Genetics and pathophysiology of systemic juvenile idiopathic arthritis and other complex autoinflammatory diseases

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

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.