An Overall View of the Functional and Structural Characterization of Glucose-6-Phosphate Dehydrogenase Variants in the Mexican Population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting an estimated 500 million people worldwide, is a genetic disorder that causes human enzymopathies. Biochemical and genetic studies have identified several variants that produce different ranges of phenotypes; thus, depending on its severity, this enzymopathy is classified from the mildest (Class IV) to the most severe (Class I). Therefore, understanding the correlation between the mutation sites of G6PD and the resulting phenotype greatly enhances the current knowledge of enzymopathies' phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments for patients with G6PD deficiency. In this review, we analyzed and compared the structural and functional data from 21 characterized G6PD variants found in the Mexican population that we previously characterized. In order to contribute to the knowledge regarding the function and structure of the variants associated with G6PD deficiency, this review aimed to determine the molecular basis of G6PD and identify how these mutations could impact the structure, stability, and function of the enzyme and its relation with the clinical manifestations of this disease.

Cut-off values for diagnosis of G6PD deficiency by flow cytometry in Thai population.

In heterozygous females, X-inactivation causes a change in glucose-6-phosphate dehydrogenase (G6PD) activity from normal to deficient. Most G6PD screening tests are used to accurately diagnose hemizygous males, but they are less reliable for diagnosing heterozygous females. This study established flow cytometric cut-off values for screening of G6PD deficiency in hemizygous males and heterozygous or homozygous females. We studied 205 (125 females, 80 males) leftover blood samples from quantitative methemoglobin reduction (MR) screening. G6PD gene mutations determined by multiplex amplification refractory mutation system-polymerase chain reaction and direct DNA sequencing were used as the gold standard reference. Accuracy of the test, including the sensitivity, specificity, and positive and negative predictive values, was analyzed using MedCalc software. The optimal cut-off values for classification of %red blood cells with normal G6PD activity or %bright cells into homozygous normal, heterozygous, and homozygous deficiency in females were 85.4-100%, 6.3-85.3%, and 0-6.2%, respectively (sensitivity 93.2%, specificity 100%). The cut-offs for classification into hemizygous normal and hemizygous deficiency in males were 76.5-100% and 0-76.4%, respectively (sensitivity 100%, specificity 96.5%). Flow cytometry can be used to differentiate heterozygous females with intermediate phenotype from homozygous females, but cannot distinguish between heterozygous females with extreme phenotype and homozygous females. By flow cytometry, heterozygous and homozygous deficiency was detected in 29.6% and 3.2% of females, respectively. Among males, hemizygous deficiency was found in 31.3%. Flow cytometry can be used to screen patients with G6PD deficiency, and reliably and efficiently identify heterozygous and homozygous females, and hemizygous males based on cellular G6PD activity.

Activity-expression profiling of glucose-6-phosphate dehydrogenase in tissues of normal and diabetic mice.

Glucose-6-phosphate dehydrogenase (G6PD) plays a principal role in the regulation of oxidative stress by modulating the nicotinamide adenine dinucleotide phosphate pool and is expected to be associated with metabolic diseases such as diabetes mellitus (DM). However, it is unclear whether hyperglycemia increases G6PD activity levels in DM because suitable assays for quantifying the activity in a high-throughput manner are lacking. Using liquid droplet arrays tailored to analyze tissue lysates, we performed G6PD activity profiling in eight tissues of normal and diabetic mice: brain, heart, kidney, liver, lung, muscle, spleen, and thyroid. Diabetic mice exhibited significantly higher G6PD activities in the kidney, liver, spleen, and thyroid than normal mice; no significant difference was found in the brain, heart, lung, or muscle. We also performed G6PD expression profiling in the eight tissues using Western blot analysis. Diabetic mice showed significantly elevated G6PD expression levels in the kidney, lung, spleen, and thyroid compared with normal mice; no significant difference was found in the brain, heart, liver, or muscle. An analysis of G6PD activity-expression profiles demonstrated tissue-specific changes in response to hyperglycemia. Thus, our approach would be helpful for understanding the role of G6PD in tissue-based pathogenesis of diabetic complications.

Subclinical reduced G6PD activity in rheumatoid arthritis and Sjögren's Syndrome patients: relation to clinical characteristics, disease activity and metabolic syndrome.

OBJECTIVE: Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway. The purpose of this study was to investigate the enzyme activity of G6PD in Rheumatoid Arthritis (RA) and Sjögren's Syndrome (SS) patients not known to be deficient in this enzyme. It was also within the scope of the aim to find the relation of G6PD to the presence of metabolic syndrome (MetS) in these patients. METHODS: Erythrocyte G6PD activity was evaluated in 40 RA patients, 30 SS patients and in 30 age- and sex-matched control. The clinical characteristics, disease activity score (DAS28), SS disease activity (SSDAI) and damage (SSDDI) indices and presence of MetS of the included patients were analyzed in relation to the enzyme level. RESULTS: The G6PD activity in RA patients (7.72 ± 3.57 U/g Hb) was significantly reduced compared to that in the SS patients (11.55 ± 3.14 U/g Hb) and control (13.23 ± 3.34 U/g Hb) especially those with MetS (4.61 ± 1.84 U/g Hb) (p < 0.001). There was a significant negative correlation of the G6PD activity with the disease duration and DAS28 (p < 0.001). CONCLUSION: The results of this study, suggest that G6PD not only does not protect against MetS in RA, but may even be considered a risk factor for the development of this disorder. The identification of regulatory tools for G6PD activity may prove promising for treating the associated metabolic disorders and chronic inflammation in RA.

Study of the antigenic characteristics of red blood cells units and their sickle cell disease recipients and the G6PD activity of transfused red blood cells units.

INTRODUCTION: Transfusion has a central place in the treatment of patients with sickle cell disease (SCD). Matching blood groups of red blood cell (RBC) units with the blood groups of the patient is essential to prevent alloimmunization and delayed hemolytic transfusion reaction. African ancestry donors have the best phenocompatibility with patients of the same origin, however their RBCs may present characteristic that can alter quality of the unit such as glucose-6-phosphate dehydrogenase (G6PD) deficiency. The objective is to analyze transfusion protocol, immunization rate and mismatch situations of SCD recipients and to evaluate the frequency of G6PD deficiency in RBCs units from African ancestry donors. METHODS: Samples of units transfused to SCD patients were analyzed. Transfusion data were collected from institutional databases. The activity of G6PD was measured in the segment of the RBC units. RESULTS: A total of 98 segments of units transfused to 37 SCD recipients in 41 transfusions episodes was collected. Among patients, 35.1% (n = 13) had no antibodies; 10.8% (n = 4) had antibodies against Fya/Fyb, Jka/Jkb, M/N, S/s; 21.6% (n = 8) against RH/K antigens. In all cases, the protocols were in line with the recommendations. G6PD deficiency was observed in 9 units, that were all collected from Afro-Caribbean donors. CONCLUSION: The transfusion protocol is established to prevent immunological reactions due to disparities in blood group antigens between donors and SCD recipients. However, the units of African ancestry donors, which allowed the best compatibility, displayed a high rate of G6PD deficiency. The storage and recovery impact of this deficiency must be evaluated.

Genetic Variants of Glucose-6-Phosphate Dehydrogenase and Their Associated Enzyme Activity: A Systematic Review and Meta-Analysis.

Low glucose-6-phosphate dehydrogenase enzyme (G6PD) activity is a key determinant of drug-induced haemolysis. More than 230 clinically relevant genetic variants have been described. We investigated the variation in G6PD activity within and between different genetic variants. In this systematic review, individual patient data from studies reporting G6PD activity measured by spectrophotometry and corresponding the G6PD genotype were pooled (PROSPERO: CRD42020207448). G6PD activity was converted into percent normal activity applying study-specific definitions of 100%. In total, 4320 individuals from 17 studies across 10 countries were included, where 1738 (40.2%) had one of the 24 confirmed G6PD mutations, and 61 observations (3.5%) were identified as outliers. The median activity of the hemi-/homozygotes with A-(c.202G>A/c.376A>G) was 29.0% (range: 1.7% to 76.6%), 10.2% (range: 0.0% to 32.5%) for Mahidol, 16.9% (range 3.3% to 21.3%) for Mediterranean, 9.0% (range: 2.9% to 23.2%) for Vanua Lava, and 7.5% (range: 0.0% to 18.3%) for Viangchan. The median activity in heterozygotes was 72.1% (range: 16.4% to 127.1%) for A-(c.202G>A/c.376A>G), 54.5% (range: 0.0% to 112.8%) for Mahidol, 37.9% (range: 20.7% to 80.5%) for Mediterranean, 53.8% (range: 10.9% to 82.5%) for Vanua Lava, and 52.3% (range: 4.8% to 78.6%) for Viangchan. A total of 99.5% of hemi/homozygotes with the Mahidol mutation and 100% of those with the Mediterranean, Vanua Lava, and Viangchan mutations had <30% activity. For A-(c.202G>A/c.376A>G), 55% of hemi/homozygotes had <30% activity. The G6PD activity for each variant spanned the current classification thresholds used to define clinically relevant categories of enzymatic deficiency.

DNA Polymerase Iota Promotes Esophageal Squamous Cell Carcinoma Proliferation Through Erk-OGT-Induced G6PD Overactivation.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers with rapid progression and a high mortality rate. Our previous study demonstrated that DNA polymerase iota (Pol ι) is overexpressed in ESCC tumors and correlates with poor prognosis. However, its role in ESCC proliferation remains obscure. We report here that Pol ι promotes ESCC proliferation and progression through Erk- O-GlcNAc transferase (OGT) regulated Glucose-6-phosphate dehydrogenase (G6PD) overactivation. Cell clonogenic ability was assessed by colony formation assay. Cell proliferation was assessed by EdU incorporation assay. Our transcriptome data was reanalyzed by GSEA and validated by analysis of cellular metabolism, G6PD activity, and cellular NADPH concentration. The level of Pol ι, OGT, G6PD and O-GlcNAcylation in ESCC cells and patient samples were analyzed. The MEK inhibitor PD98059 was applied to confirm OGT expression regulation by the Erk signaling. The G6PD inhibitor polydatin was used to examine the role of G6PD activation in Pol ι promoted proliferation. We found that Pol ι promotes ESCC proliferation. It shunted the glucose flux towards the pentose phosphate pathway (PPP) by activating G6PD through OGT-promoted O-GlcNAcylation. The expression of OGT was positively correlated with Pol ι expression and O-GlcNAcylation. Notably, elevated O-GlcNAcylation was correlated with poor prognosis in ESCC patients. Pol ι was shown to stimulate Erk signaling to enhance OGT expression, and the G6PD inhibitor polydatin attenuated Pol ι induced tumor growth in vitro and in vivo. In conclusion, Pol ι activates G6PD through Erk-OGT-induced O-GlcNAcylation to promote the proliferation and progression of ESCC, supporting the notion that Pol ι is a potential biomarker and therapeutic target of ESCC.

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.

Gene mutation analysis of glucose-6-phosphate dehydrogenase deficiency among infants in Kunming / 中华检验医学杂志

Objective:To analyze the genetic mutation characteristics of glucose-6-phosphate dehydrogenase (G6PD) deficiency among infants in Kunming.Methods:A total of 15 533 infants (7 994 males and 7 539 females) born in Kunming from January 1, 2018, to December 31, 2020, with an age range of 2 to 44 days, were selected. G6PD enzyme activity and gene mutation types were detected using fluorescence quantitative analysis, multicolor melting curve analysis (MMCA), and Sanger sequencing. Droplet digital PCR (ddPCR) was used for quantitative analysis of a newly identified variant family to determine the mutant allele proportion in family members. Meanwhile,the protein structure model and pathogenicity prediction of the novel variant were analyzed.Data analysis was conducted using SPSS 26.0. Specifically, chi-square tests were used for the detection rates of G6PD enzyme activity and gene mutations between different genders. One-way analysis of variance (ANOVA) was used for the comparison of enzyme activity among different mutation types.Results:Among 15 533 infants, 143 cases (129 males and 14 females) were tested positive for G6PD activity, with a detection rate of 0.92% (143/15 533). The difference in detection rates between males and females was statistically significant (χ 2=96.76, P<0.001). Out of 89 enzyme activity-positive cases (83 males and 6 females) underwent genetic testing, 77 (72 males and 5 females) were detected by MMCAand other 12 negative samples were underwent further Sanger sequencing, revealing mutations in 6 samples, all of which were males. Among the 83 individuals with gene mutations, 78 had heterozygous mutations, 1 had a homozygous mutation, and 4 had compound heterozygous mutations. A total of 12 mutation types were detected, with G6PD c.487G>A, c.1024C>T, c.1388G>A, and c.1376G>T being the most common, accounting for 74.70% (62/83) of all mutation types. The average G6PD enzyme activity of c.1376G>T was the lowest, and the differences were statistically significant compared to the average enzyme activity of the other three mutations ( P<0.05). One male infant with a newly identified G6PD c.242G>C mutation was detected, predicted to be pathogenic. ddPCR confirmed that the mother of the affected child was a c.242G>C mutant chimera, with a chimera proportion of 6.66%. Conclusions:In the Kunming region, the predominant G6PD deficiency gene mutation is c.487G>A, with the detection of a novel G6PD c.242G>C mutation. The application of ddPCR technology can assist in detecting the proportion of mutation chimeras.

Real-time monitoring of glucose-6-phosphate dehydrogenase activity using liquid droplet arrays and its application to human plasma samples.

Glucose-6-phosphate dehydrogenase (G6PD) regulates nicotinamide adenine dinucleotide phosphate (NADPH) levels and is related to the pathogenesis of various diseases, including G6PD deficiency, type 2 diabetes, aldosterone-induced endothelial dysfunction, and cancer. Therefore, a highly sensitive array-based assay for determining quantitative G6PD activity is required. Here, we developed an on-chip G6PD activity assay using liquid droplet fluorescence arrays. Quantitative G6PD activity was determined by calculating reduced resorufin concentrations in liquid droplets. The limit of detection (LOD) of this assay was 0.162 mU/ml (2.89 pM), which is much more sensitive than previous assays. We used our activity assay to determine kinetic parameters, including Michaelis-Menten constants (Km) and maximum rates of enzymatic reaction (Vmax) for NADP(+) and G6P, and half-maximal inhibitory concentrations (IC50). We successfully applied this new assay to determine G6PD activity in human plasma from normal healthy individuals (n=30) and patients with inflammation (n=30). The inflammatory group showed much higher G6PD activities than did the normal group (p<0.001), with a high area under the curve value of 0.939. Therefore, this new activity assay has the potential to be used for diagnosis of G6PD-associated diseases and utilizing kinetic studies.

Relationship among glucose-6-phosphate dehydrogenase (G-6-PD) activity, G-6-PD variants and reticulocytosis in neonates of northeast Thailand.

BACKGROUND: Misdiagnosis of G-6-PD deficiency in neonates, at risk of severe hemolytic episodes, extreme hyperbilirubinemia, and bilirubin encephalopathy, could possibly occur due to presence of reticulocytes, which contain higher amounts of G-6-PD than mature erythrocytes. G-6-PD mutations in the population might also affect G-6-PD activity. This study evaluated the relationship among G-6-PD activity, G-6-PD variants and reticulocytosis in northeastern Thai neonates. METHODS: Blood samples obtained from routine fluorescence spot test examination for G-6-PD deficiency were analyzed using a quantitative enzymatic assay and for common G-6-PD mutations by restriction fragment length polymorphism (RFLP)-PCR. Correlation between G-6-PD activity and percent reticulocytosis was determined. RESULTS: Among 106G-6-PD-deficient (G-6PD activity<7.0U/g Hb) neonates, no significant association is observed between G-6PD activity and percent reticulocytosis (r=0.125, p-value=0.201), but there is a weak correlation in G-6-PD-normal neonates (r=0.377, p-value=0.014). There is a high frequency of G-6-PD Viangchan in male hemizygous and female heterozygous G-6-PD-deficient and G-6-PD-normal neonates. CONCLUSIONS: A high reticulocytosis does not bias measurements of enzyme activity in G-6-PD-deficient neonates. Also, G-6-PD activity varies among female heterozygous neonates, and G-6-PD mutation analysis provides a reliable method to detect G-6-PD deficiency.

Genetic analysis of glucose-6-phosphate dehydrogenase deficiency among newborns in Zhejiang Province / 中国实用儿科杂志

OBJECTIVE: To analyze the characteristics of gene mutations of glucose-6-phosphate dehydrogenase(G6PD)deficiency in the neonates of Zhejiang Province,and discuss the genetic diversity. METHODS: A total of 2242G6PD values and blood stains on dry filter were collected from the children borned in Zhejiang Province with positive screening of G6PD deficiency between March 2015 and September 2017 in Neonatal Metabolic Screening Center of Zhejiang Province. Genomic DNA of the stains were extracted. Thirty-five gene mutation sites were detected by Mass ARRAY technology. The relationship between mutation gene sites and G6PD activity were analyzed by SPSS 22.0,taking PT,c.1388G>A,c.1024C>T,c.95 A>G,c.871G>A and c.392G>T accounted for 92.96%. There were statistically significant differences in G6PD activity of c.1376 G>T,c.1388G>A,c.1024 C>T,c.95A>G(PT has obvious regional characteristics. Using MassARRAY technology to detect specific mutation sites can be a choice as a second-level screening method for G6PD deficiency.