Diagnosis of Multisystem Inflammatory Syndrome in Children by a Whole-Blood Transcriptional Signature.

BACKGROUND: To identify a diagnostic blood transcriptomic signature that distinguishes multisystem inflammatory syndrome in children (MIS-C) from Kawasaki disease (KD), bacterial infections, and viral infections. METHODS: Children presenting with MIS-C to participating hospitals in the United Kingdom and the European Union between April 2020 and April 2021 were prospectively recruited. Whole-blood RNA Sequencing was performed, contrasting the transcriptomes of children with MIS-C (n = 38) to those from children with KD (n = 136), definite bacterial (DB; n = 188) and viral infections (DV; n = 138). Genes significantly differentially expressed (SDE) between MIS-C and comparator groups were identified. Feature selection was used to identify genes that optimally distinguish MIS-C from other diseases, which were subsequently translated into RT-qPCR assays and evaluated in an independent validation set comprising MIS-C (n = 37), KD (n = 19), DB (n = 56), DV (n = 43), and COVID-19 (n = 39). RESULTS: In the discovery set, 5696 genes were SDE between MIS-C and combined comparator disease groups. Five genes were identified as potential MIS-C diagnostic biomarkers (HSPBAP1, VPS37C, TGFB1, MX2, and TRBV11-2), achieving an AUC of 96.8% (95% CI: 94.6%-98.9%) in the discovery set, and were translated into RT-qPCR assays. The RT-qPCR 5-gene signature achieved an AUC of 93.2% (95% CI: 88.3%-97.7%) in the independent validation set when distinguishing MIS-C from KD, DB, and DV. CONCLUSIONS: MIS-C can be distinguished from KD, DB, and DV groups using a 5-gene blood RNA expression signature. The small number of genes in the signature and good performance in both discovery and validation sets should enable the development of a diagnostic test for MIS-C.

Inborn errors of OAS-RNase L in SARS-CoV-2-related multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic double-stranded RNA (dsRNA)-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the single-stranded RNA-degrading ribonuclease L (RNase L). Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNase L deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-deficient but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by mitochondrial antiviral-signaling protein (MAVS) deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.

Host genetic susceptibility underlying SARS-CoV-2-associated Multisystem Inflammatory Syndrome in Brazilian Children.

BACKGROUND: Multisystem Inflammatory Syndrome in Children (MIS-C) is a life-threatening complication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which manifests as a hyper inflammatory process with multiorgan involvement in predominantly healthy children in the weeks following mild or asymptomatic coronavirus disease 2019 (COVID-19). However, host monogenic predisposing factors to MIS-C remain elusive. METHODS: Herein, we used whole exome sequencing (WES) on 16 MIS-C Brazilian patients to identify single nucleotide/InDels variants as predisposition factors associated with MIS-C. RESULTS: We identified ten very rare variants in eight genes (FREM1, MPO, POLG, C6, C9, ABCA4, ABCC6, and BSCL2) as the most promising candidates to be related to a higher risk of MIS-C development. These variants may propitiate a less effective immune response to infection or trigger the inflammatory response or yet a delayed hyperimmune response to SARS-CoV-2. Protein-Protein Interactions (PPIs) among the products of the mutated genes revealed an integrated network, enriched for immune and inflammatory response mechanisms with some of the direct partners representing gene products previously associated with MIS-C and Kawasaki disease (KD). In addition, the PPIs direct partners are also enriched for COVID-19-related gene sets. HLA alleles prediction from WES data allowed the identification of at least one risk allele in 100% of the MIS-C patients. CONCLUSIONS: This study is the first to explore host MIS-C-associated variants in a Latin American admixed population. Besides expanding the spectrum of MIS-C-associated variants, our findings highlight the relevance of using WES for characterising the genetic interindividual variability associated with COVID-19 complications and ratify the presence of overlapping/convergent mechanisms among MIS-C, KD and COVID-19, crucial for future therapeutic management.

Distorted TCR repertoires define multisystem inflammatory syndrome in children.

While the majority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) display mild or no symptoms, rare individuals develop severe disease presenting with multisystem inflammatory syndrome (MIS-C). The reason for variable clinical manifestations is not understood. Here, we carried out TCR sequencing and conducted comparative analyses of TCR repertoires between children with MIS-C (n = 12) and mild (n = 8) COVID-19. We compared these repertoires with unexposed individuals (samples collected pre-COVID-19 pandemic: n = 8) and with the Adaptive Biotechnologies MIRA dataset, which includes over 135,000 high-confidence SARS-CoV-2-specific TCRs. We show that the repertoires of children with MIS-C are characterised by the expansion of TRBV11-2 chains with high junctional and CDR3 diversity. Moreover, the CDR3 sequences of TRBV11-2 clones shift away from SARS-CoV-2 specific T cell clones, resulting in distorted TCR repertoires. In conclusion, our study reports that CDR3-independent expansion of TRBV11-2+ cells, lacking SARS-CoV-2 specificity, defines MIS-C in children.

Genetic and Clinical Characteristics of Patients in the Middle East With Multisystem Inflammatory Syndrome in Children.

Importance: Clinical, genetic, and laboratory characteristics of Middle Eastern patients with multisystem inflammatory syndrome in children (MIS-C) have not yet been documented. Objective: To assess the genetic and clinical characteristics of patients with MIS-C of primarily Arab and Asian origin. Design, Setting, and Participants: A prospective, multicenter cohort study was conducted from September 1, 2020, to August 31, 2021, in the United Arab Emirates and Jordan. Forty-five patients with MIS-C and a matched control group of 25 healthy children with a confirmed SARS-CoV-2 infection status were recruited. Whole exome sequencing in all 70 participants was performed to identify rare, likely deleterious variants in patients with MIS-C and to correlate genetic findings with the clinical course of illness. Exposures: SARS-CoV-2. Main Outcomes and Measures: Fever, organ system complications, laboratory biomarkers, whole exome sequencing findings, treatments, and clinical outcomes were measured. The Mann-Whitney U test was used to assess the association between genetic variants and MIS-C attributes. The Fisher exact test was used to compute the genetic burden in MIS-C relative to controls. Results: A total of 45 patients with MIS-C (23 [51.1%] male; 30 [66.7%] of Middle Eastern origin; mean [SD] age, 6.7 [3.6] years) and 25 controls (17 [68.0%] male; 24 [96.0%] of Middle Eastern origin; mean [SD] age 7.4 [4.0] years) participated in the study. Key inflammatory markers were significantly dysregulated in all patients with MIS-C. Mucocutaneous and gastrointestinal manifestations were each reported in 36 patients (80.0%; 95% CI, 66.1%-89.1%), cardiac findings were reported in 22 (48.9%; 95% CI, 35.0%-63.0%), and neurologic findings were reported in 14 (31.1%; 95% CI, 19.5%-45.6%). Rare, likely deleterious heterozygous variants in immune-related genes, including TLR3, TLR6, IL22RA2, IFNB1, and IFNA6, were identified in 19 patients (42.2%; 95% CI, 29.0%-56.7%), of whom 7 had multiple variants. There was higher enrichment of genetic variants in patients relative to controls (29 vs 3, P < .001). Patients with those variants tended to have earlier disease onset (7 patients [36.8%; 95% CI, 19.1%-58.9%] with genetic findings vs 2 [7.7%; 95% CI, 2.1%-24.1%] without genetic findings were younger than 3 years at onset) and resistance to treatment (8 patients [42.1%; 95% CI, 23.1%-63.7%] with genetic findings vs 3 patients [11.5%; 95% CI, 4.0%-29.0%] without genetic findings received 2 doses of intravenous immunoglobulin). Conclusions and Relevance: The results of this cohort study suggest that rare, likely deleterious genetic variants may contribute to MIS-C disease. This finding paves the way for additional studies with larger, diverse populations to fully characterize the genetic contribution to this new disease entity.

Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19.

Pediatric Coronavirus Disease 2019 (pCOVID-19) is rarely severe; however, a minority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might develop multisystem inflammatory syndrome in children (MIS-C), with substantial morbidity. In this longitudinal multi-institutional study, we applied multi-omics (analysis of soluble biomarkers, proteomics, single-cell gene expression and immune repertoire analysis) to profile children with COVID-19 (n = 110) and MIS-C (n = 76), along with pediatric healthy controls (pHCs; n = 76). pCOVID-19 was characterized by robust type I interferon (IFN) responses, whereas prominent type II IFN-dependent and NF-κB-dependent signatures, matrisome activation and increased levels of circulating spike protein were detected in MIS-C, with no correlation with SARS-CoV-2 PCR status around the time of admission. Transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation. The association of MIS-C with the combination of HLA A*02, B*35 and C*04 alleles suggests genetic susceptibility. MIS-C B cells showed higher mutation load than pCOVID-19 and pHC. These results identify distinct immunopathological signatures in pCOVID-19 and MIS-C that might help better define the pathophysiology of these disorders and guide therapy.

The autoimmune signature of hyperinflammatory multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) manifests as a severe and uncontrolled inflammatory response with multiorgan involvement, occurring weeks after SARS-CoV-2 infection. Here, we utilized proteomics, RNA sequencing, autoantibody arrays, and B cell receptor (BCR) repertoire analysis to characterize MIS-C immunopathogenesis and identify factors contributing to severe manifestations and intensive care unit admission. Inflammation markers, humoral immune responses, neutrophil activation, and complement and coagulation pathways were highly enriched in MIS-C patient serum, with a more hyperinflammatory profile in severe than in mild MIS-C cases. We identified a strong autoimmune signature in MIS-C, with autoantibodies targeted to both ubiquitously expressed and tissue-specific antigens, suggesting autoantigen release and excessive antigenic drive may result from systemic tissue damage. We further identified a cluster of patients with enhanced neutrophil responses as well as high anti-Spike IgG and autoantibody titers. BCR sequencing of these patients identified a strong imprint of antigenic drive with substantial BCR sequence connectivity and usage of autoimmunity-associated immunoglobulin heavy chain variable region (IGHV) genes. This cluster was linked to a TRBV11-2 expanded T cell receptor (TCR) repertoire, consistent with previous studies indicating a superantigen-driven pathogenic process. Overall, we identify a combination of pathogenic pathways that culminate in MIS-C and may inform treatment.

Editorial: Multisystem Inflammatory Syndrome in Adults (MIS-A) and the Spectrum of COVID-19.

Recent studies on the pathogenesis and clinical spectrum of human disease following infection with the new human pathogen, SARS-CoV-2, have identified the varied presentations and sequelae of COVID-19. Acute 'cytokine storm' in severe COVID-19 results in multiorgan damage due to vascular hyperpermeability, edema, and hypercoagulation. The long-term consequences of infection from SARS-CoV-2 include long COVID. or post-COVID syndrome, and multisystem inflammatory syndrome in children (MIS-C). Several case reports of multisystem inflammatory syndrome in adults (MIS-A) have shown the presentation at more than four weeks after initial infection with SARS-CoV-2 in adults more than 21 years of age. In September 2021, a published systematic review of the literature identified 221 patients with MIS-A, representing the most comprehensive clinical study to date. MIS-A occurs in the post-acute COVID-19 period. The pathogenesis may involve a dysregulated antibody-mediated immune response, similar to MIS-C. Therefore, patients with MIS-A may respond to supportive therapies that control hyperinflammation. This Editorial aims to describe MIS-A and discuss COVID-19 as a spectrum of hyperinflammatory disease in terms of severity, extent, duration, and patient age.

Immunotherapy of multisystem inflammatory syndrome in children (MIS-C) following COVID-19 through mesenchymal stem cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new type of coronavirus causing coronavirus 2019 (COVID-19) that was first observed in Wuhan, China, in Dec. 2019. An inflammatory immune response targeting children appeared during the pandemic, which was associated with COVID-19 named multisystem inflammatory syndrome in children (MIS-C). Characteristics of MIS-C include the classic inflammation findings, multi-organ dysfunction, and fever as the cardinal feature. Up to now, no specific therapy has been identified for MIS-C. Currently, considerable progress has been obtained in the MIS-C treatment by cell therapy, specially Mesenchymal stem cells (MSCs). Unique properties have been reported for MSCs, such as various resources for purification of cell, high proliferation, self-renewal, non-invasive procedure, tissue regenerator, multidirectional differentiation, and immunosuppression. As indicated by a recent clinical research, MSCs have the ability of reducing disease inflammation and severity in children with MIS-C. In the present review study, the benefits and characteristics of MSCs and exosomes are discussed for treating patients with MIS-C.

Host genetics of pediatric SARS-CoV-2 COVID-19 and multisystem inflammatory syndrome in children.

PURPOSE OF REVIEW: This review is meant to describe the genetic associations with pediatric severe COVID-19 pneumonia and the postinfectious complication of the multisystem inflammatory syndrome in children (MIS-C). Multiple genetic approaches have been carried out, primarily in adults with extrapolation to children, including genome-wide association studies (GWAS), whole exome and whole genome sequencing (WES/WGS), and target gene analyses. RECENT FINDINGS: Data from adults with severe COVID-19 have identified genomic regions (human leukocyte antigen locus and 3p21.31) as potential risk factors. Genes related to viral entry into cells (ABO blood group locus, ACE2, TMPRS22) have been linked to severe COVID-19 patients by GWAS and target gene approaches. Type I interferon (e.g. IFNAR2) and antiviral gene (e.g. TLR7) associations have been identified by several genetic approaches in severe COVID-19. WES has noted associations with several immune regulatory genes (e.g. SOCS1). Target gene approaches have identified mutations in perforin-mediated cytolytic pathway genes in children and adults with severe COVID-19 and children with MIS-C. SUMMARY: Several genetic associations have been identified in individuals with severe COVID-19 and MIS-C via various genetic approaches. Broadly speaking, COVID-19 genetic associations include genes involved with antiviral functions, viral cell entry, immune regulation, chemotaxis of white blood cells, and lymphocyte cytolytic function.

Inflammasome Activation in Children With Kawasaki Disease and Multisystem Inflammatory Syndrome.

[Figure: see text].

Downregulation of exhausted cytotoxic T cells in gene expression networks of multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and pathology of multiple organs in individuals under 21 years of age in the weeks following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although an autoimmune pathogenesis has been proposed, the genes, pathways and cell types causal to this new disease remain unknown. Here we perform RNA sequencing of blood from patients with MIS-C and controls to find disease-associated genes clustered in a co-expression module annotated to CD56dimCD57+ natural killer (NK) cells and exhausted CD8+ T cells. A similar transcriptome signature is replicated in an independent cohort of Kawasaki disease (KD), the related condition after which MIS-C was initially named. Probing a probabilistic causal network previously constructed from over 1,000 blood transcriptomes both validates the structure of this module and reveals nine key regulators, including TBX21, a central coordinator of exhausted CD8+ T cell differentiation. Together, this unbiased, transcriptome-wide survey implicates downregulation of NK cells and cytotoxic T cell exhaustion in the pathogenesis of MIS-C.

Multisystem Inflammatory Syndrome in Children in February 2020 and Implications of Genomic Sequencing for SARS-CoV-2.

Multisystem inflammatory syndrome in children (MIS-C) is a newly recognized disease process that can complicate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We present what we believe to be the earliest case of MIS-C, occurring in February 2020. Our patient's SARS-CoV-2 infection was caused by an emerging lineage with the D614G variant in the spike protein. This lineage would subsequently become the predominant cause of SARS-CoV-2 outbreaks in Europe and the United States where MIS-C was first described.

Phenotype, Susceptibility, Autoimmunity, and Immunotherapy Between Kawasaki Disease and Coronavirus Disease-19 Associated Multisystem Inflammatory Syndrome in Children.

Coronavirus disease-19 (COVID-19) in children is usually mild but some are susceptible to a Kawasaki disease (KD)-like multisystem inflammatory syndrome in children (MIS-C) in the convalescent stage, posing a need to differentiate the phenotype, susceptibility, autoimmunity, and immunotherapy between KD and MIS-C, particularly in the upcoming mass vaccination of COVID-19. Patients with MIS-C are prone to gastrointestinal symptoms, coagulopathy, and shock in addition to atypical KD syndrome with fever, mucocutaneous lesions, lymphadenopathy, and/or cardiovascular events. MIS-C manifests KD-like symptoms that alert physicians to early recognize and adopt the KD treatment regimen for patients with MIS-C. MIS-C linked to COVID-19 teaches us infection-associated autoimmune vasculitis and vice versa. Studies on genetic susceptibility have identified certain human leukocyte antigen (HLA) locus and toll-like receptor (TLR) associated with KD and/or COVID-19. Certain HLA subtypes, such as HLA-DRB1 and HLA-MICA A4 are associated with KD. HLA-B*46:01 is proposed to be the risk allele of severe COVID-19 infection, and blood group O type is a protective factor of COVID-19. The autoimmune vasculitis of KD, KD shock syndrome (KDSS), or MIS-C is mediated by a genetic variant of HLA, FcγR, and/or antibody-dependent enhancement (ADE) resulting in hyperinflammation with T helper 17 (Th17)/Treg imbalance with augmented Th17/Th1 mediators: interleukin-6 (IL-6), IL-10, inducible protein-10 (IP-10), Interferon (IFNγ), and IL-17A, and lower expression of Treg-signaling molecules, FoxP3, and transforming growth factor (TGF-ß). There are certain similarities and differences in phenotypes, susceptibility, and pathogenesis of KD, KDSS, and MIS-C, by which a physician can make early protection, prevention, and precision treatment of the diseases. The evolution of immunotherapies for the diseases has shown that intravenous immunoglobulin (IVIG) alone or combined with corticosteroids is the standard treatment for KD, KDSS, and MIS-C. However, a certain portion of patients who revealed a treatment resistance to IVIG or IVIG plus corticosteroids, posing a need to early identify the immunopathogenesis, to protect hosts with genetic susceptibility, and to combat Th17/Treg imbalance by anti-cytokine or pro-Treg for reversal of the hyperinflammation and IVIG resistance. Based on physiological and pathological immunity of the diseases under genetic susceptibility and host milieu conditions, a series of sequential regimens are provided to develop a so-called "Know thyself, enemy (pathogen), and ever-victorious" strategy for the prevention and immunotherapy of KD and/or MIS-C.

HLA class I-associated expansion of TRBV11-2 T cells in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. Superantigen specificity for different Vß chains results in Vß skewing, whereby T cells with specific Vß chains and diverse antigen specificity are overrepresented in the T cell receptor (TCR) repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCRß variable gene 11-2 (TRBV11-2), with up to 24% of clonal T cell space occupied by TRBV11-2 T cells, which correlated with MIS-C severity and serum cytokine levels. Analysis of TRBJ gene usage and complementarity-determining region 3 (CDR3) length distribution of MIS-C expanded TRBV11-2 clones revealed extensive junctional diversity. Patients with TRBV11-2 expansion shared HLA class I alleles A02, B35, and C04, indicating what we believe is a novel mechanism for CDR3-independent T cell expansion. In silico modeling indicated that polyacidic residues in the Vß chain encoded by TRBV11-2 (Vß21.3) strongly interact with the superantigen-like motif of SARS-CoV-2 spike glycoprotein, suggesting that unprocessed SARS-CoV-2 spike may directly mediate TRBV11-2 expansion. Overall, our data indicate that a CDR3-independent interaction between SARS-CoV-2 spike and TCR leads to T cell expansion and possibly activation, which may account for the clinical presentation of MIS-C.

Superantigenic character of an insert unique to SARS-CoV-2 spike supported by skewed TCR repertoire in patients with hyperinflammation.

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is a newly recognized condition in children with recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These children and adult patients with severe hyperinflammation present with a constellation of symptoms that strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to T cell receptors (TCRs) and/or major histocompatibility complex class II (MHCII) molecules. Here, using structure-based computational models, we demonstrate that the SARS-CoV-2 spike (S) glycoprotein exhibits a high-affinity motif for binding TCRs, and may form a ternary complex with MHCII. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in other SARS-related coronaviruses), which is highly similar in both sequence and structure to the bacterial superantigen staphylococcal enterotoxin B. This interaction between the virus and human T cells could be strengthened by a rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like motif shared between the SARS viruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence motif on the receptor-binding domain also exhibits a high tendency to bind TCRs. Analysis of the TCR repertoire in adult COVID-19 patients demonstrates that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen activation. These data suggest that SARS-CoV-2 S may act as a superantigen to trigger the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches.

Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function.

Autoinflammatory disease can result from monogenic errors of immunity. We describe a patient with early-onset multi-organ immune dysregulation resulting from a mosaic, gain-of-function mutation (S703I) in JAK1, encoding a kinase essential for signaling downstream of >25 cytokines. By custom single-cell RNA sequencing, we examine mosaicism with single-cell resolution. We find that JAK1 transcription was predominantly restricted to a single allele across different cells, introducing the concept of a mutational "transcriptotype" that differs from the genotype. Functionally, the mutation increases JAK1 activity and transactivates partnering JAKs, independent of its catalytic domain. S703I JAK1 is not only hypermorphic for cytokine signaling but also neomorphic, as it enables signaling cascades not canonically mediated by JAK1. Given these results, the patient was treated with tofacitinib, a JAK inhibitor, leading to the rapid resolution of clinical disease. These findings offer a platform for personalized medicine with the concurrent discovery of fundamental biological principles.

Multisystem Inflammatory Syndrome in Children (MIS-C) with COVID-19: Insights from simultaneous familial Kawasaki Disease cases.

Recently, an increasing number of SARS-CoV-2 patients with COVID-19 syndrome, which overlaps with Kawasaki Disease (KD), have been reported, supporting the suggestion that infection is one of the triggers of KD. We summarized the reports of simultaneous familial KD cases to better understand the etiopathogenesis of both KD and Multisystem Inflammatory Syndrome in Children (MIS-C) related to COVID-19. Here we discuss the etiology of these syndromes from the point of view of infection and genetic susceptibility.