Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

Background: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. Methods: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. Results: Based on phenotypic testing, the prevalence of G6PD deficiency (T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. Conclusions: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population

Additional file 1 of Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

Additional file 1: Figure S1. Primers used in (A) multiplex HRM for the detection of 15 G6PD mutations and (B) G6PD gene sequencing. Table S1. Primers used in multiplex HRM assays. Table S2. Primers used for site-directed mutagenesis. Figure S2. The frequency distribution of G6PD enzyme activity in (A) males and (B) females. Figure S3. Box plot of G6PD activity for each variant among (A) malaria-positive males, (B) malaria-positive females, (C) malaria-negative males and (D) malaria-negative females. Figure S4. Secondary structure analysis of G6PD variants by circular dichroism. Table S3. Melting temperature (Tm) values of recombinant G6PD proteins by thermal shift assay. Mutations were ranked in order of stability, from most stable to least stable. Table S4. Thermal inactivation of G6PD variants as reported by T1/2. Mutations were ranked in order of stability, from most stable to least stable. Table S5. Stability of G6PD variants in the presence of Gdn-HCl as reported by C1/2. Mutations were ranked in order of stability, from most stable to least stable. Table S6. Susceptibility of G6PD variants to trypsin digestion. Mutations were ranked in order of stability, from most stable to least stable. Table S7. Structural characteristics of the dimer and tetramer interfaces (t = 100 ns). Table S8. Average values of the trajectory analyses performed on the WT and variants. Figure S5. Ligand binding pocket occupancy heatmap indicating the presence (orange) and absence (turquoise) of hydrogen bonds (t = 100 ns). Figure S6. Superimposition and structural deviations of the simulated variants against the WT (red) at the mutation site, dimer and tetramer interfaces (t = 100 ns). (A) Gaohe, (B) Valladolid, (C) Canton, (D) Viangchan, (E) Gond, (F) Gaohe + Viangchan, (G) Valladolid + Viangchan, and (H) Canton + Viangchan