis with three different methods: Gene Set Enrichment Analysis (GSEA), sequence kernel association test (SKAT) and higher criticism test. Plasma DCBLD2 levels were measured in patients with RP and healthy controls using enzyme-linked immunosorbent assay (ELISA).ResultsIn the collapsing analysis, RP was associated with higher burden of ultra-rare damaging variants in theDCBLD2gene (7.6% vs 0.1%, unadjusted odds ratio = 79.8, p = 2.93 x 10-7). Patients with RP and ultra-rare damaging variants inDCBLD2had a higher prevalence of cardiovascular manifestations. Plasma DCBLD2 protein levels were significantly higher in RP than healthy controls (5.9 vs 2.3, p < 0.001). Pathway analysis showed statistically significant enrichment of genes in the tumor necrosis factor (TNF) signaling pathway driven by rare damaging variants inRELB,RELAandRELusing higher criticism test weighted by degree and eigenvector centrality.ConclusionsThis study identified specific rare variants inDCBLD2as putative genetic risk factors for RP. Genetic variation within the TNF pathway is also potentially associated with development of RP. These findings should be validated in additional patients with RP and supported by future functional experiments.

Objective Somatic mutations in UBA1 cause a newly defined syndrome known as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome). More than 50% of patients currently identified as having VEXAS met diagnostic criteria for relapsing polychondritis (RP), but clinical features that characterize VEXAS within a cohort of patients with RP have not been defined. We undertook this study to define the prevalence of somatic mutations in UBA1 in patients with RP and to create an algorithm to identify patients with genetically confirmed VEXAS among those with RP. Methods Exome and targeted sequencing of UBA1 was performed in a prospective observational cohort of patients with RP. Clinical and immunologic characteristics of patients with RP were compared based on the presence or absence of UBA1 mutations. The random forest method was used to derive a clinical algorithm to identify patients with UBA1 mutations. Results Seven of 92 patients with RP (7.6%) had UBA1 mutations (referred to here as VEXAS-RP). Patients with VEXAS-RP were all male, were on average ≥45 years of age at disease onset, and commonly had fever, ear chondritis, skin involvement, deep vein thrombosis, and pulmonary infiltrates. No patient with VEXAS-RP had chondritis of the airways or costochondritis. Mortality was greater in VEXAS-RP than in RP (23% versus 4%; P = 0.029). Elevated acute-phase reactants and hematologic abnormalities (e.g., macrocytic anemia, thrombocytopenia, lymphopenia, multiple myeloma, myelodysplastic syndrome) were prevalent in VEXAS-RP. A decision tree algorithm based on male sex, a mean corpuscular volume >100 fl, and a platelet count

Background Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may...

Discovery of the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome demonstrates that somatic mutations in haematological precursor cells can cause adult-onset, complex inflammatory disease. Unlike germline mutations, somatic mutations occur throughout the lifespan, are restricted to specific tissue types, and may play a causal role in non-heritable rheumatological diseases, especially conditions that start in later life. Improvements in sequencing technology have enabled researchers and clinicians to detect somatic mutations in various tissue types, especially blood. Understanding the relationships between cell-specific acquired mutations and inflammation is likely to yield key insights into causal factors that underlie many rheumatological diseases. The objective of this review is to detail how somatic mutations are likely to be relevant to clinicians who care for patients with rheumatological diseases, with particular focus on the pathogenetic mechanisms of the VEXAS syndrome.

Mouth and genital ulcers with inflamed cartilage (MAGIC) syndrome is characterized by overlapping features of relapsing polychondritis (RP) and Behcet's disease (BD). To date, no studies have defined the clinical spectrum of disease in a cohort of patients with MAGIC syndrome.Adult patients within an ongoing prospective, observational cohort study in RP were clinically assessed for MAGIC syndrome. A systematic review was conducted to identify additional cases of MAGIC syndrome by searching four databases: PubMed (US National Library of Medicine), Embase (Elsevier), Scopus (Elsevier) and Web of Science: Core Collection (Clarivate Analytics). The inclusion criteria used were: [1] patients of any age or gender who were diagnosed with MAGIC syndrome, or both RP and BD; [2] case report or case series study; [3] published from 1985 - July 2020; and [4] in English language. Risk of bias was assessed using a checklist developed by the authors and based on the Consensus-based Clinical Case Reporting (CARE) Guidelines. Search results screening, article inclusion, data extraction and risk of bais assessment was performed independently by two investigators. Clinical characteristics, particularly BD-related features, were compared between patients with MAGIC syndrome and cases of non-MAGIC RP. The performance characteristics of different criteria to classify MAGIC syndrome were also evaluated.Out of 96 patients with RP, 13 (14%) patients were diagnosed with MAGIC syndrome. For the systematic review, 380 articles were retrieved of which 90 were screened at title and abstract levels. Of these screened, 60 were excluded and 30 proceeded to full text review where an additional 8 were excluded. Twenty-two articles were included in our review and from which 27 additional cases of MAGIC syndrome were identified. Pooling all 40 cases together and comparing them with non-MAGIC RP, there was a significantly higher prevalence of ocular involvement (28% vs 4%, p0.01), cutaneous involvement (35% vs 1%, p0.01), GI involvement (23% vs 4%, p0.01), and CNS involvement (8% vs 0, p = 0.04) in MAGIC syndrome. A higher prevalence of aortitis (23% vs 1%, p0.01), Raynaud's phenomenon (54% vs 11%, p0.01), and elevated anti-collagen II antibodies (50% vs 9%, p = 0.04) were observed in MAGIC syndrome. Fulfillment of either McAdam's or Damiani's Criteria for RP plus the International Criteria for Behçet's Disease had excellent sensitivity (98%) to classify cases of MAGIC syndrome.A substantial proportion of patients with RP can be clinically diagnosed with MAGIC syndrome. These patients have features of RP, BD, and other unique features including aortitis, Raynaud's phenomenon and elevated anti-collagen II antibodies.

Objective.To assess patient-reported burden of disease in pediatric patients with relapsing polychondritis (RP) and to compare those findings to adult patients.Methods.A survey based on known clinical symptoms of RP was developed and administered to patients with a pediatric diagnosis of RP. Adult patients completed a similar survey.Results.Twenty-one pediatric patients, or their parents, completed surveys. Median age at symptom onset was 6 years (interquartile range 1.8–12). Prior to diagnosis, most pediatric patients went to the emergency room (ER; 61.9%), saw > 3 physicians (57.1%), and took > 1 year to be diagnosed (61.9%). Pediatric patients were often diagnosed with asthma (42.9%), ear infections (42.9%), or sinusitis (33.3%) prior to diagnosis of RP. Symptoms prior to diagnosis included ear pain/redness (85.7%), joint pain/swelling (61.9%), and airway symptoms (38.1%). Four pediatric patients (19%) reported tracheomalacia requiring tracheostomy. Pediatric patients frequently missed school because of their disease (71.4%). Surveys from 290 adult patients were compared to pediatric patients. Pediatric patients were significantly more likely to undergo biopsy (42.9% vs 17.4%; p < 0.01) and be treated with biologics (42.9% vs 19%; p = 0.02). Adults were significantly more likely to be female (87.8% vs 28.6%; p < 0.01) and to report airway symptoms (77.9% vs 47.6%; p = 0.01). Prevalence of disease complications was not significantly different between adult and pediatric patients.Conclusions.The burden of disease in pediatric patients with RP includes missed school, diagnostic delay, ER visits, and multisystem disease, with resultant damage to cartilaginous structures. Differences in airway involvement and treatment approaches may exist between pediatric and adult patients.

Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders.We analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9-edited zebrafish were used as an in vivo model to assess gene function.We identified 25 men with somatic mutations affecting methionine-41 (p.Met41) in UBA1, the major E1 enzyme that initiates ubiquitylation. (The geneUsing a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. (Funded by the NIH Intramural Research Programs and the EU Horizon 2020 Research and Innovation Program.).

Objective Relapsing polychondritis (RP) is a systemic disease. Failure to recognize RP can lead to diagnostic delay and further complications, including death. This study was undertaken to identify clinical patterns in a prospective cohort of patients with RP. Methods Patient subgroups were identified using latent class analysis based on 8 clinical variables: saddle-nose deformity, subglottic stenosis, tracheomalacia, bronchomalacia, ear chondritis, tenosynovitis/synovitis, inflammatory eye disease, and audiovestibular disease. Model selection was based on Akaike's information criterion. Results Seventy-three patients were included in this study. Patients were classified into 1 of 3 subgroups: type 1 RP (14%), type 2 RP (29%), and type 3 RP (58%). Type 1 RP was characterized by ear chondritis (100%), tracheomalacia (100%), saddle-nose deformity (90%), and subglottic stenosis (80%). These patients had the shortest median time to diagnosis (1 year), highest disease activity, and greatest frequency of admission to the intensive care unit and tracheostomy. Type 2 RP was characterized by tracheomalacia (100%) and bronchomalacia (52%), but no saddle-nose deformity or subglottic stenosis. These patients had the longest median time to diagnosis (10 years) and highest percentage of work disability. Type 3 RP was characterized by tenosynovitis/synovitis (60%) and ear chondritis (55%). There were no significant differences in sex, race, or treatment strategies between the 3 subgroups. Conclusion Our findings indicate that there are 3 subgroups of patients with RP, with differences in time to diagnosis, clinical and radiologic characteristics, and disease-related complications. Recognizing a broader spectrum of clinical patterns in RP, beyond cartilaginous involvement of the ear and upper airway, may facilitate more timely diagnosis.

Objective To determine whether autophagy is involved in the degradation of misfolded HLA-B27 in experimental spondyloarthritis. Methods Bone marrow-derived macrophages from HLA-B27/human β2 -microglobulin (hβ2 m)-transgenic rats were incubated in the presence or absence of interferon-γ and proteasome or autophagy inhibitors. Immunoprecipitation, immunoblotting, and immunofluorescence analysis were used to measure HLA-B27 heavy chains and autophagy. Autophagy was induced using rapamycin. Macrophages from HLA-B7/hβ2 m-transgenic and wild-type rats were used as controls. Results HLA-B27-expressing macrophages showed phosphatidylethanolamine-conjugated microtubule-associated protein 1 light chain 3B levels similar to those in both control groups, before and after manipulation of autophagy. Blocking autophagic flux with bafilomycin resulted in the accumulation of misfolded HLA-B27 dimers and oligomers as well as monomers, which was comparable with the results of blocking endoplasmic reticulum-associated degradation (ERAD) with the proteasome inhibitor bortezomib. HLA-B7 monomers also accumulated after blocking each degradation pathway. The ubiquitin-to-heavy chain ratio was 2-3-fold lower for HLA-B27 than for HLA-B7. Activation of autophagy with rapamycin rapidly eliminated ~50% of misfolded HLA-B27, while folded HLA-B27 or HLA-B7 monomeric heavy chains were minimally affected. Conclusion This study is the first to demonstrate that both autophagy and ERAD play roles in the elimination of excess HLA class I heavy chains expressed in transgenic rats. We observed no evidence that HLA-B27 expression modulated the autophagy pathway. Our results suggest that impaired ubiquitination of HLA-B27 may play a role in the accumulation of misfolded disulfide-linked dimers, the elimination of which can be enhanced by activation of autophagy. Manipulation of the autophagy pathway should be further investigated as a potential therapeutic target in spondyloarthritis.

Introduction: Identifying the causes of adult-onset rheumatic diseases remains a challenge, and limits diagnosis, prognosis, and targeted treatment. We hypothesized that mutations in genes regulating the post-translational modification ubiquitin, previously implicated in two autoinflammatory diseases, may define new rheumatic disorders. Methods: We analyzed peripheral blood exome sequence data from 2,560 individuals with inflammation-related diagnoses for deleterious mutations in >800 ubiquitin-related genes. After discovering three patients with novel UBA1 mutations, we identified additional cases based on clinical similarities through screening multiple independent cohorts. Clinical evaluation of all patients combined with Sanger sequencing, digital droplet PCR, immunoblotting, immunohistochemistry, flow cytometry, and transcriptome/cytokine profiling were performed. CRISPR/Cas9 edited zebrafish provided an in vivo model to assess UBA1 gene function. Results: Twenty-eight adult males were identified with somatic mutations at methionine 41 in UBA1, an X-linked gene, encoding the major E1 enzyme that initiates ubiquitylation. Methionine 41 is highly conserved in UBA1, and these mutations were not observed in exome sequences from over 80,000 healthy controls. Among affected individuals, mutations were found in more than half of hematopoietic stem cells, exclusively in peripheral blood myeloid cells, and not in lymphocytes or fibroblasts. The variant allele fraction of UBA1 p.Met41 mutations in peripheral blood ranged from 20-95%. Patients developed an often fatal, treatment-refractory inflammatory syndrome in late adulthood, with fever, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis and some individuals met clinical criteria for relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, or giant cell arteritis. In addition, the majority of subjects developed myelodysplastic bone marrow with cytopenias, characteristic vacuoles in myeloid and erythroid precursors cells, progressive bone marrow failure and thromboembolic disease, and some fulfilled clinical criteria for myelodysplastic syndrome or plasma cell dyscrasia. Transformation into MDS with excess blasts or acute myeloid leukemia did not occur in any case. Mutations at p.Met41, the initiation start site of the canonical cytoplasmic isoform, caused loss of this protein and expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral blood cells exhibited decreased ubiquitylation and activated innate immune pathways. Knockout of the zebrafish cytoplasmic UBA1 isoform homologue, but not the nuclear isoform, caused systemic inflammation. These results identify somatic mutations in UBA1 as the cause, and not the consequence, of this inflammatory disease. Conclusions: We have defined a novel disorder, VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic), which connects seemingly unrelated adult-onset inflammatory and hematologic diseases. Our work also reveals somatic mutations in a hematopoietic stem cell as a cause of adult-onset rheumatic syndromes that overlap with hematologic conditions. Identification of UBA1 mutations as a cause of these diseases has important implications for classification, prognosis, and treatment of patients, and for the role of somatic mutations of hematopoietic origin in multi-organ pathophysiology. Figure Disclosures Young: Novartis: Research Funding.

Somatic mutations in UBA1 in hematopoietic stem cells and myeloid cells have recently been described and are associated with adult-onset severe autoinflammatory diseases including relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, and giant cell arteritis. This newly defined syndrome is named VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic). We performed clinicopathologic, cytogenetic, flow cytometric and molecular bone marrow assessment of 15 patients diagnosed with VEXAS and peripheral cytopenias. All patients were male, with a median age of 64y (IQR 45-80) and all had somatic missense mutations affecting the p.Met41 residue in UBA1 with variant allele frequencies (VAF) over 20% from peripheral blood or bone marrow samples and with lineage restriction to mature myeloid cells. Germline mutations at this residue are not reported in any public databases. Peripheral blood findings included macrocytic anemia in all patients (100%), thrombocytopenia in 9/15, and neutropenia in 1/15. All bone marrow aspirates (15/15; 100%) showed prominent vacuolization of both myeloid and erythroid precursors with few vacuoles noted in mature cells. Of the 9 patients tested for serum copper levels all were normal or borderline low. Marrow was hypercellular with granulocytic and megakaryocytic hyperplasia in 12/15 (80%). Definitive WHO criteria for MDS was met in 5/15 (40%) with 3 cases of MDS-MLD, and 2 of MDS-SLD. The remaining cases were suspicious for MDS with low or borderline levels of dyspoiesis that did not exceed greater than 10% of cells in respective lineages meeting criteria for clonal cytopenia of undetermined significance (CCUS). Of the MDS cases, dysplasia was seen in megakaryocytes (5/5), myeloid (2/5) and erythroid (1/5) precursors. Cytogenetic abnormalities were detected in 2/5 MDS patients involving t(3;12)(q21;q13) in one case, and del (5q)/del (13q) in another case. Next generation sequencing analyses were performed in 9/15 patients and showed mutations in DNMT3A in 2/5 MDS cases (VAF 43% and 44%), CSF1R in 1/5 MDS (VAF 3.1%), GNA11 in 1/5 MDS (VAF3.3%) and EZH2 mutation (VAF 22%) in one of the patients without overt MDS. Clonal plasma cell or B-cell populations were diagnosed in 6/15 patients with 3 plasma cell dyscrasias, and 2 cases of monoclonal B-cell lymphocytosis. The most common abnormalities detected by bone marrow flow cytometry analysis were severely reduced or absent B-cell precursors, inverted CD4:CD8 ratios and abnormal expression of CD56 on monocytes. In summary, hematologic disorders were identified in 10/15 VEXAS patients, including both myeloid and lymphoid clonal disease comprising MDS, plasma cell dyscrasias, and monoclonal B cell lymphocytosis. Based on the presence of UBA1 somatic mutations in hematopoietic cells of all VEXAS patients, the patients without evidence of overt neoplasia in this study met criteria for CCUS. Importantly, all patients had characteristic vacuoles in myeloid and erythroid precursors in the marrow without evidence of copper deficiency. These findings suggest that vacuolization of hematopoietic precursors in marrow of cytopenic patients, particularly in male patients and in the setting of systemic inflammation and normal copper levels, should prompt evaluation for somatic UBA1 mutations. In addition to severe autoinflammatory disease manifestations, VEXAS patients appear to confer increased risk for development of both myeloid and lymphoid/plasma cell neoplasia and require surveillance for disease progression. Figure Disclosures Young: Novartis: Research Funding.

Background:Somatic mutations in ubiquitin activating enzyme 1 (UBA1) cause a newly defined syndrome known as VEXAS. [1] More than fifty percent of patients currently identified with VEXAS meet diagnostic criteria for relapsing polychondritis (RP).Objectives:To determine the prevalence VEXAS within a cohort of patients with RP, to compare their clinical, laboratory, and immunologic features and to develop a clinical algorithm to inform genetic screening for VEXAS among patients with RP.Methods:Exome and targeted sequencing of the UBA1 gene was performed in a prospective observational cohort of patients with RP. Clinical and immunological characteristics of patients with RP were compared based on presence or absence of UBA1 mutations. Random forest was used to derive a clinical algorithm to identify patients with UBA1 mutations. Immune populations were quantified by multipanel flow cytometry. Categorical and continuous variables were compared using the chi square or Kruskal-Wallis test. PResults:Seven of 92 patients with RP (7.6%) were confirmed to have UBA1 mutations (VEXAS-RP). Six additional patients with VEXAS-RP from other cohorts were included for subsequent analyses. Patients with VEXAS-RP were all male, older at disease onset, and commonly had fever, ear chondritis, skin involvement, deep vein thrombosis, and pulmonary infiltrates. Patients with RP as compared with VEXAS-RP had a significantly higher prevalence of airway chondritis, costochondritis and tenosynovitis/arthralgias. (Table). Mortality was significantly greater in VEXAS-RP than RP (27% vs 2% p=0.01). Maximum ESR, CRP, and mean corpuscular volume (MCV) values were significantly greater in VEXAS-RP. Absolute monocyte, lymphocyte, and platelet counts were significantly lower in VEXAS-RP. A decision tree based on male sex, MCV>100 fl and Platelet countTable 1.Clinical Characteristics of patients with RP vs VEXAS-RPAll Patientsn=98RPn=85VEXAS-RPn=13p valueDemographic CharacteristicsRace, White n (%)90 (92)77 (91)13 (100)0.59Sex, Male n (%)26 (27)13 (15)13 (100)Age, Symptom onset, years, Median (IQR)38 (30-47)37 (28-43)56 (54-64)Clinical SymptomsFever n (%)33 (34)20 (24)13 (100)Ear chondritis n (%)61 (62)48 (56)13 (100)0.0015Nose chondritis n (%)83 (85)71 (84)12 (92)0.68Airway chondritis n (%)37 (38)37 (44)0 (0)0.0015Tenosynovitis/arthalgias n (%)83 (85)77 (91)6 (46)0.0005Skin involvement n (%)33 (34)22 (2611 (85)Laboratory ValuesESR, mm/hr, median (IQR)12 (6-22)11 (5-19)66.5 (42-110)CRP, mg/L, median (IQR)2.9 (0.8-9.6)1.9 (0.6-6.3)17.7 (9.6-99.5)Platelet count (k/uL)246(201-299)258 (227-312)145 (100-169)MCV fL93.05 (90-98)92.2 (89-95)105 (102-115)Absolute lymphocyte count1.6 (1.1-2.3)1.78(1.4-2.4)0.92 (0.5-1.2)CT scan abnormalitiesPulmonary infiltrates n (%)16 (16.33)6 (7.06)10 (77)ComplicationsDeath n (%)6 (6)3 (4)3 (23)0.029Unprovoked DVT12 (12)4 (5)8 (62)N number; IQR = interquartile rangeConclusion:Mutations in UBA1 are causal for disease in a subset of patients with RP. These patients are defined by disease onset in the fifth decade of life or later, male sex, ear/nose chondritis and hematologic abnormalities. Early identification is important in VEXAS given the associated high mortality rate.References:[1]Beck DB, Ferrada MA, Sikora KA, Ombrello AK, Collins JC, Pei W, Balanda N, Ross DL, Ospina Cardona D, Wu Z et al: Somatic Mutations in UBA1 and Severe Adult-Onset Autoinflammatory Disease. N Engl J Med 2020, 383(27):2628-2638.Disclosure of Interests:None declared

Objective To assess patient-reported symptoms and burden of disease in relapsing polychondritis (RP). Methods Patients with RP completed a disease-specific online survey to identify symptoms attributed to illness. Patients were divided into subgroups based upon presence or absence of ear/nose, airway, or joint involvement. Pathway to diagnosis, treatment, and disease-related complications were assessed within each subgroup. Results Data from 304 respondents were included in this analysis. Prior to diagnosis, most patients with RP went to the emergency room (54%), saw >3 physicians (54%), and had symptoms for >5 years (64%). A concomitant diagnosis of fibromyalgia and absence of ear/nose or joint involvement was associated with diagnostic delay >1 year. Common diagnoses prior to RP diagnosis included asthma in patients with airway involvement (35% vs 22%, p=0.03) and ear infection in patients with ear/nose involvement (51% vs 6%, p

Purpose of review Systemic juvenile idiopathic arthritis (SJIA) is an inflammatory condition characterized by fever, lymphadenopathy, rash, arthritis, and serositis. Although the ultimate cause of this disorder remains elusive, recent work defining cytokine effector mechanisms has led to a new treatment paradigm for this condition. In this review, we describe the recent immunological reclassification of SJIA as an autoinflammatory disorder as well as detailing the dramatic changes in its treatment. Recent findings SJIA is an autoinflammatory disorder in which defects of innate immune system pathways lead to significant inflammation. Recent studies of the pathophysiology, as well as successful treatment trials, have established interleukin-1β (IL-1β) and IL-6 as key cytokines in the pathogenesis of this condition. As a result, their inhibition has become the centerpiece of the current SJIA treatment paradigm. Summary There has been a major shift away from the traditional treatments of SJIA towards therapeutics that inhibit IL-1β and IL-6. In fact, the IL-1 blocker anakinra is now regarded as standard of care for SJIA patients with systemic symptoms, while the IL-6 inhibitor tocilizumab shows great potential. Future research holds promise for the development of more efficient cytokine inhibition as well a more comprehensive knowledge of the innate cytokine networks in this disease.

Genetic and genomic investigations are a starting point for the study of human disease, seeking to discover causative variants relevant to disease pathophysiology. Over the past 5 years, massively parallel, high-throughput, next-generation sequencing techniques have revolutionized genetics and genomics, identifying the causes of many Mendelian diseases. The application of whole-genome sequencing and whole-exome sequencing to large populations has produced several publicly available sequence datasets that have revealed the scope of human genetic variation and have contributed to important methodological advances in the study of both common and rare genetic variants in genetically complex diseases. The importance of noncoding genetic variation has been highlighted by the Encyclopedia of DNA Elements (ENCODE) project and National Institutes of Health (NIH) Roadmap Epigenomics Program and integrated analyses of these datasets, together with disease-specific datasets, will provide an important and powerful tool for determining the mechanisms through which disease-associated, noncoding variation influences disease risk.

Systemic juvenile idiopathic arthritis (SJIA) is an autoinflammatory disease characterized by spiking fevers, arthritis, polyserositis, evanescent rash, and lymphadenopathy (1). Evidence suggests that one of the effector cells in the pathogenesis of this disease is of the monocyte/macrophage lineage. Patients with active SJIA not only have an increased proportion of CD14+ monocytes (2), but also increased myelomonocytic precursors within their peripheral blood (3,4). In addition, one of the feared complications of SJIA is the macrophage activation syndrome (MAS) where affected patients experience widespread activation and expansion of T lymphocytes and cells of the monocyte-macrophage lineage, leading to unremitting fever, hyperferritinemia, hypertriglyceridemia, hepatosplenomegaly, a consumptive coagulopathy, and cytopenias. One of its prominent characteristics is an expansion of macrophages actively engaged in the hemophagocytosis of multiple cellular elements within the bone marrow and other lymphoid tissue (5,6). Yet, many of these features can be appreciated in severe SJIA too, in an intermediate phenotype called subclinical or occult MAS, thus suggesting MAS and SJIA may be different extremes of the same clinical spectrum (7,8). The activated macrophage phenotype can be divided into two general groups, M1 and M2, depending on the stimuli they receive and their subsequent differentiation pathway (9–11). In this model, M1 macrophage differentiation is mainly driven by interferon-γ (IFN-γ) and M2 macrophages result from mostly anti-inflammatory signals, such as IL-4, IL-10, M-CSF, TGF-β, or glucocorticoids. M1 macrophages excel at intracellular pathogen eradication, producing high amounts of IL-12 and TNF-α. Due to their highly phagocytic properties, M2 macrophages are mostly scavengers that are heavily involved in wound healing and tissue remodeling. In contrast to M1 macrophages, M2 macrophages are a prominent source of IL-10. Currently, the activated macrophage in SJIA and MAS has yet to be characterized into the M1/M2 polar model. SJIA and MAS are both inflammatory conditions, thus it’s likely that the macrophages therein are of the M1 phenotype. However, the data supporting IFN-γ, which is the key facilitator of M1 macrophage differentiation, as a mediator in active SJIA is scant. Gattorno et al. demonstrated elevated serum levels of both IFN-γ and IP-10, regardless of disease activity (12). In contrast, Lasiglie at al. showed that upon overnight stimulation of PBMC populations, no difference in the absolute number of IFN-γ-producing cells between active SJIA patients and healthy controls could be appreciated (13). Congruent with this finding, three independent gene expression studies have failed to detect an IFN-induced genetic signature within the peripheral blood mononuclear cells (PBMCs) of active SJIA patients (3,14,15). In contrast to SJIA, IFN-γ is believed to have a prominent role in the pathogenesis of hemophagocytic lymphohistiocytosis (HLH), both a hereditary and acquired hemophagocytic syndrome clinically similar to MAS, where IFN-γ is found to be highly elevated in the serum (16–18). In fact, blockade of IFN-γ has been suggested as a potential treatment for both inherited and acquired forms of active HLH (19). To this end, the goals of this study were to characterize the status of an IFN-induced signature within affected tissues and to gauge the integrity of IFN-signaling pathways within the peripheral monocytes of patients with SJIA. IFN-γ is a pleotropic cytokine that canonically signals via the Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway, machinery that is common to the many different cytokines (20). Specifically, IFN-γ will lead to the recruitment of Jak1 and Jak2 to the intracellular portion of the IFN-γ receptor, which then becomes phosphorylated on a tyrosine residue. This allows recruitment, docking, and subsequent phosphorylation of STAT1. In its active form, STAT1 will then form a homodimer and translocate into the nucleus in order to bind to a promoter region IFN-γ activation site (GAS) to initiate specific IFN-responsive genes. Given the inflammatory nature of active SJIA, it seems paradoxical that an IFN-induced genetic signature cannot be detected within the PBMCs of active SJIA. In this study, we explore the role of IFN-γ in the pathogenesis of SJIA. We confirm a restricted IFN-induced genetic signature in not only the peripheral mononuclear cells, but also isolated peripheral monocytes therein. Furthermore, we demonstrate a markedly constrained expression of IFN-responsive genes within peripherally affected SJIA synovial tissue in comparison to extended oligoarticular JIA, a subtype of JIA that is known to express an IFN-induced genetic signature within its synovial cells (21). Upon exploring the ability of SJIA peripheral monocytes to even respond to IFN-γ, we did not find their overall reactivity to be significantly different from healthy control peripheral monocytes, which suggests that the absence of an IFN-induced genetic signature is not due to monocyte hyporesponsiveness to IFN-γ, but likely due to a limited exposure to it. Finally, we also report an increased basal interferon signal and augmented reactivity to IFN-γ within the peripheral monocytes of inactive SJIA patients on an IL-1β blocking agent, a finding that may have an impact on the ability of these patients to appropriately respond to viral illnesses.