Introduction: Charcot-Marie-Tooth (CMT) is a disorder that encompasses a group of hereditary neuropathies. The clinical features of CMT is variable due to genetic heterogeneity. This study aimed to evaluate the utility of targeted next-generation sequencing (NGS) panels in the molecular diagnosis of CMT in routine clinical practice and to determine the causative genetic variants. Methods: NGS was used to detect the causative single nucleotide variants in 55 genes associated with CMT alongside multiplex ligation probe amplification (MLPA) for copy number variation (CNV) analysis of the PMP22 gene. The period from the first clinical findings to genetic testing was considered as the “diagnostic odyssey.” Results: A total of 58 patients with suspected CMT were analyzed. MLPA was performed on 54 patients, while 4 underwent direct NGS due to suspicion of other CMT types. MLPA revealed pathogenic CNVs in 21 patients (36.2%). Among the 33 patients with negative MLPA results, 24 underwent NGS. Pathogenic/likely pathogenic variants were identified in 17 (60.7%) of total 28 patients. Overall, the diagnostic yield was 65.5% (38/58). Moreover, a novel likely pathogenic variant (c.637C>T) was detected in the NDRG1 gene. The average diagnostic odyssey in diagnosed patients was 5.6 years. Conclusion: Targeted NGS panels are a highly effective tool for the genetic diagnosis of CMT and increase the diagnostic rates when used in conjunction with MLPA. Molecular genetic diagnosis is critical for CMT patients, particularly in light of ongoing research into targeted therapies. Furthermore, presence of undiagnosed cases underscores the likelihood that additional causative genes and mechanisms in CMT etiology remain to be discovered.

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