Methylomic Analysis of Nasal Brushings Reveals Two Subgroups in Pediatric Acute Respiratory Distress Syndrome

Methylomic Analysis of Nasal Brushings Reveals Two Subgroups in Pediatric Acute Respiratory Distress Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Methylomic Analysis of Nasal Brushings Reveals Two Subgroups in Pediatric Acute Respiratory Distress Syndrome James G. Williams, Akhilesh Kaushal, Nirmeen Elmadany, Rashika Joshi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8042030/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Pediatric acute respiratory distress syndrome (PARDS) is a significant cause of mortality in the pediatric intensive care unit (PICU), and supportive care remains the mainstay of treatment. The biological heterogeneity of PARDS hampers the development of new therapies. One source of heterogeneity is the epigenetic regulation of gene expression via methylation. We hypothesized that PARDS patients could be classified into at least two subgroups defined by differential methylation of immune-related genes. We conducted a prospective, single-center cohort study of PARDS and control patients under 18 years of age admitted to the PICU. Nasal brushings were obtained on day 1 for methylomic analysis, and clinical information and outcomes were recorded until discharge. We identified two groups of PARDS subjects using PCA and hierarchical clustering, which were defined by the differential methylation of promoters and bodies of genes involved in immune, repair, and regeneration processes. One group trended toward worse clinical outcomes. The other group had a methylation pattern very similar to control subjects. PARDS patients can be divided into two subgroups based on patterns of differential methylation around genes involved in immune, repair, and regeneration processes. These findings, if confirmed, could represent potential targets for future therapies. Health sciences/Biomarkers Health sciences/Diseases Biological sciences/Genetics Biological sciences/Immunology Health sciences/Medical research Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementalTable1.docx SupplementalFigure1.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 23 Apr, 2026 Reviews received at journal 12 Apr, 2026 Reviewers agreed at journal 03 Apr, 2026 Reviews received at journal 16 Mar, 2026 Reviewers agreed at journal 04 Mar, 2026 Reviewers invited by journal 04 Mar, 2026 Editor invited by journal 10 Nov, 2025 Editor assigned by journal 07 Nov, 2025 Submission checks completed at journal 07 Nov, 2025 First submitted to journal 05 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8042030","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":561604423,"identity":"82bbe218-39a2-465f-9504-61e82cfa1681","order_by":0,"name":"James G. Williams","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBAC9gbGxgMPDEDMwwegYgn4tTA2MDYcSABpYTwGU0pQCwPDAbAa5jMGRGqZkQy0pcAuWr7tzDeJnzl1DPzsOQYEtCSCHJacu+HM2W2SvdsOM0j2vCGgZTZYC3PuBomz2yR4tx1gMLhByBaIlvrc+fPfPJP8u62OwZ6QFkGIlsO5DQfOsEnzbmNmMJAgoEVa/iFIy/HcDQeOGVvLbjvMI3HmWQFeLXw8xx8++PCnOnd+w+GHN99uq5Pjb0/egFcLBuAhTfkoGAWjYBSMAqwAAFbTUd46EBDRAAAAAElFTkSuQmCC","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"James","middleName":"G.","lastName":"Williams","suffix":""},{"id":561604424,"identity":"7df22df3-2698-421c-a096-c35f4e491a98","order_by":1,"name":"Akhilesh Kaushal","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Akhilesh","middleName":"","lastName":"Kaushal","suffix":""},{"id":561604425,"identity":"86a7f8c3-ef45-4f33-b67c-bca2c97c14db","order_by":2,"name":"Nirmeen Elmadany","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nirmeen","middleName":"","lastName":"Elmadany","suffix":""},{"id":561604426,"identity":"427f5e9f-f0fc-4095-a8c2-9be1f32a80fb","order_by":3,"name":"Rashika Joshi","email":"","orcid":"","institution":"University of Cincinnati, Cincinnati Children’s Hospital Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Rashika","middleName":"","lastName":"Joshi","suffix":""},{"id":561604427,"identity":"9f7f7b10-73ca-4bb6-9777-1ad9c93acc5b","order_by":4,"name":"Rhonda Jones","email":"","orcid":"","institution":"University of Cincinnati, Cincinnati Children’s Hospital Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Rhonda","middleName":"","lastName":"Jones","suffix":""},{"id":561604428,"identity":"24470f87-b26c-4c93-b4d1-0b505ed36a85","order_by":5,"name":"Nathan Gregor","email":"","orcid":"","institution":"University of Cincinnati, Cincinnati Children’s Hospital Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Nathan","middleName":"","lastName":"Gregor","suffix":""},{"id":561604429,"identity":"ddc44bcf-f2b6-4456-bd19-7ba8acddd8b8","order_by":6,"name":"Patrick Lahni","email":"","orcid":"","institution":"University of Cincinnati, Cincinnati Children’s Hospital Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Lahni","suffix":""},{"id":561604430,"identity":"9b325af2-3916-44b5-aceb-030cfaaa964f","order_by":7,"name":"Steven W. Standage","email":"","orcid":"","institution":"University of Cincinnati, Cincinnati Children’s Hospital Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"W.","lastName":"Standage","suffix":""},{"id":561604431,"identity":"19583451-78c3-4824-a709-21179190ddfe","order_by":8,"name":"Brian M. Varisco","email":"","orcid":"","institution":"University of Arkansas for Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Brian","middleName":"M.","lastName":"Varisco","suffix":""}],"badges":[],"createdAt":"2025-11-05 22:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8042030/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8042030/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100949912,"identity":"d3e78f12-ba68-4f21-881d-61d4e5c8dcf8","added_by":"auto","created_at":"2026-01-23 07:06:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92962,"visible":true,"origin":"","legend":"","description":"","filename":"MetholomicAnalysisofNasalBrushingsRevealTwoSubgroupsinPediatricAcuteRespiratoryDistressSyndromeFINAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/2abe2f6e884b9ca34d859bda.docx"},{"id":100866073,"identity":"44e0b23c-ddbb-4780-956d-56adb8219d43","added_by":"auto","created_at":"2026-01-22 08:28:10","extension":"json","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9835,"visible":true,"origin":"","legend":"","description":"","filename":"171d83a976474963baa5a1d83f12be91.json","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/56ea0bbd8e1b7e1a04effe69.json"},{"id":100949860,"identity":"6fa7cd7e-65b2-48f5-85f4-752f07d4bc85","added_by":"auto","created_at":"2026-01-23 07:06:03","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15745,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/37d2d672eec9d0d2db5eabfb.docx"},{"id":100866081,"identity":"c2150d00-c489-47ce-8da5-f9a8fbff22a0","added_by":"auto","created_at":"2026-01-22 08:28:12","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16604,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/0fb2668b80d3075157318345.docx"},{"id":100866079,"identity":"7922e73f-1d54-4c14-bb43-a095a0a83b85","added_by":"auto","created_at":"2026-01-22 08:28:11","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99999,"visible":true,"origin":"","legend":"","description":"","filename":"171d83a976474963baa5a1d83f12be911enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/0c3bd30461e95bcc0ddfb9a0.xml"},{"id":100866074,"identity":"53cc270b-0517-42b7-a385-055741626e9a","added_by":"auto","created_at":"2026-01-22 08:28:10","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":590373,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/b4cd514d95fac473dadec9e7.pdf"},{"id":100866080,"identity":"190c3115-6636-4112-bd34-4d6dcf719240","added_by":"auto","created_at":"2026-01-22 08:28:11","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44666,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/cd1045742a6249099704dfb9.pdf"},{"id":100949747,"identity":"f85e1339-10cb-48a4-811b-1528bd2db3d4","added_by":"auto","created_at":"2026-01-23 07:05:19","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":672262,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/c20560c9eebba3684706fc96.pdf"},{"id":100866072,"identity":"b1f7c152-0139-4f72-8573-b9abd358df34","added_by":"auto","created_at":"2026-01-22 08:28:10","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":561258,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/1ca77756cbd2675c6cdae08f.pdf"},{"id":100866083,"identity":"b91c1d00-a816-4195-8e2b-6cec46a17165","added_by":"auto","created_at":"2026-01-22 08:28:16","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97863,"visible":true,"origin":"","legend":"","description":"","filename":"171d83a976474963baa5a1d83f12be911structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/4a215194a79c2dcd4af34270.xml"},{"id":100866075,"identity":"7a09d7fd-4070-4aff-97fe-96652e4c6693","added_by":"auto","created_at":"2026-01-22 08:28:11","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114602,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/1e65c12db961f3717c8841f6.html"},{"id":100953369,"identity":"746f984d-c1b7-4b8d-b563-43ef457a1e02","added_by":"auto","created_at":"2026-01-23 07:21:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2297060,"visible":true,"origin":"","legend":"","description":"","filename":"MetholomicAnalysisofNasalBrushingsRevealTwoSubgroupsinPediatricAcuteRespiratoryDistressSyndromeFINAL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1_covered_876c342d-1aa1-4912-8b1c-83df98f175d0.pdf"},{"id":100866056,"identity":"26b10f49-8448-4641-b6e6-924f5f4a0169","added_by":"auto","created_at":"2026-01-22 08:28:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16604,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/92262421870e95d3bbaebd2d.docx"},{"id":100866078,"identity":"8a1fa723-695d-428f-b8aa-c2590506bf85","added_by":"auto","created_at":"2026-01-22 08:28:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15745,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8042030/v1/d82c5ea576ab250ff9d9de5b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Methylomic Analysis of Nasal Brushings Reveals Two Subgroups in Pediatric Acute Respiratory Distress Syndrome","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8042030/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8042030/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePediatric acute respiratory distress syndrome (PARDS) is a significant cause of mortality in the pediatric intensive care unit (PICU), and supportive care remains the mainstay of treatment. The biological heterogeneity of PARDS hampers the development of new therapies. One source of heterogeneity is the epigenetic regulation of gene expression via methylation. We hypothesized that PARDS patients could be classified into at least two subgroups defined by differential methylation of immune-related genes. We conducted a prospective, single-center cohort study of PARDS and control patients under 18 years of age admitted to the PICU. Nasal brushings were obtained on day 1 for methylomic analysis, and clinical information and outcomes were recorded until discharge. We identified two groups of PARDS subjects using PCA and hierarchical clustering, which were defined by the differential methylation of promoters and bodies of genes involved in immune, repair, and regeneration processes. One group trended toward worse clinical outcomes. The other group had a methylation pattern very similar to control subjects. PARDS patients can be divided into two subgroups based on patterns of differential methylation around genes involved in immune, repair, and regeneration processes. These findings, if confirmed, could represent potential targets for future therapies.\u003c/p\u003e","manuscriptTitle":"Methylomic Analysis of Nasal Brushings Reveals Two Subgroups in Pediatric Acute Respiratory Distress Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 08:27:27","doi":"10.21203/rs.3.rs-8042030/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-23T07:16:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T12:55:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284805767146965649105836944480831190953","date":"2026-04-03T11:57:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T20:06:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324937068591787264761940738658958573436","date":"2026-03-04T13:47:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-04T06:21:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-10T18:10:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-07T10:46:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-07T10:45:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-05T22:29:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1dc1e09c-e03b-4398-b13d-9e007cd4df1b","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":59796067,"name":"Health sciences/Biomarkers"},{"id":59796068,"name":"Health sciences/Diseases"},{"id":59796069,"name":"Biological sciences/Genetics"},{"id":59796070,"name":"Biological sciences/Immunology"},{"id":59796071,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-04-23T07:24:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 08:27:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8042030","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8042030","identity":"rs-8042030","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}