Optimizing a multiplex high resolution melting curve to diagnose G6PD deficiency based on viangchan and canton mutations

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency which is caused by mutation on G6PD gene  is the most common enzyme disorder in human. There have been 184 discovered mutations among which Viangchan [Val²⁹¹Met] and Canton mutation [Arg⁴⁵⁹Leu] are the most common variants in Vietnamese. Due to the severity of this disease, several methods  have been devised for diagnostics. However, time-consuming, low sensitivity and expensiveness are major problems of those techniques. Recently, High Resolution Melting (HRM) has been developed and proven to be an effective method for DNA genotyping, mutation scanning and sequence matching. Hence, in this study a multiplex HRM has been developed aiming at detecting these two mutations concurrently. At first, a singleplex HRM was designed for each mutation. Then, conditions for these singleplex assay were combined and optimized again in order to get the optimal condition for multiplex HRM.  Although this method showed a promising potential with a high accuracy, sensitivity, and specificity, it is impractial to continue developing this method because of the lack of controls. However, the optimized singleplex PCR-HRM in this study still can be used for single mutation detection and serve as the background to develop PCR-HRM for other G6PD mutations in Vietnamese-Kinh population.