Objectives: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide. Different kinds of G6PD mutations may result in variable severity of clinical onset in G6PD-deficient individuals. In this study, a reliable molecular diagnostic method was developed for rapid detection of G6PD gene mutation.
Design and methods: Primers were designed to amplify G6PD gene fragments that were subjected to mutation screening using denaturing high-performance liquid chromatography (DHPLC) analysis. Mutations were identified by their distinct elution peak patterns and were confirmed by DNA sequencing. The assay was further validated against 29 samples from individuals with G6PD deficiency.
Results: A DHPLC-based assay for G6PD mutation detection was established. The 9 common G6PD mutations in the Taiwanese and Chinese population could be distinguished through the analysis of DNA elution patterns. During the validation test with the 29 G6PD deficiency specimens, two additional rare mutations, T517C and C519G, were unveiled. Overall, the DHPLC-based mutation detection was 100% concordant with the DNA sequencing results.
Conclusion: Compared to other genotyping techniques, this method requires significantly less technical time to perform and has a greatly increased throughput capacity. Hence, the DHPLC method represents a major technical advance for G6PD genotyping and should benefit G6PD-deficient individuals for proper clinical care.