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Benchmarking Nanopore Sequencing for CLN2 (TPP1) Mutation Detection: Integrating Rapid Genomics and Orthogonal
Betül Teker1, Gökce Akan2, Hasan Hüseyin Kazan3
1Institute of Health Sciences, Istanbul University, 34452 Fatih, Türkiye.
International Journal of Molecular Sciences
|June 13, 2025
Summary
Oxford Nanopore sequencing accurately detects TPP1 gene mutations in CLN2 disease patients. This method, combined with enzymatic assays, enhances diagnostic precision for timely treatment of this rare neurodegenerative disorder.
Area of Science:
- Genetics and Genomics
- Biochemistry
- Rare Diseases
Background:
- CLN2 disease, a rare lysosomal storage disorder, stems from TPP1/CLN2 gene mutations, leading to neuronal ceroid lipofuscinosis.
- Impaired tripeptidyl peptidase 1 (TPP1) activity due to these mutations causes progressive neurodegeneration.
- Early diagnosis and enzyme replacement therapy are crucial for managing CLN2 disease progression.
Purpose of the Study:
- To evaluate Oxford Nanopore long-read sequencing (ONT-LRS) for detecting TPP1 gene mutations in Turkish CLN2 patients.
- To assess the concordance of ONT-LRS with Sanger sequencing and TPP1 enzymatic activity assays.
- To expand the understanding of TPP1 mutation spectrum in the Turkish CLN2 cohort.
Main Methods:
- Targeted sequencing of the entire TPP1 gene (6846 bp) using Oxford Nanopore MinIon platform with a custom primer panel.
- Validation of detected variants through Sanger sequencing.
- Correlation of genetic findings with TPP1 enzyme activity measured in leukocytes and dried blood spots.
Main Results:
- Four pathogenic/likely pathogenic TPP1 variants (c.622C>T, c.857A>G, c.1204G>T, c.225A>G) and 14 benign variants were identified.
- Novel findings include the homozygous state of c.857A>G and compound heterozygous c.225A>G/c.622C>T variants.
- Significantly reduced TPP1 enzymatic activity was observed in all affected individuals (p < 0.0001).
Conclusions:
- ONT-LRS provides a robust and cost-effective method for high-resolution TPP1 gene analysis.
- Integrating molecular and biochemical data improves diagnostic accuracy for CLN2 disease.
- This approach supports timely interventions, especially in regions with consanguinity and limited diagnostic resources.
