Single-myofiber RNA-sequencing reveals myofiber-level transcriptomic heterogeneity and DUX4 activation in FSHD muscle biopsies

Single-myofiber RNA-sequencing reveals myofiber-level transcriptomic heterogeneity and DUX4 activation in FSHD muscle biopsies | 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 Single-myofiber RNA-sequencing reveals myofiber-level transcriptomic heterogeneity and DUX4 activation in FSHD muscle biopsies Silvère van der Maarel, Anita van den Heuvel, Dongxu Zheng, Svetlana Pasteuning - Vuhman, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7715788/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Although expression of the transcription factor DUX4 in skeletal muscle is widely regarded as the primary driver of facioscapulohumeral muscular dystrophy (FSHD), its assumed sporadic and transient nature complicates full characterization of its transcriptional impact. Cellular heterogeneity within FSHD muscles remains only partially understood. Here, we applied single-myofiber RNA-sequencing to FSHD muscle biopsies to address this heterogeneity. Our data revealed myofiber heterogeneity, with DUX4-signature expression detected in only a small subset of myofibers. We identified 119 genes differentially expressed in DUX4-positive myofibers, of which most were downregulated. These genes showed similar but attenuated expression patterns in bulk muscle RNA-seq data, highlighting the benefit of single-myofiber RNA-sequencing over the masking-affected bulk approaches, especially for downregulated genes. DUX4 signature-positive myofibers showed a reduction in factors involved in RNA processing and surveillance and several factors of the mediator complex, perhaps reflecting an overall response to the cellular stress these myofibers endure. Our study demonstrates the potential value of single-myofiber transcriptomics for studying transcriptional heterogeneity in FSHD, but may also provide insights into challenges in capturing disease-affected myofibers during quality control procedures. Biological sciences/Biological techniques/Gene expression analysis Biological sciences/Molecular biology/Transcriptomics Biological sciences/Cell biology/Mechanisms of disease Full Text Additional Declarations Yes there is potential Competing Interest. All authors declare to have no competing interests. S.v.d.M. has acted as consultant and/or is a member of the advisory board for Avidity Biosciences, Dyne Therapeutics and Fulcrum Therapeutics, is a Board member for Renogenyx and is coinventor of several FSHD-related patent applications. Supplementary Files SupplementalTable1vandenHeuveletal2025CommsBio.xlsx Supplemental Table 1 SupplementalTable2vandenHeuveletal2025CommsBio.xlsx Supplemental Table 2 Cite Share Download PDF Status: Under Review Version 1 posted 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-7715788","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":524330036,"identity":"72e1713f-4243-460a-bbbe-19988e48e1f5","order_by":0,"name":"Silvère van der Maarel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIie2RsYrCQBCG5xzYFO6erdd4rzCd2MRXCQSuivgACgoBrbTWwneITerIQNLcA1gcXGzsBG3kKnFzQbC4DSmv2K/6YfjYf2YBLJZ/TfIIDu7K7NRWUHhlxvqKpErlfdr0UI6/4DVb5I392B22UJ5ZBS50DQolKkKZHuHtMyMMUr+3DlXEKvahFxoUUNHLSjDQ/gMwEEjEv0oCxKZihXLTyvdRK7cJ9VnmlQroYnCZFa8IwMGMiVBCpVLUSC5L1rvoYoNlRm0WtNvEvjQWmy+2uXdlfbFUF7uOqDUPD/kpdjuUTf92sPyRiR47P88DadjEYrFYLDW4A5P8WYwStkChAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8103-711X","institution":"Leiden University Medical Centre","correspondingAuthor":true,"prefix":"","firstName":"Silvère","middleName":"van der","lastName":"Maarel","suffix":""},{"id":524330037,"identity":"6e6e60ec-7e7c-429c-b79e-ff4ee398c727","order_by":1,"name":"Anita van den Heuvel","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Anita","middleName":"van den","lastName":"Heuvel","suffix":""},{"id":524330038,"identity":"1c97526f-1a6e-4082-ad6c-e74809caf323","order_by":2,"name":"Dongxu Zheng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dongxu","middleName":"","lastName":"Zheng","suffix":""},{"id":524330039,"identity":"a4c7f29f-d542-45da-88bc-f0765be8659e","order_by":3,"name":"Svetlana Pasteuning - 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S.v.d.M. has acted as consultant and/or is a member of the advisory board for Avidity Biosciences, Dyne Therapeutics and Fulcrum Therapeutics, is a Board member for Renogenyx and is coinventor of several FSHD-related patent applications.","formattedTitle":"Single-myofiber RNA-sequencing reveals myofiber-level transcriptomic \r\nheterogeneity and DUX4 activation in FSHD muscle biopsies","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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7715788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7715788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Although expression of the transcription factor DUX4 in skeletal muscle is widely regarded as the primary driver of facioscapulohumeral muscular dystrophy (FSHD), its assumed sporadic and transient nature complicates full characterization of its transcriptional impact. Cellular heterogeneity within FSHD muscles remains only partially understood. Here, we applied single-myofiber RNA-sequencing to FSHD muscle biopsies to address this heterogeneity. Our data revealed myofiber heterogeneity, with DUX4-signature expression detected in only a small subset of myofibers. We identified 119 genes differentially expressed in DUX4-positive myofibers, of which most were downregulated. These genes showed similar but attenuated expression patterns in bulk muscle RNA-seq data, highlighting the benefit of single-myofiber RNA-sequencing over the masking-affected bulk approaches, especially for downregulated genes. DUX4 signature-positive myofibers showed a reduction in factors involved in RNA processing and surveillance and several factors of the mediator complex, perhaps reflecting an overall response to the cellular stress these myofibers endure. 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