Glucose-6-phosphate dehydrogenase mutations in malaria endemic area of Thailand by multiplexed high-resolution melting curve analysis.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy in humans, is prevalent in tropical and subtropical areas where malaria is endemic. Anti-malarial drugs, such as primaquine and tafenoquine, can cause haemolysis in G6PD-deficient individuals. Hence, G6PD testing is recommended before radical treatment against vivax malaria. Phenotypic assays have been widely used for screening G6PD deficiency, but in heterozygous females, the random lyonization causes difficulty in interpreting the results. Over 200 G6PD variants have been identified, which form genotypes associated with differences in the degree of G6PD deficiency and vulnerability to haemolysis. This study aimed to assess the frequency of G6PD mutations using a newly developed molecular genotyping test. METHODS: A multiplexed high-resolution melting (HRM) assay was developed to detect eight G6PD mutations, in which four mutations can be tested simultaneously. Validation of the method was performed using 70 G6PD-deficient samples. The test was then applied to screen 725 blood samples from people living along the Thai-Myanmar border. The enzyme activity of these samples was also determined using water-soluble tetrazolium salts (WST-8) assay. Then, the correlation between genotype and enzyme activity was analysed. RESULTS: The sensitivity of the multiplexed HRM assay for detecting G6PD mutations was 100 % [95 % confidence interval (CI): 94.87-100 %] with specificity of 100 % (95 % CI: 87.66-100 %). The overall prevalence of G6PD deficiency in the studied population as revealed by phenotypic WST-8 assay was 20.55 % (149/725). In contrast, by the multiplexed HRM assay, 27.17 % (197/725) of subjects were shown to have G6PD mutations. The mutations detected in this study included four single variants, G6PD Mahidol (187/197), G6PD Canton (4/197), G6PD Viangchan (3/197) and G6PD Chinese-5 (1/197), and two double mutations, G6PD Mahidol + Canton (1/197) and G6PD Chinese-4 + Viangchan (1/197). A broad range of G6PD enzyme activities were observed in individuals carrying G6PD Mahidol, especially in females. CONCLUSIONS: The multiplexed HRM-based assay is sensitive and reliable for detecting G6PD mutations. This genotyping assay can facilitate the detection of heterozygotes, which could be useful as a supplementary approach for high-throughput screening of G6PD deficiency in malaria endemic areas before the administration of primaquine and tafenoquine.

The integrity and stability of specimens under different storage conditions for glucose-6-phosphate dehydrogenase deficiency screening using WST-8.

Accurate measurement of glucose-6-phosphate dehydrogenase (G6PD) activity is critical for malaria treatment as misclassification of G6PD deficiency could cause serious harm to patients. G6PD activity should be assessed in blood samples on the day of collection. Otherwise, specimens should be stored under suitable conditions to prevent loss of G6PD activity. Here, we assessed stability and integrity of G6PD testing in samples from normal controls, heterozygous females, and G6PD deficient individuals using water-soluble tetrazolium salts (WST-8) assay. Specimens were stored as ethylenediaminetetraacetic acid (EDTA) whole blood and dried blood spots (DBS) at various temperatures (37 °C, room temperature, 4 °C and -20 °C) and under different humidity conditions (with and without desiccant). G6PD normal samples were stable for up to 1 year when stored at -20 °C under controlled conditions, with 85% and 91% G6PD activity in EDTA whole blood and DBS in the presence of desiccant, respectively. Specimens from heterozygous females showed greater G6PD activity when stored as DBS, with 85% enzyme activity after 1 year of storage at -20 °C under controlled conditions in the presence of desiccant. G6PD deficient samples rapidly lost enzyme activity in all storage conditions tested. However, the reduction in G6PD enzyme activity in G6PD deficient samples did not interfere with G6PD classification. Samples stored under suitable conditions for G6PD testing will allow accurate measurement of enzyme activity, prevent misclassification of G6PD deficiency and enable safe and effective use of antimalarial drugs such as primaquine and tafenoquine.

Controlled reversible assembly of gold nanoparticles as a new colorimetric and sensitive detection of glucose-6-phosphate dehydrogenase deficiency.

Recently, several studies have examined possible applications of nanoparticles for the development of electronic and optical sensors. The plasmon absorbance of gold nanoparticles has been used extensively to study biomolecular processes, including nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate-dependent enzymatic reactions. In this report, we describe the development of gold nanoparticles as a new colorimetric and sensitive detection method of glucose-6-phosphate dehydrogenase deficiency by means of controlled reversible assembly of gold nanoparticles. 3-nm polyvinylpyrrolidone/N,N'-dimethylaminopyridine-stabilized gold nanoparticles were synthesized, characterized and applied for an in vitro activity assay of 11 recombinant human glucose-6-phosphate dehydrogenase variants. Differences in the activity of the glucose-6-phosphate dehydrogenase variants from different deficiency classes were readily detected using the synthesized gold nanoparticles. The developed method can be easily distinguished with color change by naked eye for the detection of glucose-6-phosphate dehydrogenase deficiency. Moreover, we are the first to propose the segregation mechanism of polyvinylpyrrolidone/N,N'-dimethylaminopyridine-stabilized gold nanoparticles by reduced nicotinamide adenine dinucleotide phosphate. The method enables visual detection of glucose-6-phosphate dehydrogenase deficiency, which could be further developed for diagnostic testing of glucose-6-phosphate dehydrogenase deficiency.

Functional and structural analysis of double and triple mutants reveals the contribution of protein instability to clinical manifestations of G6PD variants.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common polymorphism and enzymopathy in humans, affecting approximately 400 million people worldwide. Over 200 point mutations have been identified in g6pd and the molecular mechanisms underlying the severity of G6PD variants differ. We report the detailed functional and structural characterization of 11 recombinant human G6PD variants: G6PD Asahi, G6PD A, G6PD Guadalajara, G6PD Acrokorinthos, G6PD Ananindeua, G6PD A-(202), G6PD Sierra Leone, G6PD A-(680), G6PD A-(968), G6PD Mount Sinai and G6PD No name. G6PD Guadalajara, G6PD Mount Sinai and G6PD No name are inactive variants and, correlating with the observed clinical manifestations, exhibit complete loss of enzyme activity. Protein structural instability, causing a reduction in catalytic efficiency, contributes to the clinical phenotypes of all variants. In double and triple mutants sharing the G6PD A mutation, we observed cooperative interaction between two and three mutations to cause protein dysfunction. The G6PD A (Asn126Asp) mutation exhibits no effect on protein activity and stability, indicating that the additional mutations in these G6PD variants significantly contribute to enzyme deficiency. We provide insight into the molecular basis of G6PD deficiency, which can explain the severity of clinical manifestations observed in individuals with G6PD deficiency.