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Fulltext Computational analysis of dimer G6PD structure to elucidate pathogenicity of G6PD variants

Chandran S, Louis NE, Amran SI, Ab Latif N, Hamza MA, Alonazi M

Biomedicine (Taipei), 2024;14(1):47-59.
PMID: 38533298 DOI: 10.37796/2211-8039.1431

An inherent genetic enzyme disorder in humans, known as glucose-6-phosphate dehydrogenase (G6PD) deficiency, arises due to specific mutations. While the prevailing approach for investigating G6PD variants involves biochemical analysis, the intricate structural details remain limited, impeding a comprehensive understanding of how different G6PD variants of varying classes impact their functionality. This study 22 examined the dynamic properties of G6PD wild types and six G6PD variants from 23 different classes using molecular dynamic simulation (MDS). The wild-type and variant 24 G6PD structures unveil high fluctuations within the amino acid range of 274-515, the structural NADP+ binding site, pivotal for enzyme dimerization. Specifically, two variants, G6PDZacatecas (R257L) and G6PDDurham (K238R), demonstrate compromised structural stability at the dimer interface, attributable to the disruption of a salt bridge involving Glu 206 and Lys 407, along with the disturbance of hydrogen bonds formed by Asp 421 at the βN-βN sheets. Consequently, this impairment cascades to affect the binding affinity of crucial interactions, such as Lys 171-Glucose-6-Phosphate (G6P) and Lys 171-catalytic NADP+, leading to diminished enzyme activity. This study underscores the utility of computational in silico techniques in predicting the structural alterations and flexibility of G6PD variants. This insight holds promise for guiding future endeavors in drug development targeted at mitigating the impacts of G6PD deficiency.
Fulltext Preliminary Study of Structural Changes of Glucose-6-Phosphate Dehydrogenase Deficiency Variants

Louis NE, Hamza MA, Baharuddin PNEB, Chandran S, Latif NA, Alonazi MA, et al.

Biomedicine (Taipei), 2022;12(3):12-19.
PMID: 36381187 DOI: 10.37796/2211-8039.1355

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency disorder affecting over 400 million individuals worldwide. G6PD protects red blood cells (RBC) from the harmful effects of oxidative substances. There are more than 400 G6PD mutations, of which 186 variants have shown to be linked to G6PD deficiency by decreasing the activity or stability of the enzyme. Different variants manifest different clinical phenotypes which complicate comprehending the mechanism of the disease. In order to carry out computational approaches to elucidate the structural changes of different G6PD variants that are common to the Asian population, a complete G6PD monomer-ligand complex was constructed using AutoDock 4.2, and the molecular dynamics simulation package GROMACS 4.6.7 was used to study the protein dynamics. The G410D and V291M variants were chosen to represent classes I and II respectively and were created by in silico site-directed mutagenesis. Results from the Root mean square deviation (RMSD), Root mean square fluctuation (RMSF) and Radius of gyration (Rg) analyses provided insights on the structure - function relationship for the variants. G410D indicated impaired dimerization and structural NADP binding while the impaired catalytic activity for V291M was indicated by a conformational change at its mutation site.
Fulltext Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

Boonyuen U, Jacob BAC, Wongwigkan J, Chamchoy K, Singha-Art N, Pengsuk N, et al.

Malar J, 2024 Feb 02;23(1):38.
PMID: 38308253 DOI: 10.1186/s12936-024-04864-8

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.
Improved genetic screening with zygosity detection through multiplex high-resolution melting curve analysis and biochemical characterisation for G6PD deficiency

Boonyuen U, Jacob BAC, Chamchoy K, Pengsuk N, Talukam S, Petcharat C, et al.

Trop Med Int Health, 2025 Mar 13.
PMID: 40078033 DOI: 10.1111/tmi.14105

Accurate diagnosis of glucose-6-phosphate dehydrogenase (G6PD) deficiency is crucial for relapse malaria treatment using 8-aminoquinolines (primaquine and tafenoquine), which can trigger haemolytic anaemia in G6PD-deficient individuals. This is particularly important in regions where the prevalence of G6PD deficiency exceeds 3%-5%, including Southeast Asia and Thailand. While quantitative phenotypic tests can identify women with intermediate activity who may be at risk, they cannot unambiguously identify heterozygous females who require appropriate counselling. This study aimed to develop a genetic test for G6PD deficiency using high-resolution melting curve analysis, which enables zygosity identification of 15 G6PD alleles. In 557 samples collected from four locations in Thailand, the prevalence of G6PD deficiency based on indirect enzyme assay was 6.10%, with 8.08% exhibiting intermediate deficiency. The developed high-resolution melting assays demonstrated excellent performance, achieving 100% sensitivity and specificity in detecting G6PD alleles compared with Sanger sequencing. Genotypic variations were observed across four geographic locations, with the combination of c.1311C>T and c.1365-13T>C being the most common genotype. Compound mutations, notably G6PD Viangchan (c.871G>A, c.1311C>T and c.1365-13T>C), accounted for 15.26% of detected mutations. The high-resolution melting assays also identified the double mutation G6PD Chinese-4 + Canton and G6PD Radlowo, a variant found for the first time in Thailand. Biochemical and structural characterisation revealed that these variants significantly reduced catalytic activity by destabilising protein structure, particularly in the case of the Radlowo mutation. The refinement of these high-resolution melting assays presents a highly accurate and high-throughput platform that can improve patient care by enabling precise diagnosis, supporting genetic counselling and guiding public health efforts to manage G6PD deficiency-especially crucial in malaria-endemic regions where 8-aminoquinoline therapies pose a risk to deficient individuals.
Fulltext Spectrum of glucose-6-phosphate dehydrogenase (G6PD) mutations and trends in hemoglobin levels among adult dengue patients in Thailand

Chamnanchanunt S, Jacob BAC, Thanachartwet V, Desakorn V, Singha-Art N, Sahassananda D, et al.

PLoS One, 2025;20(9):e0332039.
PMID: 40966243 DOI: 10.1371/journal.pone.0332039

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans that may exacerbate clinical outcomes during viral infections such as dengue, particularly in regions where both conditions are endemic. This study aimed to characterize the spectrum of G6PD mutations and explore trends in hemoglobin levels among adult dengue patients in Thailand. Samples from 231 adult patients diagnosed with dengue were analyzed. G6PD deficiency was identified in 24 individuals (10.4%), while G6PD mutations were detected in 111 patients (48.1%). The most frequently observed mutations include a combination of synonymous and intronic mutations (c.1311C > T and c.1365-13T > C), compound mutation of G6PD Viangchan (c. 871G > A, c.1311C > T and c.1365-13T > C), and a deletion variant (c.486-34delT). Additionally, a novel variant, c.1439T > C, was identified and named "G6PD Phaya Thai". Patients carrying G6PD mutations exhibited different hemoglobin level trends compared to those without mutations. Specifically, while hemoglobin levels increased from the febrile to critical phase in patients without mutations, a significant decline was observed in mutation carriers. Median hemoglobin levels differed significantly between the two groups during both the febrile and critical phases (p = 0.02 and p