Duration of contact sports play associated with aberrant DNA methylation in human frontal cortex

Duration of contact sports play associated with aberrant DNA methylation in human frontal cortex | 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 Duration of contact sports play associated with aberrant DNA methylation in human frontal cortex Kerry Breen, Nurgul Aytan, Samantha Hawkins, Raymond Nicks, Victor Alvarez, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7861173/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Repetitive head impacts (RHI), primarily through contact sports play and military service, are a recognized risk factor for cognitive and behavioral symptoms, as well as progressive neurodegenerative diseases such as chronic traumatic encephalopathy. While altered DNA methylation has been linked to environmental exposures and neurodegeneration, its association with RHI remains unknown. In this study, we investigated whether duration of contact sports play in a community-based aging cohort is associated with altered DNA methylation patterns. Reduced representation bisulfite sequencing on human dorsolateral frontal cortex identified 461 genome-wide significant CpG sites associated with duration of contact sports play, spanning 13 genes of which the majority were hypomethylated. The hypomethylation pattern was largely replicated in an independent cohort. Notably, CAMK2B, B4GALT6, and TLR2, were hypomethylated and upregulated in the cortical sulcus of the DLFC in individuals with RHI exposure from contact sports. Furthermore, alterations of CAMK2B and B4GALT6 were observed in CTE cases. Together, these findings reveal distinct, region-specific epigenetic changes associated with contact sports exposure and provide new insights into the molecular mechanisms underlying RHI-related sequalae. Biological sciences/Neuroscience/Epigenetics in the nervous system Health sciences/Diseases/Neurological disorders/Brain injuries Biological sciences/Genetics/Epigenetics/DNA methylation Figures Figure 1 Figure 2 Figure 3 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1 SupplementaryTable2.xlsx Supplementary Table 2 SupplementaryTable3.xlsx Supplementary Table 3 Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-7861173","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":553238494,"identity":"5c3b2fe4-7f2d-4b85-85d9-a946e1d054b1","order_by":0,"name":"Kerry Breen","email":"","orcid":"https://orcid.org/0000-0002-1177-3493","institution":"Boston Chobanian \u0026 Avedisian University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kerry","middleName":"","lastName":"Breen","suffix":""},{"id":553238495,"identity":"e4cd630d-b3d3-44e4-bcce-c42fec845dcf","order_by":1,"name":"Nurgul 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Created in BioRender. Breen, K. (2025) https://BioRender.com/czxsqhy (B) Manhattan plot displaying the association between aberrant DNA methylation and duration of contact sports play (years). Genome-wide significance line (p \u0026lt; 5 x 10\u003csup\u003e-8\u003c/sup\u003e) is denoted by the red line. (C) Bar plot showing the number of aberrantly methylated CpG sites in each GWS DMR. (D) Pie chart showing percent of hypo- and hypermethylated CpG sites (top) and bar plot displaying genomic and CpG island (CGI) annotations of CpG sites (bottom). (E) Gene ontology analysis on hypomethylated gene list (left) and displaying which GO terms included \u003cem\u003eCAMK2B\u003c/em\u003e, \u003cem\u003eB4GALT6\u003c/em\u003e, and \u003cem\u003eTLR2\u003c/em\u003e (right). D-E performed with genes/CpG sites with a p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7861173/v2/d5386d3ea4e4e71761552168.jpg"},{"id":99734601,"identity":"40ce4e46-749f-4815-bae7-784f55b45aff","added_by":"auto","created_at":"2026-01-07 19:02:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased cortical sulcus expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCAMK2B, B4GALT6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTLR2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in individuals with RHI exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of \u003cem\u003ein situ\u003c/em\u003ehybridization of \u003cem\u003eCAMK2B\u003c/em\u003e (green), \u003cem\u003eB4GALT6\u003c/em\u003e (yellow), and \u003cem\u003eTLR2\u003c/em\u003e(orange). Left scale bar 50μm, right scale bar 10μm. (B) Bar plot of quantifications of \u003cem\u003eCAMK2B, B4GALT6\u003c/em\u003e, and \u003cem\u003eTLR2\u003c/em\u003e transcript copies/puncta. *p\u0026lt;0.05 **p\u0026lt;0.01 ***p\u0026lt;0.005 ****p\u0026lt;0.0005 (C) Scatter plot showing \u003cem\u003eCAMK2B, B4GALT6\u003c/em\u003eand \u003cem\u003eTLR2\u003c/em\u003e copies across duration of play groups. Dots depict individual samples; line represents general linear regression. (D) Bar plot of quantifications of \u003cem\u003eCAMK2B, B4GALT6\u003c/em\u003e, and \u003cem\u003eTLR2\u003c/em\u003e cellular density. *p\u0026lt;0.05 ****p\u0026lt;0.0005\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7861173/v2/b7335d232f17781faac88e75.png"},{"id":99734603,"identity":"dda54bc2-b377-4879-ad11-df62d6dab475","added_by":"auto","created_at":"2026-01-07 19:02:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":689541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell-type localization and regional alterations of CAMK2B, B4GALT6, and TLR2 in control, RHI, and CTE.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of mIF of CAMK2B (yellow) and HuC/HuD (orange), red asterisks indicate depth of the sulcus. Scale bar 1mm. (B) Representative density heatmaps of CAMK2B+/DAPI (red) and CAMK2B-/DAPI (blue). (C) Bar plot of quantifications of CAMK2B positive cells. *p\u0026lt;0.05 **p\u0026lt;0.01 (D) Representative images of mIF of B4GALT6 (green), GFAP (orange), and HuC/HuD (red) at the depths of the cortical sulcus, solid arrow in RHI shows a B4GALT6+/HuC/HuD+ cell and solid arrow in high CTE shows a B4GALT6+/GFAP+ cell. Scale bar 50μm. (E) Bar plot of quantifications of B4GALT6 positive cells. **p\u0026lt;0.01 ***p\u0026lt;0.005 ****p\u0026lt;0.0001 (F) Representative images of mIF of TLR2 (orange), Iba1 (yellow), and CD3 (green) at the depths of the cortical sulcus, solid white arrow shows a TLR2+/Iba1+ cell and solid red arrow shows a TLR2+/CD3+ cell. Scale bar 100μm. \u0026nbsp;(E) Bar plot of quantifications of TLR2 positive cells.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7861173/v2/b8a3df8f7868fee5715defa0.png"},{"id":100356382,"identity":"7254a91c-f2d1-4888-a672-dfd740ef9521","added_by":"auto","created_at":"2026-01-16 07:06:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1333869,"visible":true,"origin":"","legend":"","description":"","filename":"RHIDNAMethylationv2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7861173/v2_covered_389458e2-41aa-4e4b-87ec-4e31c2ff8d35.pdf"},{"id":99734605,"identity":"49b33611-89ba-476c-ae2c-a9f7d9210b22","added_by":"auto","created_at":"2026-01-07 19:02:41","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6180447,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1\u003c/p\u003e","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7861173/v2/5e0b95a8d07565ef3bfc51f6.xlsx"},{"id":99797959,"identity":"e7e83b7a-c4d0-4b9a-bda8-f79962d86f64","added_by":"auto","created_at":"2026-01-08 13:46:59","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":46386,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7861173/v2/d2ff6fa80719a3ff7ea852ec.xlsx"},{"id":99798090,"identity":"6e0147cf-7c88-47a3-bd78-030f15daf5cf","added_by":"auto","created_at":"2026-01-08 13:47:14","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":52954,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3\u003c/p\u003e","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7861173/v2/2b7847cce16bf84eac91aa22.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Duration of contact sports play associated with aberrant DNA methylation in human frontal cortex","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7861173/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7861173/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Repetitive head impacts (RHI), primarily through contact sports play and military service, are a recognized risk factor for cognitive and behavioral symptoms, as well as progressive neurodegenerative diseases such as chronic traumatic encephalopathy. While altered DNA methylation has been linked to environmental exposures and neurodegeneration, its association with RHI remains unknown. In this study, we investigated whether duration of contact sports play in a community-based aging cohort is associated with altered DNA methylation patterns. Reduced representation bisulfite sequencing on human dorsolateral frontal cortex identified 461 genome-wide significant CpG sites associated with duration of contact sports play, spanning 13 genes of which the majority were hypomethylated. The hypomethylation pattern was largely replicated in an independent cohort. Notably, CAMK2B, B4GALT6, and TLR2, were hypomethylated and upregulated in the cortical sulcus of the DLFC in individuals with RHI exposure from contact sports. Furthermore, alterations of CAMK2B and B4GALT6 were observed in CTE cases. Together, these findings reveal distinct, region-specific epigenetic changes associated with contact sports exposure and provide new insights into the molecular mechanisms underlying RHI-related sequalae.","manuscriptTitle":"Duration of contact sports play associated with aberrant DNA methylation in human frontal cortex","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-01-07 19:02:37","doi":"10.21203/rs.3.rs-7861173/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2025-12-01 08:18:04","doi":"10.21203/rs.3.rs-7861173/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ba25a0f-5d41-4a23-97e3-c161bc64560d","owner":[],"postedDate":"January 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58863313,"name":"Biological sciences/Neuroscience/Epigenetics in the nervous system"},{"id":58863314,"name":"Health sciences/Diseases/Neurological disorders/Brain injuries"},{"id":58863315,"name":"Biological sciences/Genetics/Epigenetics/DNA methylation"}],"tags":[],"updatedAt":"2025-12-18T14:09:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-07 19:02:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-7861173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7861173","identity":"rs-7861173","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}