{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:19:11Z","timestamp":1760059151005,"version":"build-2065373602"},"reference-count":55,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T00:00:00Z","timestamp":1747958400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Istanbul University","award":["39456"],"award-info":[{"award-number":["39456"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>CLN2 disease (neuronal ceroid lipofuscinosis type 2) is an ultra-rare lysosomal storage disorder caused by mutations in the TPP1\/CLN2 gene, resulting in impaired tripeptidyl peptidase 1 (TPP1) activity. The timely initiation of enzyme replacement therapy is pivotal for attenuating progressive and irreversible neurodegeneration. This study aimed to benchmark the performance of Oxford Nanopore long-read sequencing (ONT-LRS) for targeted TPP1 mutation detection in a Turkish CLN2 cohort and to assess its concordance with orthogonal validation methods, including Sanger sequencing and enzymatic activity assays. Using a custom-designed primer panel, the entire TPP1 gene (6846 bp) was sequenced on the Oxford Nanopore (ONT) MinIon platform in seven clinically confirmed CLN2 index patients and sixteen unaffected family members. Detected variants were validated via Sanger sequencing and correlated with TPP1 enzyme activity in leucocytes and dried blood spots. Four pathogenic or likely pathogenic TPP1 variants were identified: c.622C&gt;T (p.Arg208*), c.857A&gt;G (p.Asn286Ser), c.1204G&gt;T (p.Glu402*), and c.225A&gt;G (p.Gln75=), along with fourteen additional benign variants. Variant allele frequencies were 50% for c.622C&gt;T, 28.6% for c.1204G&gt;T, 14.3% for c.857A&gt;G, and 7.1% for c.225A&gt;G. Notably, this is the first report to document the homozygous state of the c.857A&gt;G variant and the compound heterozygous configuration of the c225A&gt;G and c.622C&gt;T variants in CLN2 patients, thereby expanding the known mutational landscape. In contrast, the globally common variant c.509-1G&gt;C was not observed, suggesting regional variation in TPP1 mutation patterns. Consistent with the prior Turkish studies, c.622C&gt;T (p.Arg208*) was the most prevalent variant, followed by c.1204G&gt;T (p.Glu402*). TPP1 enzymatic activity was significantly reduced in all affected individuals (p &lt; 0.0001), supporting the functional relevance of the identified variants. ONT-LRS offers a robust, cost-effective platform for high-resolution analysis of the TPP1 gene. Integrating molecular and biochemical data improves diagnostic precision and supports timely, targeted interventions for CLN2 disease, particularly in regions with high consanguinity and limited diagnostic infrastructure.<\/jats:p>","DOI":"10.3390\/ijms26115037","type":"journal-article","created":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T09:58:09Z","timestamp":1747994289000},"page":"5037","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Benchmarking Nanopore Sequencing for CLN2 (TPP1) Mutation Detection: Integrating Rapid Genomics and Orthogonal Validation for Precision Diagnostics"],"prefix":"10.3390","volume":"26","author":[{"given":"Bet\u00fcl","family":"Teker","sequence":"first","affiliation":[{"name":"Institute of Health Sciences, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"G\u00f6kce","family":"Akan","sequence":"additional","affiliation":[{"name":"Research Center of Experimental Health Sciences, Near East University, 99138 Mersin, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hasan H\u00fcseyin","family":"Kazan","sequence":"additional","affiliation":[{"name":"Department of Medical Biology, Gulhane Faculty of Medicine, University of Health Sciences, 06018 Ankara, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00d6zge","family":"\u00d6zgen","sequence":"additional","affiliation":[{"name":"Rare Diseases Research Laboratory, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-4401-896X","authenticated-orcid":false,"given":"Suzin","family":"Tatonyan","sequence":"additional","affiliation":[{"name":"Institute of Health Sciences, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mehmet Cihan","family":"Balci","sequence":"additional","affiliation":[{"name":"Rare Diseases Research Laboratory, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"},{"name":"Division of Nutrition and Metabolism, Department of Pediatrics, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meryem","family":"Karaca","sequence":"additional","affiliation":[{"name":"Rare Diseases Research Laboratory, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"},{"name":"Division of Nutrition and Metabolism, Department of Pediatrics, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fulya","family":"Kurekci","sequence":"additional","affiliation":[{"name":"Division of Neurology, Department of Pediatrics, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Edibe Pembeg\u00fcl","family":"Y\u0131ld\u0131z","sequence":"additional","affiliation":[{"name":"Division of Neurology, Department of Pediatrics, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Olcay","family":"G\u00fcngor","sequence":"additional","affiliation":[{"name":"Neurology Unit, Department of Pediatrics, Pamukkale University, 20160 Pamukkale, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adnan","family":"Deniz","sequence":"additional","affiliation":[{"name":"Rare Diseases Research Laboratory, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"},{"name":"Neurology Unit, Department of Pediatrics, Medical School, Kocaeli University, 41001 Kocaeli, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7121-6077","authenticated-orcid":false,"given":"Asuman","family":"Gedikbasi","sequence":"additional","affiliation":[{"name":"Department of Pediatric Basic Sciences, Institute of Child Health, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1793-9220","authenticated-orcid":false,"given":"Fatmahan","family":"Atalar","sequence":"additional","affiliation":[{"name":"Rare Diseases Research Laboratory, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"},{"name":"Department of Rare Diseases, Institute of Child Health, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3726-5726","authenticated-orcid":false,"given":"G\u00fclden Fatma","family":"Gokcay","sequence":"additional","affiliation":[{"name":"Rare Diseases Research Laboratory, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"},{"name":"Division of Nutrition and Metabolism, Department of Pediatrics, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1957-6072","authenticated-orcid":false,"given":"Mehves","family":"Poda","sequence":"additional","affiliation":[{"name":"Rare Diseases Research Laboratory, Istanbul Medical Faculty, Istanbul University, 34452 Fatih, T\u00fcrkiye"},{"name":"Department of Rare Diseases, Institute of Child Health, Istanbul University, 34452 Fatih, T\u00fcrkiye"},{"name":"Department of Genetics, Aziz Sancar Institute of Experimental Medicine, Istanbul University, 34452 Fatih, T\u00fcrkiye"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2237","DOI":"10.1016\/j.bbadis.2015.05.011","article-title":"Genetics of the neuronal ceroid lipofuscinoses (Batten disease)","volume":"1852","author":"Mole","year":"2015","journal-title":"Biochim. 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