Isocitrate dehydrogenase (IDH) is an enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate and is one of the enzymes of the Krebs cycle. The reaction catalyzed by isocitrate dehydrogenase also represents one of the major metabolic pathways for the formation of NADPH, an important cellular reducing agent. Variants in the IDH genes are predominantly somatic and are located at key arginine residues in the active site responsible for binding the substrate isocitrate—namely R132 in IDH1 and R140 and R172 in IDH2. The mutated isocitrate dehydrogenase enzyme acquires a neomorphic function, catalyzing the conversion of α-ketoglutarate into the oncometabolite 2-hydroxyglutarate. The consequences of this include metabolic alterations, DNA and histone hypermethylation, and redox imbalance. Variants in IDH1 or IDH2 occur in 5–7 % of patients with myelodysplastic syndromes and in 20 % of patients with acute myeloid leukemia. They occur predominantly in older patients, have a negative prognostic impact, and are associated with poorer overall survival. At the same time, variants in the IDH genes also have therapeutic significance, as in addition to standard treatment, targeted therapy with specific small-molecule inhibitors is available for these patients—ivosidenib for IDH1 and enasidenib for IDH2. Because in the diagnosis of acute myeloid leukemia it is crucial to initiate treatment as soon as possible, a rapid method for detecting these variants is required. Sanger sequencing is suitable for this purpose, as results can be obtained within a few days. Therefore, within the framework of the master’s thesis, we performed a validation of the Sanger sequencing method for the detection of the R132 variant in the IDH1 gene and the R140 and R172 variants in the IDH2 gene, focusing on a comparison of our method with the previously used next-generation sequencing method, which is too time-consuming for this purpose. We demonstrated 100 % analytical sensitivity, 100 % analytical specificity, 100 % within-run accuracy, 100 % between-run accuracy, and 100 % inter-instrument reproducibility. The limit of detection was set at 15 % mutated DNA in the sample, which is appropriate with regard to clinical applicability, as patients at the time of diagnosis of acute myeloid leukemia or myelodysplastic syndromes typically have a significantly higher proportion of mutated DNA. The validation was successful, as we demonstrated that the method is suitable for its intended clinical purpose and is therefore ready for use in routine diagnostics.
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