Epigenome-Wide Association Study in Asian Cohort Identifies Novel DNA Methylation Markers for Carotid Intima-Media Thickness

Epigenome-Wide Association Study in Asian Cohort Identifies Novel DNA Methylation Markers for Carotid Intima-Media Thickness | 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 Research Article Epigenome-Wide Association Study in Asian Cohort Identifies Novel DNA Methylation Markers for Carotid Intima-Media Thickness Konstanze Tan, Sarah E Harris, Jane Maddock, Darwin Tay, Pritesh R Jain, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9046066/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Carotid-intima media-thickness (cIMT) predicts cardiovascular events and informs mechanistic research on cardiovascular diseases. However, cardiovascular disease research remains Eurocentric despite etiological differences across ancestries. Incorporating Asian populations who face substantial cardiovascular disease burden with distinct etiological landscape can enhance our understanding of cIMT biology and subclinical processes linked to CVD. This study aims to elucidate methylation-based mechanisms of cIMT through DNA methylation profiling integrated with multi-omics data and clinically informative cIMT thresholds, leveraging an Asian cohort to enhance discovery. Methods We conducted an epigenome-wide association study (EWAS) of cIMT using peripheral blood DNA methylation at ~ 850,000 CpG sites in the Asian Health for Life in Singapore (HELIOS) cohort (n = 1,357), followed by targeted trans-ancestry meta-analysis with European cohorts (overall n = 2,765). Causal inference analyses (summary data-based Mendelian Randomisation [SMR] and colocalisation) evaluated methylation-mediated effects on cIMT, cardiovascular disease and proximal gene expression. We derived a methylation risk score (MRS) and tested its association with cIMT thresholds indicative of elevated cardiovascular risk (≥ 75th percentile for age, sex and ethnicity). Results Three novel CpG-cIMT associations were identified (P < 9.35E-07). Causal analyses supported cg08227773 methylation-mediated effects on both coronary artery disease risk (P SMR =2.91E-05, coloc PP.H4 = 0.91) and NBEAL2 (Neurobeachin-like 2) expression (P SMR =9.13E-08, coloc PP.H4 = 0.69), a gene implicated in immune dysregulation. MRS of cIMT aggregating the three sentinel CpGs was associated with clinically-informative cIMT elevation (Odds Ratio = 2.75 for Q4 vs Q1, 95% CI: 1.47–5.13). Conclusions Through Asian-led discovery, this study identifies three novel DNA methylation markers for cIMT that are linked to cIMT elevation above clinically meaningful risk thresholds. Causal inference analyses suggest methylation-mediated coronary artery disease risk via NBEAL2 regulation, nominating biologically relevant targets while underscoring the need for larger multi-omics resources to refine mechanisms. Carotid intima-media thickness (cIMT) DNA methylation Epigenome-wide association study (EWAS) Asian atherosclerosis Full Text Additional Declarations No competing interests reported. Supplementary Files 20260226additionalfile1.xlsx 20260226additionalfile3.docx 20260226additionalfile2.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 13 Mar, 2026 Editor assigned by journal 09 Mar, 2026 Submission checks completed at journal 09 Mar, 2026 First submitted to journal 05 Mar, 2026 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-9046066","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":605617815,"identity":"5bdaa69f-6def-4756-b5f3-6641056415f4","order_by":0,"name":"Konstanze Tan","email":"","orcid":"","institution":"Lee Kong Chian School of Medicine, Nanyang Technological University","correspondingAuthor":false,"prefix":"","firstName":"Konstanze","middleName":"","lastName":"Tan","suffix":""},{"id":605617816,"identity":"e3a95575-a41e-43ba-a709-a229003dea65","order_by":1,"name":"Sarah E Harris","email":"","orcid":"","institution":"University of 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Thickness","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"clinical-epigenetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clep","sideBox":"Learn more about [Clinical Epigenetics](http://clinicalepigeneticsjournal.biomedcentral.com/)","snPcode":"13148","submissionUrl":"https://submission.nature.com/new-submission/13148/3","title":"Clinical Epigenetics","twitterHandle":"@OAgenetics","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Carotid intima-media thickness (cIMT), DNA methylation, Epigenome-wide association study (EWAS), Asian, atherosclerosis","lastPublishedDoi":"10.21203/rs.3.rs-9046066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9046066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCarotid-intima media-thickness (cIMT) predicts cardiovascular events and informs mechanistic research on cardiovascular diseases. However, cardiovascular disease research remains Eurocentric despite etiological differences across ancestries. Incorporating Asian populations who face substantial cardiovascular disease burden with distinct etiological landscape can enhance our understanding of cIMT biology and subclinical processes linked to CVD. This study aims to elucidate methylation-based mechanisms of cIMT through DNA methylation profiling integrated with multi-omics data and clinically informative cIMT thresholds, leveraging an Asian cohort to enhance discovery.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e We conducted an epigenome-wide association study (EWAS) of cIMT using peripheral blood DNA methylation at ~\u0026thinsp;850,000 CpG sites in the Asian Health for Life in Singapore (HELIOS) cohort (n\u0026thinsp;=\u0026thinsp;1,357), followed by targeted trans-ancestry meta-analysis with European cohorts (overall n\u0026thinsp;=\u0026thinsp;2,765). Causal inference analyses (summary data-based Mendelian Randomisation [SMR] and colocalisation) evaluated methylation-mediated effects on cIMT, cardiovascular disease and proximal gene expression. We derived a methylation risk score (MRS) and tested its association with cIMT thresholds indicative of elevated cardiovascular risk (\u0026ge;\u0026thinsp;75th percentile for age, sex and ethnicity).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThree novel CpG-cIMT associations were identified (P\u0026thinsp;\u0026lt;\u0026thinsp;9.35E-07). Causal analyses supported cg08227773 methylation-mediated effects on both coronary artery disease risk (P\u003csub\u003eSMR\u003c/sub\u003e=2.91E-05, coloc PP.H4\u0026thinsp;=\u0026thinsp;0.91) and \u003cem\u003eNBEAL2\u003c/em\u003e (Neurobeachin-like 2) expression (P\u003csub\u003eSMR\u003c/sub\u003e=9.13E-08, coloc PP.H4\u0026thinsp;=\u0026thinsp;0.69), a gene implicated in immune dysregulation. MRS of cIMT aggregating the three sentinel CpGs was associated with clinically-informative cIMT elevation (Odds Ratio\u0026thinsp;=\u0026thinsp;2.75 for Q4 vs Q1, 95% CI: 1.47\u0026ndash;5.13).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThrough Asian-led discovery, this study identifies three novel DNA methylation markers for cIMT that are linked to cIMT elevation above clinically meaningful risk thresholds. Causal inference analyses suggest methylation-mediated coronary artery disease risk via \u003cem\u003eNBEAL2\u003c/em\u003e regulation, nominating biologically relevant targets while underscoring the need for larger multi-omics resources to refine mechanisms.\u003c/p\u003e","manuscriptTitle":"Epigenome-Wide Association Study in Asian Cohort Identifies Novel DNA Methylation Markers for Carotid Intima-Media Thickness","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 17:24:37","doi":"10.21203/rs.3.rs-9046066/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-03-13T11:07:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-09T11:47:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-09T04:03:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Epigenetics","date":"2026-03-06T04:39:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-epigenetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clep","sideBox":"Learn more about [Clinical Epigenetics](http://clinicalepigeneticsjournal.biomedcentral.com/)","snPcode":"13148","submissionUrl":"https://submission.nature.com/new-submission/13148/3","title":"Clinical Epigenetics","twitterHandle":"@OAgenetics","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84eb7715-bcf2-47ef-ad24-534eb0b15261","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T17:24:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 17:24:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9046066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9046066","identity":"rs-9046066","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}