Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C

Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with acute COVID-19 (n=70) or MIS-C (n=141) across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between these two disease states, with increased heterogeneity and multi-organ involvement in MIS-C encompassing diverse cell types such as endothelial and neuronal Schwann cells. Whole blood RNA profiling reveals upregulation of similar pro-inflammatory signaling pathways in COVID-19 and MIS-C, but also MIS-C specific downregulation of T cell-associated pathways. Profiling of plasma cell-free RNA and whole blood RNA in paired samples yields different yet complementary signatures for each disease state. Our work provides a systems-level, multi-analyte view of immune responses and tissue damage in COVID-19 and MIS-C and informs the future development of new disease biomarkers. ### Competing Interest Statement I.D.V. is a member of the Scientific Advisory Board of Karius Inc., Kanvas Biosciences and GenDX. C.Y.C. is a founder and a member of the Scientific Advisory Board of Delve Bio. A.P.C. is listed as an inventor on submitted patents pertaining to cell-free DNA (US patent applications 63/237,367, 63/056,249, 63/015,095, 16/500,929) and receives consulting fees from Eurofins Viracor. C.A.R.s institution has received funding to conduct clinical research unrelated to this manuscript from BioFire Inc., GSK, MedImmune, Micron, Merck, Novavax, PaxVax, Regeneron, Pfizer, and Sanofi-Pasteur. She is co-inventor of patented RSV vaccine technology (International PCT Application No. PCT/US2016/058976, filed 12/28/2016 by Emory University), which has been licensed to Meissa Vaccines, Inc. with royalties received. Her institution has received funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines. E.J.A has consulted for Pfizer, Sanofi Pasteur, GSK, Janssen, and Medscape, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Sanofi-Pasteur, Janssen, and Micron. He also serves on a safety monitoring board for Kentucky BioProcessing, Inc. and Sanofi Pasteur. He serves on a data adjudication board for WCG and ACI Clinical. His institution has also received funding from NIH to conduct clinical trials of Moderna and Janssen COVID-19 vaccines. Atul Butte is a co-founder and consultant to Personalis and NuMedii; consultant to Mango Tree Corporation, and in the recent past, Samsung, 10x Genomics, Helix, Pathway Genomics, and Verinata (Illumina); has served on paid advisory panels or boards for Geisinger Health, Regenstrief Institute, Gerson Lehman Group, AlphaSights, Covance, Novartis, Genentech, and Merck, and Roche; is a shareholder in Personalis and NuMedii; is a minor shareholder in Apple, Meta (Facebook), Alphabet (Google), Microsoft, Amazon, Snap, 10x Genomics, Illumina, Regeneron, Sanofi, Pfizer, Royalty Pharma, Moderna, Sutro, Doximity, BioNtech, Invitae, Pacific Biosciences, Editas Medicine, Nuna Health, Assay Depot, and Vet24seven, and several other non-health related companies and mutual funds; and has received honoraria and travel reimbursement for invited talks from Johnson and Johnson, Roche, Genentech, Pfizer, Merck, Lilly, Takeda, Varian, Mars, Siemens, Optum, Abbott, Celgene, AstraZeneca, AbbVie, Westat, and many academic institutions, medical or disease specific foundations and associations, and health systems. Atul Butte receives royalty payments through Stanford University, for several patents and other disclosures licensed to NuMedii and Personalis. Atul Buttes research has been funded by NIH, Peraton (as the prime on an NIH contract), Genentech, Johnson and Johnson, FDA, Robert Wood Johnson Foundation, Leon Lowenstein Foundation, Intervalien Foundation, Priscilla Chan and Mark Zuckerberg, the Barbara and Gerson Bakar Foundation, and in the recent past, the March of Dimes, Juvenile Diabetes Research Foundation, California Governors Office of Planning and Research, California Institute for Regenerative Medicine, LOreal, and Progenity. The authors have declared that none of these companies or competing interests had any role in this work or manuscript. ### Funding Statement This work was supported by the National Institutes of Health (NIH) / National Institute of Child Health and Human Development (NICHD) grant R61HD105618 (R.D., C.A.R., I.D.V., and C.Y.C.). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The protocols for this study were approved locally at each site by the University of California, San Francisco (UCSF) Institutional Review Board (IRB) (#21-33403), San Francisco, CA; Emory University IRB (STUDY00000723), Atlanta, GA; Children's National Medical Center IRB (Pro00010632), Washington, DC; and Cornell University IRB for Human Participants (2012010003), New York, NY. The protocols at UCSF and Children's National Medical Center were "no subject contact" sample biobanking protocols under which data was extracted from the medical chart and consent was not obtained. The protocol at Emory University IRB was a prospective enrollment study under which parents provided consent and children assent as appropriate for age. De-identified samples and patient information were shared with collaborating institutions for sample processing (UCSF and Cornell University) and analysis. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data and Code Availability All code will be made available on Github. Processed sequencing data will be deposited in the National Institutes of Health (NIH) / National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) and Gene Expression Omnibus (GEO) repositories under restricted access via Database for Genotypes and Phenotypes (dbGAP).