Optimize Hrm Method For Diagnosis Of G6pd Deficiency In Kinh-Vietnamese Via Viangchan Mutation

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme disorder affects human beings. This disease has more than 184 discovered point mutations and there are several methods have been applied for diagnosing this disease. However, time-consuming, low sensitivity and expensiveness are the major problems of those techniques. Recently, High Resolution Melting (HRM) has been studied and proven to be effective for DNA genotyping, mutation scanning and sequence matching. Therefore, HRM has been chosen for diagnosing G6PD deficiency via Viangchan mutation. This point mutation is caused by a substitution of guanine (G) by adenine (A) at the position 13031 and categorized into class 2 or 3 of G6PD which causes severe deficiency or mild deficiency respectively. In this study, we used Umelt software to design primers and then optimized annealing temperature and MgCl2 concentration for HRM conditions. Validating of this method was performed by comparing to ARMS-PCR genotyping results of 50 G6PD samples. Following after that, the genotyping results were confirmed by sequencing. Amazingly, both sensitivity and specificity of this technique reached 100%. Base on the accurate outcome, we have successfully optimized the HRM conditions for diagnosing 50 G6PD samples. This was such an essential preconditions that showed HRM could be applied for other types of G6PD through other type of mutation such as Canton or developed for HRM-Multiplex reactions. Keywords: G6PD Viangchan HRM Melting curves analysis