Akan Gökçe, Balcı Mehmet Cihan, Tuncel Gülten, Karaca Meryem, Kazan Hasan Hüseyin, Özketen Ahmet Çağlar, Özgen Özge, Gökçay Gülden Fatma, Atalar Fatmahan
DESAM Institute, Near East University, Mersin 10, Türkiye.
Division of Pediatric Nutrition and Metabolism, Istanbul Medical Faculty, Istanbul University, Istanbul, Türkiye.
Orphanet J Rare Dis. 2025 May 26;20(1):250. doi: 10.1186/s13023-025-03775-4.
Carnitine transport and cycle disorders (CCD) are a group of metabolic disorders characterized by either carnitine depletion or dysfunction in the carnitine cycle, a critical process for the transport of fatty acids into the mitochondria and their subsequent β-oxidation. Clinically, CCD can manifest with a wide range of symptoms, including hypoketotic hypoglycemia, which may be accompanied by signs of liver dysfunction, hepatic steatosis, myopathy and cardiomyopathy. Biochemical diagnosis typically involves measuring carnitine and acylcarnitine levels in blood, alongside organic acid profiling in urine. However, due to phenotypic overlaps with other metabolic disorders, precise molecular diagnosis is essential for accurate disease classification and subtype determination. The present study aimed to develop and clinically validate a novel CCD panel, specifically designed for Oxford Nanopore Technologies (ONT) platform compatibility. The panel targeted four key CCD related genes (CPT-1, CPT-2, SLC22A5 and SLC25A20). An amplification-based library preparation method pooling 21 primers specific to the CCD-related genes into two tubes was optimized. The panel was then applied to screen 20 patients previously diagnosed with CCD via second-generation sequencing platform. Comparative analysis of results from both platforms revealed a 100% concordance in detecting pathogenic, likely pathogenic, and variants of unknown significance associated with CCD. In silico analysis was also performed to predict the pathogenic potential of the variants of unknown significance. Here we report the development and clinical validation of a multi-gene diagnostic panel for ONT platform. The results demonstrated the feasibility of ONT-based genetic testing for CCD and set the stage for the development of similar diagnostic panels for other genetic disorders, offering a streamlined and putatively cost-effective alternative to current sequencing methodologies.
肉碱转运和循环障碍（CCD）是一组代谢紊乱疾病，其特征是肉碱缺乏或肉碱循环功能障碍，而肉碱循环是脂肪酸转运至线粒体及其后续β氧化的关键过程。临床上，CCD可表现出广泛的症状，包括低酮性低血糖，可能伴有肝功能障碍、肝脂肪变性、肌病和心肌病的体征。生化诊断通常包括测量血液中的肉碱和酰基肉碱水平，以及尿液中的有机酸谱分析。然而，由于与其他代谢紊乱存在表型重叠，精确的分子诊断对于准确的疾病分类和亚型确定至关重要。本研究旨在开发并临床验证一种新型的CCD检测组合，该组合专门设计用于与牛津纳米孔技术（ONT）平台兼容。该检测组合针对四个与CCD相关的关键基因（CPT-1、CPT-2、SLC22A5和SLC25A20）。优化了一种基于扩增的文库制备方法，将21条与CCD相关基因特异的引物合并到两个管中。然后将该检测组合应用于通过第二代测序平台先前诊断为CCD的20名患者的筛查。对两个平台的结果进行比较分析发现，在检测与CCD相关的致病、可能致病和意义未明的变异方面，一致性为100%。还进行了计算机分析以预测意义未明变异的致病潜力。在此我们报告了一种用于ONT平台的多基因诊断检测组合的开发和临床验证。结果证明了基于ONT的CCD基因检测的可行性，并为开发其他遗传疾病的类似诊断检测组合奠定了基础，为当前测序方法提供了一种简化且可能具有成本效益的替代方案。
