A genome-wide association study suggests new susceptibility loci for primary antiphospholipid syndrome.

OBJECTIVES: Primary antiphospholipid syndrome (PAPS) is a rare autoimmune disease characterized by the presence of antiphospholipid antibodies and the occurrence of thrombotic events and pregnancy complications. Our study aimed to identify novel genetic susceptibility loci associated with PAPS. METHODS: We performed a genome-wide association study comprising 5,485 individuals (482 affected individuals) of European ancestry. Significant and suggestive independent variants from a meta-analysis of approximately 7 million variants were evaluated for functional and biological process enrichment. The genetic risk variability for PAPS in different populations was also assessed. Hierarchical clustering, Mahalanobis distance, and Dirichlet Process Mixtures with uncertainty clustering methods were used to assess genetic similarities between PAPS and other immune-mediated diseases. RESULTS: We revealed genetic associations with PAPS in a regulatory locus within the HLA class II region near HLA-DRA and in STAT1-STAT4 with a genome-wide level of significance. 34 additional suggestive genetic susceptibility loci for PAPS were also identified. The disease risk allele near HLA-DRA is associated with overexpression of HLA-DRB6, HLA-DRB9, HLA-DQA2 and HLA-DQB2 in immune cells, vascular tissue, and nervous tissue. This association is independent of the association between PAPS and HLA-DRB1*1302. Functional analyses highlighted immune-related pathways in PAPS-associated loci. The comparison with other immune-mediated diseases revealed a close genetic relatedness to neuromyelitis optica, systemic sclerosis, and Sjögren's syndrome, suggesting colocalized causal variations close to STAT1-STAT4, TNPO3, and BLK. CONCLUSIONS: This study represents a comprehensive large-scale genetic analysis for PAPS and provides new insights into the genetic basis and pathophysiology of this rare disease.

Rare Turner syndrome and lupus coexistence with insights from DNA methylation patterns.

Systemic lupus erythematosus (SLE or lupus) is a complex autoimmune disease that can affect multiple organs. While the exact disease etiology remains incompletely understood, there is a suggested influence of X-chromosome dosage in the pathogenesis of lupus. Here, we report a rare case of a female patient diagnosed with mosaic Turner syndrome and subsequently presenting with juvenile-onset SLE. DNA methylation patterns were analyzed in this patient and compared with age-matched female SLE controls, revealing higher methylation levels in interferon-regulated genes previously shown to be hypomethylated in SLE. These data provide a potential link between a gene-dose effect from the X-chromosome and the lupus-defining epigenotype. We hypothesize that the attenuated demethylation in interferon-regulated genes might provide a protective effect explaining the rarity of SLE in Turner syndrome.

Neutrophil glucose flux as a therapeutic target in antiphospholipid syndrome.

Neutrophil hyperactivity and neutrophil extracellular trap release (NETosis) appear to play important roles in the pathogenesis of the thromboinflammatory autoimmune disease known as antiphospholipid syndrome (APS). The understanding of neutrophil metabolism has advanced tremendously in the past decade, and accumulating evidence suggests that a variety of metabolic pathways guide neutrophil activities in health and disease. Our previous work characterizing the transcriptome of APS neutrophils revealed that genes related to glycolysis, glycogenolysis, and the pentose phosphate pathway (PPP) were significantly upregulated. Here, we found that APS patient neutrophils used glycolysis more avidly than healthy control neutrophils, especially when the neutrophils were from APS patients with a history of microvascular disease. In vitro, inhibiting either glycolysis or the PPP tempered phorbol myristate acetate- and APS IgG-induced NETosis, but not NETosis triggered by a calcium ionophore. In mice, inhibiting either glycolysis or the PPP reduced neutrophil reactive oxygen species production and suppressed APS IgG-induced NETosis ex vivo. When APS-associated thrombosis was evaluated in mice, inhibiting either glycolysis or the PPP markedly suppressed thrombosis and circulating NET remnants. In summary, these data identify a potential role for restraining neutrophil glucose flux in the treatment of APS.

Thymic gene expression analysis reveals a potential link between HIF-1A and Th17/Treg imbalance in thymoma associated myasthenia gravis.

Myasthenia gravis (MG) is an immune-mediated disease frequently associated with thymic changes. Increased T helper 17 (Th17) cell activity and dysfunctional regulatory T (Treg) cells have been demonstrated in subgroups of MG. On the other hand, hypoxia-inducible factor 1 (HIF-1) has been shown to regulate the Th17/Treg balance by inducing Th17 differentiation while attenuating Treg development. To identify the underlying mechanisms of different thymic pathologies in MG development, we evaluated thymic samples from thymoma-associated myasthenia gravis (TAMG), MG with hyperplasia (TFH-MG) and thymoma without MG (TOMA) patients. Differential gene expression analysis revealed that TAMG and TFH-MG cells are associated with different functional pathways. A higher RORC/FOXP3 ratio provided evidence for Th17/Treg imbalance in TAMG potentially related to increased HIF1A. The hypoxic microenvironment in thymoma may be a driver of TAMG by increasing HIF1A. These findings may lead to new therapeutic approaches targeting HIF1A in the development of TAMG.

Intestinal permeability correlates with disease activity and DNA methylation changes in lupus patients.

OBJECTIVE: Systemic lupus erythematosus (SLE or lupus) is a chronic autoimmune disease that can involve various organ systems. Several studies have suggested that increased intestinal permeability may play a role in the pathogenesis of lupus. The aim of this study was to elucidate the relationship between intestinal permeability, disease activity, and epigenetic changes in lupus patients. METHODS: A total of 25 female lupus patients were included in this study. Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores were used as indicator of disease activity. Plasma zonulin levels were measured, using an ELISA, as a marker of intestinal permeability. Genome-wide DNA methylation patterns were assessed in neutrophils for 19 of the lupus patients using the Infinium MethylationEPIC array. Linear regression and Pearson's correlation were used to evaluate the correlation between zonulin concentrations and SLEDAI scores. The relationship between DNA methylation levels and zonulin concentrations was assessed using beta regression, linear regression, and Pearson's correlation, adjusting for age and race. RESULTS: Intestinal permeability positively correlated with disease activity in lupus patients (p-value = 7.60 × 10-3, r = 0.53). DNA methylation levels in 926 CpG sites significantly correlated with intestinal permeability. The highest correlation was identified in LRIG1 (cg14159396, FDR-adjusted p-value = 1.35 × 10-12, adjusted r2 = 0.92), which plays a role in intestinal homeostasis. Gene Ontologies related to cell-cell adhesion were enriched among the genes that were hypomethylated with increased intestinal permeability in lupus. CONCLUSION: Our data suggest a correlation between increased intestinal permeability and disease activity in lupus patients. Further, increased intestinal permeability might be associated with epigenetic changes that could play a role in the pathogenesis of lupus.

Inducible deletion of Ezh2 in CD4+ T cells inhibits kidney T cell infiltration and prevents interstitial nephritis in MRL/lpr lupus-prone mice.

Systemic lupus erythematosus is a remitting relapsing autoimmune disease characterized by autoantibody production and multi-organ involvement. T cell epigenetic dysregulation plays an important role in the pathogenesis of lupus. We have previously demonstrated upregulation of the key epigenetic regulator EZH2 in CD4+ T cells isolated from lupus patients. To further investigate the role of EZH2 in the pathogenesis of lupus, we generated a tamoxifen-inducible CD4+ T cell Ezh2 conditional knockout mouse on the MRL/lpr lupus-prone background. We demonstrate that Ezh2 deletion abrogates lupus-like disease and prevents T cell differentiation. Single-cell analysis suggests impaired T cell function and activation of programed cell death pathways in EZH2-deficient mice. Ezh2 deletion in CD4+ T cells restricts TCR clonal repertoire and prevents kidney-infiltrating effector CD4+ T cell expansion and tubulointerstitial nephritis, which has been linked to end-stage renal disease in patients with lupus nephritis.

Clinical trait-specific genetic analysis in Behçet's disease identifies novel loci associated with ocular and neurological involvement.

Behçet's disease is a complex inflammatory vasculitis with a broad spectrum of clinical manifestations. The purpose of this study was to investigate the genetics underlying specific clinical features of Behçet's disease. A total of 436 patients with Behçet's disease from Turkey were studied. Genotyping was performed using the Infinium ImmunoArray-24 BeadChip. After imputation and quality control measures, logistic regressions adjusting for sex and the first five principal components were performed for each clinical trait using a case-case genetic analysis approach. A weighted genetic risk score was calculated for each clinical feature. Genetic association analyses of previously identified susceptibility loci in Behçet's disease revealed a genetic association between ocular lesions and HLA-B/MICA (rs116799036: OR = 1.85 [95% CI = 1.35-2.52], p-value = 1.1 × 10-4). The genetic risk score was significantly higher in Behçet's disease patients with ocular lesions compared to those without ocular involvement, which is explained by the genetic variation in the HLA region. New genetic loci predisposing to specific clinical features in Behçet's disease were suggested when genome-wide variants were evaluated. The most significant associations were observed in ocular involvement with SLCO4A1 (rs6062789: OR = 0.41 [95% CI = 0.30-0.58], p-value = 1.92 × 10-7), and neurological involvement with DDX60L (rs62334264: OR = 4.12 [95% CI 2.34 to 7.24], p-value = 8.85 × 10-7). Our results emphasize the role of genetic factors in predisposing to specific clinical manifestations in Behçet's disease, and might shed additional light into disease heterogeneity, pathogenesis, and variability of Behçet's disease presentation across populations.

Phenotype Risk Score but Not Genetic Risk Score Aids in Identifying Individuals With Systemic Lupus Erythematosus in the Electronic Health Record.

OBJECTIVE: Systemic lupus erythematosus (SLE) poses diagnostic challenges. We undertook this study to evaluate the utility of a phenotype risk score (PheRS) and a genetic risk score (GRS) to identify SLE individuals in a real-world setting. METHODS: Using a de-identified electronic health record (EHR) database with an associated DNA biobank, we identified 789 SLE cases and 2,261 controls with available MEGAEX genotyping. A PheRS for SLE was developed using billing codes that captured American College of Rheumatology SLE criteria. We developed a GRS with 58 SLE risk single-nucleotide polymorphisms (SNPs). RESULTS: SLE cases had a significantly higher PheRS (mean ± SD 7.7 ± 8.0 versus 0.8 ± 2.0 in controls; P < 0.001) and GRS (mean ± SD 12.2 ± 2.3 versus 11.0 ± 2.0 in controls; P < 0.001). Black individuals with SLE had a higher PheRS compared to White individuals (mean ± SD 10.0 ± 10.1 versus 7.1 ± 7.2, respectively; P = 0.002) but a lower GRS (mean ± SD 9.0 ± 1.4 versus 12.3 ± 1.7, respectively; P < 0.001). Models predicting SLE that used only the PheRS had an area under the curve (AUC) of 0.87. Adding the GRS to the PheRS resulted in a minimal difference with an AUC of 0.89. On chart review, controls with the highest PheRS and GRS had undiagnosed SLE. CONCLUSION: We developed a SLE PheRS to identify established and undiagnosed SLE individuals. A SLE GRS using known risk SNPs did not add value beyond the PheRS and was of limited utility in Black individuals with SLE. More work is needed to understand the genetic risks of SLE in diverse populations.

Ezh2 Knockout in B Cells Impairs Plasmablast Differentiation and Ameliorates Lupus-like Disease in MRL/lpr Mice.

OBJECTIVES: EZH2 regulates B cell development and differentiation. We previously demonstrated increased EZH2 expression in peripheral blood mononuclear cells from lupus patients. The goal of this study was to evaluate the role of EZH2 expression in B cells in the pathogenesis of lupus. METHODS: We generated an MRL/lpr mouse with floxed Ezh2, which was crossed with CD19-Cre mice to examine the effect of B cell EZH2 deficiency in MRL/lpr lupus-prone mice. Differentiation of B cells was assessed using flow cytometry. Single-cell RNA sequencing and single-cell B cell receptor sequencing were performed. In vitro B cell culture with an X-box binding protein 1 (XBP1) inhibitor was performed. EZH2 and XBP1 messenger RNA levels in CD19+ B cells isolated from lupus patients and healthy controls were analyzed. RESULTS: We show that Ezh2 deletion in B cells significantly decreased autoantibody production and improved glomerulonephritis. B cell development was altered in the bone marrow and spleen of EZH2-deficient mice. Differentiation of germinal center B cells and plasmablasts was impaired. Single-cell RNA sequencing showed that XBP1, a key transcription factor in B cell development, is down-regulated in the absence of EZH2. Inhibiting XBP1 in vitro impairs plasmablast development similar to EZH2 deficiency in mice. Single-cell B cell receptor RNA sequencing revealed defective immunoglobulin class-switch recombination in EZH2-deficient mice. In human lupus B cells, we observed a strong correlation between EZH2 and XBP1 messenger RNA expression levels. CONCLUSION: EZH2 overexpression in B cells contributes to disease pathogenesis in lupus.

Identification of new risk loci shared across systemic vasculitides points towards potential target genes for drug repurposing.

OBJECTIVES: The number of susceptibility loci currently associated with vasculitis is lower than in other immune-mediated diseases due in part to small cohort sizes, a consequence of the low prevalence of vasculitides. This study aimed to identify new genetic risk loci for the main systemic vasculitides through a comprehensive analysis of their genetic overlap. METHODS: Genome-wide data from 8467 patients with any of the main forms of vasculitis and 29 795 healthy controls were meta-analysed using ASSET. Pleiotropic variants were functionally annotated and linked to their target genes. Prioritised genes were queried in DrugBank to identify potentially repositionable drugs for the treatment of vasculitis. RESULTS: Sixteen variants were independently associated with two or more vasculitides, 15 of them representing new shared risk loci. Two of these pleiotropic signals, located close to CTLA4 and CPLX1, emerged as novel genetic risk loci in vasculitis. Most of these polymorphisms appeared to affect vasculitis by regulating gene expression. In this regard, for some of these common signals, potential causal genes were prioritised based on functional annotation, including CTLA4, RNF145, IL12B, IL5, IRF1, IFNGR1, PTK2B, TRIM35, EGR2 and ETS2, each of which has key roles in inflammation. In addition, drug repositioning analysis showed that several drugs, including abatacept and ustekinumab, could be potentially repurposed in the management of the analysed vasculitides. CONCLUSIONS: We identified new shared risk loci with functional impact in vasculitis and pinpointed potential causal genes, some of which could represent promising targets for the treatment of vasculitis.

Regulation of Photosensitivity by the Hippo Pathway in Lupus Skin.

OBJECTIVE: Photosensitivity is one of the most common manifestations of systemic lupus erythematosus (SLE), yet its pathogenesis is not well understood. The normal-appearing epidermis of patients with SLE exhibits increased ultraviolet B (UVB)-driven cell death that persists in cell culture. Here, we investigated the role of epigenetic modification and Hippo signaling in enhanced UVB-induced apoptosis seen in SLE keratinocytes. METHODS: We analyzed DNA methylation in cultured keratinocytes from SLE patients compared to keratinocytes from healthy controls (n = 6/group). Protein expression was validated in cultured keratinocytes using immunoblotting and immunofluorescence. An immortalized keratinocyte line overexpressing WWC1 was generated via lentiviral vector. WWC1-driven changes were inhibited using a large tumor suppressor kinase 1/2 (LATS1/2) inhibitor (TRULI) and small interfering RNA (siRNA). The interaction between the Yes-associated protein (YAP) and the transcriptional enhancer associate domain (TEAD) was inhibited by overexpression of an N/TERT cell line expressing a tetracycline-inducible green fluorescent protein-tagged protein that inhibits YAP-TEAD binding (TEADi). Apoptosis was assessed using cleaved caspase 3/7 and TUNEL staining. RESULTS: Hippo signaling was the top differentially methylated pathway in SLE versus control keratinocytes. SLE keratinocytes (n = 6) showed significant hypomethylation (Δß = -0.153) and thus overexpression of the Hippo regulator WWC1 (P = 0.002). WWC1 overexpression increased LATS1/2 kinase activation, leading to YAP cytoplasmic retention and altered proapoptotic transcription in SLE keratinocytes. Accordingly, UVB-mediated apoptosis in keratinocytes could be enhanced by WWC1 overexpression or YAP-TEAD inhibition, mimicking SLE keratinocytes. Importantly, inhibition of LATS1/2 with either the chemical inhibitor TRULI or siRNA effectively eliminated enhanced UVB-apoptosis in SLE keratinocytes. CONCLUSION: Our work unravels a novel driver of photosensitivity in SLE: overactive Hippo signaling in SLE keratinocytes restricts YAP transcriptional activity, leading to shifts that promote UVB apoptosis.

Physician Perspectives on Vaccination in Patients With Autoimmune Inflammatory Rheumatic Diseases: An International Survey.

OBJECTIVE: To evaluate the perspective of physicians who care for patients with autoimmune inflammatory rheumatic disease (AIIRD) toward vaccination. METHODS: Physicians who care for patients with AIIRD were invited to participate in an online survey regarding their vaccination perspectives in adult patients with AIIRD. RESULTS: Survey responses of 370 physicians from Asia (41.1%), North America (41.6%), Europe (13.8%), and other countries (3.5%) were analyzed. Participants stated that rheumatologists (58.2%) should be primarily responsible for vaccination coverage, followed by general internists (19.3%) and family medicine practitioners (12.8%). Additionally, 96.7% of participants considered vaccination very important (≥ 4/5 rating) for patients with AIIRD. Despite these sentiments, only one-third (37%) reported vaccinating the majority (≥ 60%) of their patients. CONCLUSION: Physicians who care for patients with AIIRD agree that vaccines are effective and safe in patients with AIIRD. Unfortunately, they often do not ensure that their patients are adequately vaccinated. Further studies are needed to investigate how to improve vaccination coverage for this high-risk patient population.