A gene expression-based diagnostic classifier for identification of severe COVID-19 and multisystem inflammatory syndrome in children (MIS-C)

MIS-C is a severe hyperinflammatory condition with involvement of multiple organs that occurs in children who had COVID-19 infection. Accurate diagnostic tests are needed to guide management and appropriate treatment and to inform clinical trials of experimental drugs and vaccines, yet the diagnosis of MIS-C is highly challenging due to overlapping clinical features with other acute syndromes in hospitalized patients. Here we developed a gene expression-based classifier for MIS-C by RNA-Seq transcriptome profiling and machine learning based analyses of 195 whole blood RNA and 76 plasma cell-free RNA samples from 191 subjects, including 95 MIS-C patients, 66 COVID-19 infected patients with moderately severe to severe disease, and 30 uninfected controls. We divided the group into a training set (70%) and test set (30%). After selection of the top 300 differentially expressed genes in the training set, we simultaneously trained 13 classification models to distinguish patients with MIS-C and COVID-19 from controls using five-fold cross-validation and grid search hyperparameter tuning. The final optimal classifier models had 100% diagnostic accuracy for MIS-C (versus non-MIS-C) and 85% accuracy for severe COVID-19 (versus mild/asymptomatic COVID-19). Orthogonal validation of a random subset of 11 genes from the final models using quantitative RT-PCR confirmed the differential expression and ability to discriminate MIS-C and COVID-19 from controls. These results underscore the utility of a gene expression classifier for diagnosis of MIS-C and severe COVID-19 as specific and objective biomarkers for these conditions. ### 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. I.D.V. and A.P.C. are 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 receive consulting fees from Eurofins Viracor. C.A.R. 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. A.B. 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. A.B. receives royalty payments through Stanford University, for several patents and other disclosures licensed to NuMedii and Personalis. Research from A.B. 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 Governor 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 University of California, San Francisco (UCSF) Institutional Review Board (IRB) (#21-33403), San Francisco, CA; Emory University IRB (STUDY00000723), Atlanta, GA; Childrens National Medical Center (CNMC) IRB (Pro00010632), Washington, DC; and Cornell University IRB for Human Participants (2012010003), New York, NY each approved the protocols for this study. All samples and patient information were de-identified for analysis and sharing with collaboration institutions. At Emory University the IRB approved protocol was a prospective enrollment study under which parents provided consent and children assent as appropriate for age. At CNMC and UCSF, the IRB protocols were no subject contact sample biobanking protocols under which content was not obtained and data was extracted from medical charts. 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 All code will be made available on Github. Processed sequencing data will be deposited in the National Institutes of Health (NIH) and 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).