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Principal Investigator: Michael Ombrello
Organization: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
Fiscal Year: 2023
Award: $974,635
Funding agency: National Institute of Arthritis and Musculoskeletal and Skin Diseases
In the current reporting period, we have continued to employ genetic and genomic approaches to discover causes of or risk factors for Stills disease/systemic juvenile idiopathic arthritis (sJIA).
We have continued working on an Immunochip association study of 889 children with sJIA and >16,000 control subjects. This has identified a strong association between sJIA and CXCR4, the gene that encodes the C-X-C chemokine receptor type 4. We observed a 125kb haplotype allele that is more common in children with sJIA than in healthy people (p = 4.3 times 10 to the negative 8; odds ratio 1.7; 95% confidence interval 1.5 to 1.9). Homozygosity for the risk haplotype was an even greater risk factor, with over 5% of children with sJIA carrying 2 risk alleles (odds ratio 5.1; 95% confidence interval 2.8 to 9.3). We next established that the risk haplotype has a biologic effect, regulating CXCR4 expression and downstream signaling. The sJIA-associated region encodes the CXCR4 gene and important regulatory elements that govern its expression, including a lymphocyte-specific super-enhancer. Using lymphoblastoid cell line RNA sequencing from 373 European Geuvadis Project participants, we showed that the risk haplotype allele leads to higher CXCR4 expression than the protective allele (p = 0.0004). To evaluate the effect of the risk allele on CXCR4-dependent signaling, we quantified migration of freshly isolated primary leukocytes from 12 genotypically-ascertained healthy subjects towards the CXCR4 ligand, CXCL12, using a flow cytometry-based migration assay. We observed enhanced migration of primary monocytes (p = 0.0091), basophils (p = 0.0097) and neutrophils (p = 0.0462) from risk haplotype homozygotes, relative to those from protective haplotype homozygotes. Finally, we demonstrated that CXCR4 pathway activation is a feature of sJIA that is present in both the active and inactive disease states. In bulk monocyte RNA sequencing data (GSE147608), CXCR4 expression was increased in children with sJIA, relative to healthy children (p = 0.00092), regardless of disease activity. Gene set enrichment analysis of GSE147608 demonstrated upregulation of CXCR4 signaling, upregulation of MAP kinase and NF-KB signaling, and upregulation of mTORC1 signaling and autophagy in sJIA monocytes, regardless of disease activity. As an independent indicator of CXCL12-CXCR4 pathway activity, we measured serum levels of CXCL12 and soluble CXCR4 (sCXCR), the product of surface cleavage, in sJIA patients from our clinic and healthy subjects. Serum sCXCR4 and CXCL12 were both higher in patients with sJIA than in healthy subjects, regardless of disease activity. This study suggests that increased CXCR4 expression and enhanced CXCR4-mediated signaling are hallmarks of sJIA monocytes, independent of disease activity. As such, CXCR4 represents a potentially unique therapeutic target in sJIA/AOSD. A manuscript describing this work is in preparation.
We have continued to search for genetic or transcriptomic biomarkers predictive of disease course, complications or therapeutic responses in Stills disease. Several studies have described enhanced expression of interferon (IFN) stimulated genes (ISG) in children with recalcitrant courses of sJIA, but whether/how IFNs are involved in sJIA is unclear. To investigate this, we performed trio whole exome sequencing and quantified ISG expression with custom Nanostring assays in 59 consecutive Stills disease patients in our clinic. The distribution of ISG scores among Stills patients revealed negative skewing with an upper quartile that was separate from the remainder of the cohort. Lung disease with hypersensitivity (LD/DHR) was more common in the top quartile than the bottom quartile (0.54, 0.07; p=0.02), but the rates of active disease, interleukin-18 levels over 15 ng/mL, HLA-DRB1*15 carriage and history of MAS did not differ significantly between the groups. To determine whether genetic variation was contributing to their elevated ISG expression, we used Web-based Gene Set Analysis Toolkit (webgestalt.org) to perform pathway-based over-representation analysis (ORA) of the full set of genes that harbor rare, protein-altering Mendelian candidate mutations in the 15 subjects in the top quartile (n=83). ORA revealed significant (false discovery rate < 0.05) enrichment of mutated genes involved in macrophage activation (ratio = 21.7), type I IFN biosynthetic process (ratio = 77.9) and myeloid leukocyte activation (ratio = 5.0). ORA of the candidate gene list from individuals in the bottom ISG quartile revealed no enrichment of any pathways. This enrichment of potentially causative variation in overlapping innate immune pathways suggests that rare de novo or recessive variation contribute to the pathophysiology of Stills disease, promoting enhanced IFN signaling and potentially predisposing to LD/DHR. A preliminary description of this work will be presented as an oral presentation at the 2023 American College of Rheumatology Convergence. A manuscript describing these findings is in preparation.
We have invested time and effort to establish a framework for collaborative genetic studies with the Childhood Arthritis and Rheumatology Research Alliance (CARRA), including a large family-based sequencing study of sJIA and a pharmacogenomics study of participants in the now complete FiRst Line Options for Systemic JIA Treatment (FROST) Study.
We also performed an anonymous online survey of Still's disease patients between June and August 2022 to learn about their experiences with COVID-19 vaccines and infection. We received 150 valid responses, including 99 patients who received at least 1 dose of the vaccine and 51 unvaccinated patients. Vaccination was well tolerated by respondents, with only 6 patients (6%) reporting a disease flare and 2 patients who reported cardiac side effects (one myocarditis, one atrial fibrillation). Thirty-three patients contracted COVID after 2 doses of the vaccine, and 46 patients contracted the infection without full immunization. The only COVID infection requiring ICU admission occurred in the non-immunized group. A manuscript describing this study is under revision at Pediatric Rheumatology.
Beyond sJIA, we have continued to study patients with immune dysregulation and mutations of phospholipase C gamma 2 (PLCG2). We have previously described autosomal dominant forms of immune dysregulation associated with dominant negative and hypermorphic mutations of PLCG2. Now we have described 60 new variants of PLCG2 observed in patients with immune dysregulation. PLCG2 variants were generated by mutagenesis of eGFP-PLCG2 plasmid, which was overexpressed in a Plcg2-deficient DT-40 B-cell line. B-cell receptor induced calcium flux and ERK phosphorylation were assayed by flow cytometry. Stimulation-induced calcium flux and natural killer cell degranulation were each measured in primary cells from a subset of patients. Three-fourths of PLCG2 variants led to functional alteration of B-cell activation, in vitro, including 13 gain-of-function (GOF) variants. Surprisingly, 29 of the PLCG2 variants led to loss-of-function (LOF), with these patients and their mutations defining a new class of immune dysregulation caused by PLCG2 LOF. Clinically, susceptibility to infection and autoinflammation were common with both GOF and LOF variants, while a new phenotypic cluster consisting of humoral immune deficiency, autoinflammation, susceptibility to herpes virus infection and natural killer (NK) cell dysfunction was observed in association with heterozygous LOF variants detected in both familial and sporadic cases. This work defines a novel form of PLCG2-associated immune dysregulation caused by monoallelic loss of function variants. A manuscript describing this work is in press in the Journal of Allergy and Clinical Immunology.
Terms: <0-11 years old><16 year old><16 years of age><2019-nCoV vaccine><Admission><Admission activity><Adult-Onset Diabetes Mellitus><Age of Onset><Alleles><Allelic Loss><Allelomorphs><Allergy><American><Antimorphic mutation><Arthritis><Assay><Atheroscleroses><Atherosclerosis><Atherosclerotic Cardiovascular Disease><Atrial Fibrillation><Auricular Fibrillation><Autoimmune Status><Autoimmunity><Autophagocytosis><B blood cells><B cell><B cell receptor><B cells><B-Cell Activation><B-Cell Antigen Receptor><B-Cells><B-Lymphocytes><B-cell><Basophilic Granulocyte><Basophils><Bio-Informatics><Bioassay><Biochemical><Bioinformatics><Biologic Assays><Biological><Biological Assay><Biology><Blood Basophil><Blood Neutrophil><Blood Polymorphonuclear Neutrophil><Blood Serum><Blood leukocyte><Blood monocyte><C-X-C Chemokine Receptor Type 4><CD184 Antigen><COVID><COVID-19 infection><COVID-19 vaccine><COVID-19 virus infection><COVID19 infection><COVID19 vaccine><CXC-R4><CXCL12><CXCL12 gene><CXCL12 protein><CXCR-4><CXCR4><CXCR4 Receptors><CXCR4 gene><Candidate Disease Gene><Candidate Gene><Cardiac><Causality><Cell Body><Cell Communication and Signaling><Cell Degranulation><Cell Line><Cell Migration Assay><Cell Signaling><CellLine><Cells><Chemokine (C-X-C Motif) Ligand 12><Chemokine (C-X-C Motif) Receptor 4><Chemokine, CXC Motif, Receptor 4><Child><Child Youth><Childhood><Children (0-21)><Chronic><Chronic Childhood Arthritis><Clinic><Clinical><Clinical Immunology><CoV disease><Collaborations><Collection><Complex><Confidence Intervals><Contracting Opportunities><Contracts><Cross-Product Ratio><Custom><Cytotoxic cell><D2S201E><Data><Disease><Disorder><Dominant Negative><Dominant-Negative Mutant><Dominant-Negative Mutation><Dose><Dysfunction><Enhancers><Environmental Factor><Environmental Risk Factor><Etiology><European><Exanthem><Exanthema><FB22><Family><Fever><Flare><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Frequencies><Functional disorder><Fusin><Gene Expression><Gene set enrichment analysis><Gene variant><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Diversity><Genetic Variation><Genetic defect><Genetic study><Genetics-Mutagenesis><Genomic approach><Genomics><Genotype><Goals><HLA-DRB1><HLA-DRB1 antigen><HM89><HSY3RR><Haplotypes><Hemophagocytic Lymphohistiocytoses><Hereditary Periodic Fever Syndromes><Herpes infection><Herpesviridae Infections><Herpesviridae disease><Herpesvirus Infections><Heterogeneity><History><Homozygote><Hypersensitivity><IFN><IFN-Gamma-Inducing Factor Gene><IFN-gamma-Inducing Factor><IGIF><IGIF Gene><IL-1 Gamma><IL-1 Gamma Gene><IL-18><IL-18 Gene><IL-1g><IL-1g Gene><IL18><IL18 Protein><IL18 gene><IL1F4><IL1F4 Gene><Immune><Immune Cell Activation><Immunes><Immunization><Immunoglobulin Enhancer-Binding Protein><In Vitro><Individual><Infection><Inflammation><Inflammatory><Inflammatory Arthritis><Interferon Type I><Interferon-Gamma-Inducing Factor Gene><Interferon-gamma-Inducing Factor><Interferons><Interleukin 18 (Interferon-Gamma-Inducing Factor)><Interleukin 18 (Interferon-Gamma-Inducing Factor) Gene><Interleukin 18 Proprotein><Interleukin 18 Proprotein Gene><Interleukin-1 Gamma><Interleukin-1 Gamma Gene><Interleukin-18><Interleukin-18 Precursor><Interleukin-18 Precursor Gene><International><Intracellular Communication and Signaling><Intramural Research><Investigation><Investments><Joints><Journals><Juvenile Chronic Arthritis><Juvenile Chronic Polyarthritis><Juvenile Rheumatoid Arthritis><Juvenile-Onset Still's Disease><K lymphocyte><Ketosis-Resistant Diabetes Mellitus><LAP3><LCR1><LESTR><LESTR Receptor><LPS-Associated Protein 3><Learning><Lesion><Leukocyte-Derived Seven-Transmembrane Domain Receptor><Leukocytes><Leukocytes Reticuloendothelial System><Life><Ligands><Lipopolysaccharide-Associated Protein 3><Loss of Heterozygosity><Lung><Lung Respiratory System><Lung diseases><Lymphatic cell><Lymphocyte><Lymphocytic><MAP kinase><MGC12320><MGC12320 Gene><Macrophage Activation><Macrophage activation syndrome><Magazine><Manuscripts><Marrow Neutrophil><Marrow leukocyte><Marrow monocyte><Maturity-Onset Diabetes Mellitus><Measures><Mediating><Mendelian disease><Mendelian disorder><Mendelian genetic disorder><Migration Assay><Mitogen-Activated Protein Kinases><Mutagenesis><Mutagenesis Molecular Biology><Mutate><Mutation><Myelogenous><Myeloid><Myocarditis><NF-kB><NF-kappa B><NF-kappaB><NFKB><NIDDM><NIH><NK Cells><NPY3R><NPYR><NPYRL><NPYY3R><National Institutes of Health><Natural Killer Cells><Neuropeptide Y Receptor Y3><Neutrophilic Granulocyte><Neutrophilic Leukocyte><Non-Insulin Dependent Diabetes><Non-Insulin-Dependent Diabetes Mellitus><Noninsulin Dependent Diabetes><Noninsulin Dependent Diabetes Mellitus><Nuclear Factor kappa B><Nuclear Transcription Factor NF-kB><Odds Ratio><On-Line Systems><Online Systems><Oral><PBSF><PLCG2><PLCG2 gene><Participant><Pathogenesis><Pathway interactions><Patients><Pattern><Persons><Pharmacogenomics><Phenotype><Phosphatidylinositol-Specific Phospholipase C><Phospholipase C Gamma 2><Phosphorylation><Physiopathology><Plasmids><Polymorphonuclear Cell><Polymorphonuclear Leukocytes><Polymorphonuclear Neutrophils><Population><Pre-B Cell Growth Stimulating Factor><Predisposition><Preparation><Prevalence><Process><Protein Phosphorylation><Proteins><Protocol><Protocols documentation><Pulmonary Diseases><Pulmonary Disorder><Pyrexia><RNA Seq><RNA sequencing><RNAseq><Rash><Recording of previous events><Regulatory Element><Relative Odds><Reporting><Research><Research Resources><Resources><Respondent><Rheumatology><Risk><Risk Factors><Risk Ratio><Risk-associated variant><SARS-CoV-2 infection><SARS-CoV-2 vaccine><SARS-CoV2 infection><SARS-CoV2 vaccine><SARS-coronavirus-2 vaccine><SCYB12><SDF-1><SDF-1 Receptor><SDF-1A><SDF-1B><SDF-1alpha><SDF1><SDF1/PBSF Receptor CXCR4><SDF1A><SDF1B><Salmon><Sdf1 protein><Serositis><Serum><Severe Acute Respiratory Syndrome CoV 2 vaccine><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 vaccine><Signal Transduction><Signal Transduction Systems><Signaling><Skin Rash><Slow-Onset Diabetes Mellitus><Spleen Seven-Transmembrane-Segment Receptor><Stable Diabetes Mellitus><Still's disease><Strains Cell Lines><Stromal Cell-Derived Factor 1><Stromal Cell-Derived Factor 1 Receptor><Surface><Survey Instrument><Surveys><Susceptibility><T2 DM><T2D><T2DM><TLSF-A><TLSF-B><TPAR1><Time><Transcription Factor NF-kB><Type 2 Diabetes Mellitus><Type 2 diabetes><Type II Diabetes Mellitus><Type II diabetes><United States National Institutes of Health><Up-Regulation><Upregulation><Vaccination><Vaccines><Variant><Variation><White Blood Cells><White Cell><Work><activated B cells><adult onset diabetes><age 16 years><allele variant><allelic variant><arthritic><atheromatosis><atherosclerotic disease><atherosclerotic vascular disease><autoinflammation><autoinflammatory diseases><autoinflammatory disorders><autophagy><autosome><biologic><biological signal transduction><boys><calcium flux><calcium mobilization><cardiac inflammation><causation><cohort><college><collegiate><corona virus disease><corona virus disease 2019 vaccine><coronavirus disease><coronavirus disease 2019 infection><coronavirus disease 2019 vaccine><coronavirus disease-19 vaccine><cultured cell line><customs><disease causation><disease of the lung><disorder of the lung><environmental risk><exome sequencing><exome-seq><experience><familial hemophagocytic lymphohistiocytosis><febrile><febris><flow cytophotometry><gain of function><genetic approach><genetic strategy><genetic variant><genome mutation><genomic effort><genomic strategy><genomic variant><girls><hIRH><hemophagocytic lymphohistiocytosis><histories><immune activation><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><innate immune pathways><juvenile arthritis><juvenile idiopathic arthritis><kappa B Enhancer Binding Protein><ketosis resistant diabetes><kids><leukocyte activation><loss of function><lung disorder><lymph cell><lymphoblastoid cell line><lymphocyte hyperplasia><lymphoid hyperplasia><maturity onset diabetes><migration><monocyte><monogenic disease><monogenic disorder><nCoV vaccine><nCoV-19 vaccine><nCoV19 vaccine><nano-string><nanostring><neutrophil><non vaccinated><not vaccinated><novel><nuclear factor kappa beta><online computer><overexpress><overexpression><pathophysiology><pathway><patient subgroups><patient subpopulations><patient subsets><patient subtypes><pediatric><predictive biomarkers><predictive marker><predictive molecular biomarker><preparations><protective allele><protective variant><prototype><pulmonary><recruit><release of sequestered calcium ion into cytoplasm><response><response to therapy><response to treatment><risk allele><risk gene><risk genotype><risk loci><risk locus><risk variant><side effect><single-gene disease><single-gene disorder><sixteen year old><sixteen years of age><skin color><stromal cell-derived factor-1alpha><systemic juvenile idiopathic arthritis><systemic onset juvenile idiopathic arthritis><therapeutic response><therapeutic target><therapy response><transcriptome sequencing><transcriptomic sequencing><transcriptomics><treatment response><type 2 DM><type II DM><type two diabetes><unvaccinated><vaccine against 2019-nCov><vaccine against SARS-CoV-2><vaccine against SARS-CoV2><vaccine against SARS-coronavirus-2><vaccine against Severe Acute Respiratory Syndrome CoV 2><vaccine against Severe acute respiratory syndrome coronavirus 2><vaccine for novel coronavirus><variant detection><web based><white blood cell><white blood corpuscle><youngster>