Generation of a novel mouse model of nemaline myopathy due to recurrent NEB exon 55 deletion

Generation of a novel mouse model of nemaline myopathy due to recurrent NEB exon 55 deletion | 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 Generation of a novel mouse model of nemaline myopathy due to recurrent NEB exon 55 deletion Zachary Coulson, Justin Kolb, Nesrin Sabha, Esmat Karimi, Zaynab Hourani, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5456324/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Mar, 2025 Read the published version in Skeletal Muscle → Version 1 posted 10 You are reading this latest preprint version Abstract Biallelic pathogenic variants in the nebulin ( NEB ) gene lead to the congenital muscle disease nemaline myopathy. In-frame deletion of exon 55 (ΔExon55) is the most common disease-causing variant in NEB . Previously, a mouse model of Neb ΔExon55 was developed; however, it presented an uncharacteristically severe phenotype with a near complete reduction in Neb transcript expression that is not observed in NEB exon 55 patients. We identified by RNA sequencing that the cause of this unexpectedly severe presentation in mice is the generation of a pseudoexon containing two premature termination codons (and promoting nonsense mediated decay) at the Neb exon 55 deletion site. To prove that this is the cause of the loss of Neb transcript, and to generate a more faithful model of the human disease, we used CRISPR gene editing to remove the pseudoexon sequence and replace it with human intron 54 sequence containing a validated cas9 gRNA protospacer. The resulting “hmz” mice have a significant reduction in pseudoexon formation (93.6% reduction), and a re-introduction of stable Neb transcript expression. This new model has the characteristic features of nemaline myopathy at the physiological, histological, and molecular levels. Importantly, unlike the existing exon 55 deletion mice (which die by age 7 days), it survives beyond the first months and exhibits obvious signs of neuromuscular dysfunction. It thus provides a new, robust model for studying pathomechanisms and developing therapies for NEB related nemaline myopathy. nebulin nemaline myopathy pseudoexon transcript stabilization CRISPR phenotyping Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 20 Mar, 2025 Read the published version in Skeletal Muscle → Version 1 posted Editorial decision: Revision requested 20 Dec, 2024 Reviews received at journal 11 Dec, 2024 Reviews received at journal 10 Dec, 2024 Reviewers agreed at journal 22 Nov, 2024 Reviewers agreed at journal 21 Nov, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers invited by journal 20 Nov, 2024 Editor assigned by journal 20 Nov, 2024 Submission checks completed at journal 17 Nov, 2024 First submitted to journal 14 Nov, 2024 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. 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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-5456324","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":389136063,"identity":"141ee0e3-eba8-4eb7-8bfd-ec798d0c5181","order_by":0,"name":"Zachary Coulson","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIie2QsWrDMBCGzwTkRYlXhRTlFRQE7dCXaTDYiwsZM5WAQV4Mnf0W7tahg8KBu+gRCk0XTRmSLYEMdcEhHUTaboHqGw4J3cd/JwCP5zIhx0Og28L/pMCXIru7+L0yXfykjAu0q/0LQlSUYrmbv6X1e95sts8HDiGuXEpQJjeT0iIwYwRSY+/rhsRVZYQEmjijepARRjWCYJnAQGGrUNnrKzFdMPd0JFqT4aFTlnuFqTgp4calUJaR0TFF9xXefVOoM4Uxez260illppm1u+CkapI4qJSUhGYz5489xna41rc8KvKnj90cxwNEhK3iPApfa5fSkVPHmmf6Wx7OP3s8Hs//5hPrj11Lel95UAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Toronto","correspondingAuthor":true,"prefix":"","firstName":"Zachary","middleName":"","lastName":"Coulson","suffix":""},{"id":389136064,"identity":"78678202-e37c-45a3-8e72-b8b00bad2233","order_by":1,"name":"Justin Kolb","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Justin","middleName":"","lastName":"Kolb","suffix":""},{"id":389136065,"identity":"94a15d5f-1af6-4f02-a54c-bd15dfedef35","order_by":2,"name":"Nesrin Sabha","email":"","orcid":"","institution":"Hospital for Sick Children","correspondingAuthor":false,"prefix":"","firstName":"Nesrin","middleName":"","lastName":"Sabha","suffix":""},{"id":389136066,"identity":"ac662750-15f7-4c0e-9155-301d2f177324","order_by":3,"name":"Esmat Karimi","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Esmat","middleName":"","lastName":"Karimi","suffix":""},{"id":389136067,"identity":"5d12c3fc-bf94-4004-a7d2-bdaa53020ae8","order_by":4,"name":"Zaynab Hourani","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Zaynab","middleName":"","lastName":"Hourani","suffix":""},{"id":389136068,"identity":"0758623c-2e25-49dc-b044-94bee571a0ae","order_by":5,"name":"Coen Ottenheijm","email":"","orcid":"","institution":"Amsterdam UMC Location VUmc","correspondingAuthor":false,"prefix":"","firstName":"Coen","middleName":"","lastName":"Ottenheijm","suffix":""},{"id":389136069,"identity":"57ca2fac-a819-41db-8c5f-a41a14bc5f0f","order_by":6,"name":"Henk Granzier","email":"","orcid":"","institution":"University of Arizona","correspondingAuthor":false,"prefix":"","firstName":"Henk","middleName":"","lastName":"Granzier","suffix":""},{"id":389136070,"identity":"fc77f49a-6e18-40bd-a4ef-8417466857ad","order_by":7,"name":"James J. 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In-frame deletion of exon 55 (ΔExon55) is the most common disease-causing variant in \u003cem\u003eNEB\u003c/em\u003e. Previously, a mouse model of \u003cem\u003eNeb\u003c/em\u003e\u003csup\u003eΔExon55\u003c/sup\u003e was developed; however, it presented an uncharacteristically severe phenotype with a near complete reduction in \u003cem\u003eNeb\u003c/em\u003e transcript expression that is not observed in \u003cem\u003eNEB\u003c/em\u003e exon 55 patients.\u003c/p\u003e \u003cp\u003eWe identified by RNA sequencing that the cause of this unexpectedly severe presentation in mice is the generation of a pseudoexon containing two premature termination codons (and promoting nonsense mediated decay) at the \u003cem\u003eNeb\u003c/em\u003e exon 55 deletion site. To prove that this is the cause of the loss of \u003cem\u003eNeb\u003c/em\u003e transcript, and to generate a more faithful model of the human disease, we used CRISPR gene editing to remove the pseudoexon sequence and replace it with human intron 54 sequence containing a validated cas9 gRNA protospacer. The resulting \u0026ldquo;hmz\u0026rdquo; mice have a significant reduction in pseudoexon formation (93.6% reduction), and a re-introduction of stable \u003cem\u003eNeb\u003c/em\u003e transcript expression. This new model has the characteristic features of nemaline myopathy at the physiological, histological, and molecular levels. Importantly, unlike the existing exon 55 deletion mice (which die by age 7 days), it survives beyond the first months and exhibits obvious signs of neuromuscular dysfunction. It thus provides a new, robust model for studying pathomechanisms and developing therapies for \u003cem\u003eNEB\u003c/em\u003e related nemaline myopathy.\u003c/p\u003e","manuscriptTitle":"Generation of a novel mouse model of nemaline myopathy due to recurrent NEB exon 55 deletion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-17 12:29:48","doi":"10.21203/rs.3.rs-5456324/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-20T09:52:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-11T15:50:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-10T13:35:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143654430971190777036972877251802404879","date":"2024-11-23T02:53:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171295537040681507281066962532519288365","date":"2024-11-21T15:42:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115827476240964435919995779890250200531","date":"2024-11-20T13:19:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-20T09:45:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-20T09:34:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-18T01:50:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Skeletal Muscle","date":"2024-11-14T20:37:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"skeletal-muscle","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"skem","sideBox":"Learn more about [Skeletal Muscle](http://skeletalmusclejournal.biomedcentral.com/)","snPcode":"13395","submissionUrl":"https://submission.nature.com/new-submission/13395/3","title":"Skeletal Muscle","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"97cdcc89-b5f1-4d11-a2ef-1f802e24ed3d","owner":[],"postedDate":"December 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T16:02:43+00:00","versionOfRecord":{"articleIdentity":"rs-5456324","link":"https://doi.org/10.1186/s13395-025-00378-2","journal":{"identity":"skeletal-muscle","isVorOnly":false,"title":"Skeletal Muscle"},"publishedOn":"2025-03-20 15:57:52","publishedOnDateReadable":"March 20th, 2025"},"versionCreatedAt":"2024-12-17 12:29:48","video":"","vorDoi":"10.1186/s13395-025-00378-2","vorDoiUrl":"https://doi.org/10.1186/s13395-025-00378-2","workflowStages":[]},"version":"v1","identity":"rs-5456324","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5456324","identity":"rs-5456324","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}