{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:46:39Z","timestamp":1760060799556,"version":"build-2065373602"},"reference-count":38,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2025,9,18]],"date-time":"2025-09-18T00:00:00Z","timestamp":1758153600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"\u201cInGene 2.0\u201d","award":["\u201cBando Ricerca Salute 2018\u201d, Decree n. 15397, 26 September 2018, Tuscany Region"],"award-info":[{"award-number":["\u201cBando Ricerca Salute 2018\u201d, Decree n. 15397, 26 September 2018, Tuscany Region"]}]},{"name":"Italian Ministry of Health, Ricerca Corrente 2024","award":["\u201cBando Ricerca Salute 2018\u201d, Decree n. 15397, 26 September 2018, Tuscany Region"],"award-info":[{"award-number":["\u201cBando Ricerca Salute 2018\u201d, Decree n. 15397, 26 September 2018, Tuscany Region"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Background: The TWNK gene encodes a protein that colocalizes with mitochondrial DNA (mtDNA) in mitochondrial nucleoids. It acts as mtDNA helicase during replication, thus playing a pivotal role in the replication and maintenance of mtDNA stability. TWNK mutations are associated with a wide spectrum of clinical phenotypes and a marked heterogeneity. However, heterozygous nonsense variants in the gene have never been described in association with disease. Methods: We analyzed a next-generation sequencing (NGS) targeted gene panel in a cohort including 40 patients with high clinical suspicion of mitochondrial disorders. Selected patients underwent a complete neurological examination, electrophysiology tests, and muscle biopsy. Segregation analysis was performed in available family members. The 3D structure of twinkle was visualized and analyzed using Swiss Model and Pymol version 3.1.6.1. Results: We found four TWNK-mutated subjects from two unrelated families. They exhibited a variable clinical spectrum, ranging from asymptomatic individuals to subjects with psychiatric disorder, chronic progressive external ophthalmoplegia (CPEO), and CPEO-plus. All the subjects shared the heterozygous TWNK p.Glu665Ter variant. Discussion and Conclusions: We describe the clinical phenotype and muscle biopsy findings associated with the first reported heterozygous nonsense TWNK variant, thus expanding the current knowledge of Twinkle-related disorders. Our findings are in line with the high intrafamilial clinical variability associated with TWNK mutations. Although PEO and skeletal muscle involvement remain hallmarks of the disease, extra-muscular features should be carefully assessed.<\/jats:p>","DOI":"10.3390\/biom15091337","type":"journal-article","created":{"date-parts":[[2025,9,18]],"date-time":"2025-09-18T14:45:46Z","timestamp":1758206746000},"page":"1337","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The First Heterozygous TWNK Nonsense Mutation Associated with Progressive External Ophthalmoplegia: Evidence for a New Piece in the Puzzle of Mitochondrial Diseases"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2256-0482","authenticated-orcid":false,"given":"Diego","family":"Lopergolo","sequence":"first","affiliation":[{"name":"Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, Italy"},{"name":"UOC Neurologia, Azienda Ospedaliero-Universitaria Senese, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gianna","family":"Berti","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, Italy"},{"name":"UOC Neurologia, Azienda Ospedaliero-Universitaria Senese, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gian Nicola","family":"Gallus","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Silvia","family":"Bianchi","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Filippo Maria","family":"Santorelli","sequence":"additional","affiliation":[{"name":"Neurobiology and Molecular Medicine, IRCCS Fondazione Stella Maris, 56128 Pisa, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alessandro","family":"Malandrini","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, Italy"},{"name":"UOC Neurologia, Azienda Ospedaliero-Universitaria Senese, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicola","family":"De Stefano","sequence":"additional","affiliation":[{"name":"Department of Medicine, Surgery and Neurosciences, University of Siena, 53100 Siena, Italy"},{"name":"UOC Neurologia, Azienda Ospedaliero-Universitaria Senese, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1038\/290457a0","article-title":"Sequence and Organization of the Human Mitochondrial Genome","volume":"290","author":"Anderson","year":"1981","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1619","DOI":"10.1212\/WNL.0b013e3181df099f","article-title":"The Clinical, Histochemical, and Molecular Spectrum of PEO1 (Twinkle)-Linked AdPEO","volume":"74","author":"Fratter","year":"2010","journal-title":"Neurology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1038\/90058","article-title":"Human Mitochondrial DNA Deletions Associated with Mutations in the Gene Encoding Twinkle, a Phage T7 Gene 4-like Protein Localized in Mitochondria","volume":"28","author":"Spelbrink","year":"2001","journal-title":"Nat. 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