Teker Betül, Akan Gökce, Kazan Hasan Hüseyin, Özgen Özge, Tatonyan Suzin, Balci Mehmet Cihan, Karaca Meryem, Kurekci Fulya, Yıldız Edibe Pembegül, Güngor Olcay, Deniz Adnan, Gedikbasi Asuman, Atalar Fatmahan, Gokcay Gülden Fatma, Poda Mehves
Institute of Health Sciences, Istanbul University, 34452 Fatih, Türkiye.
Research Center of Experimental Health Sciences, Near East University, 99138 Mersin, Türkiye.
Int J Mol Sci. 2025 May 23;26(11):5037. doi: 10.3390/ijms26115037.
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 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 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 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 ( < 0.0001), supporting the functional relevance of the identified variants. ONT-LRS offers a robust, cost-effective platform for high-resolution analysis of the 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.
CLN2病（2型神经元蜡样脂褐质沉积症）是一种极为罕见的溶酶体贮积症，由TPP1/CLN2基因突变引起，导致三肽基肽酶1（TPP1）活性受损。及时开始酶替代疗法对于减轻进行性和不可逆的神经退行性变至关重要。本研究旨在对牛津纳米孔长读长测序（ONT-LRS）在土耳其CLN2队列中进行靶向突变检测的性能进行基准测试，并评估其与包括桑格测序和酶活性测定在内的正交验证方法的一致性。使用定制设计的引物组，在牛津纳米孔（ONT）MinIon平台上对7例临床确诊的CLN2索引患者和16名未受影响的家庭成员的整个TPP1基因（6846 bp）进行了测序。通过桑格测序对检测到的变异进行验证，并与白细胞和干血斑中的TPP1酶活性相关联。鉴定出4种致病或可能致病的变异：c.622C>T（p.Arg208*）、c.857A>G（p.Asn286Ser）、c.1204G>T（p.Glu402*）和c.225A>G（p.Gln75=），以及另外14种良性变异。c.622C>T的变异等位基因频率为50%，c.1204G>T为28.6%，c.857A>G为14.3%，c.225A>G为7.1%。值得注意的是，这是第一份记录CLN2患者中c.857A>G变异的纯合状态以及c225A>G和c.622C>T变异的复合杂合构型的报告，从而扩展了已知的突变图谱。相比之下，未观察到全球常见的变异c.509-1G>C，表明突变模式存在区域差异。与之前的土耳其研究一致，c.622C>T（p.Arg208*）是最常见的变异，其次是c.1204G>T（p.Glu402*）。所有受影响个体的TPP1酶活性均显著降低（<0.0001），支持所鉴定变异的功能相关性。ONT-LRS为该基因的高分辨率分析提供了一个强大、经济高效的平台。整合分子和生化数据可提高诊断准确性，并支持对CLN2病进行及时、有针对性的干预，特别是在近亲结婚率高且诊断基础设施有限的地区。
