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Benchmarking Nanopore Sequencing for CLN2 (TPP1) Mutation Detection: Integrating Rapid Genomics and Orthogonal Validation for Precision Diagnostics.

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  • Additional Information
    • Source:
      Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE
    • Publication Information:
      Original Publication: Basel, Switzerland : MDPI, [2000-
    • Subject Terms:
      Neuronal Ceroid-Lipofuscinoses*/genetics ; Neuronal Ceroid-Lipofuscinoses*/diagnosis ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases*/genetics ; Aminopeptidases*/genetics ; Serine Proteases*/genetics ; Nanopore Sequencing*/methods ; Genomics*/methods ; Mutation*; Precision Medicine/methods ; Humans ; Tripeptidyl-Peptidase 1 ; Male ; Female ; Child ; Adult ; Adolescent ; Child, Preschool ; Benchmarking ; Young Adult ; Middle Aged
    • Abstract:
      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>T (p.Arg208*), c.857A>G (p.Asn286Ser), c.1204G>T (p.Glu402*), and c.225A>G (p.Gln75=), along with fourteen additional benign variants. Variant allele frequencies were 50% for c.622C>T, 28.6% for c.1204G>T, 14.3% for c.857A>G, and 7.1% for c.225A>G. Notably, this is the first report to document the homozygous state of the c.857A>G variant and the compound heterozygous configuration of the c225A>G and c.622C>T variants in CLN2 patients, thereby expanding the known mutational landscape. In contrast, the globally common variant c.509-1G>C was not observed, suggesting regional variation in TPP1 mutation patterns. Consistent with the prior Turkish studies, c.622C>T (p.Arg208*) was the most prevalent variant, followed by c.1204G>T (p.Glu402*). TPP1 enzymatic activity was significantly reduced in all affected individuals (p < 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.
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    • Grant Information:
      ADEP grant (39456) Istanbul University
    • Contributed Indexing:
      Keywords: CLN2; DNA sequencing; TPP1; TPP1 enzyme activity; comprehensive care; long-read sequencing
    • Accession Number:
      EC 3.4.14.9 (TPP1 protein, human)
      EC 3.4.14.- (Dipeptidyl-Peptidases and Tripeptidyl-Peptidases)
      EC 3.4.11.- (Aminopeptidases)
      EC 3.4.- (Serine Proteases)
      0 (Tripeptidyl-Peptidase 1)
      0 (Tpp1 protein, rat)
    • Subject Terms:
      Ceroid Lipofuscinosis, Neuronal, 2
    • Publication Date:
      Date Created: 20250613 Date Completed: 20250613 Latest Revision: 20250615
    • Publication Date:
      20260130
    • Accession Number:
      PMC12155472
    • Accession Number:
      10.3390/ijms26115037
    • Accession Number:
      40507848