Prevalence and molecular characterization of Glucose-6-Phosphate dehydrogenase deficient variants among the Kurdish population of Northern Iraq

Background  

Glucose-6-Phosphate dehydrogenase (G6PD) is a key enzyme of the pentose monophosphate pathway, and its deficiency is the most common inherited enzymopathy worldwide. G6PD deficiency is common among Iraqis, including those of the Kurdish ethnic group, however no study of significance has ever addressed the molecular basis of this disorder in this population. The aim of this study is to determine the prevalence of this enzymopathy and its molecular basis among Iraqi Kurds.     

Cytochemical flow analysis of intracellular G6PD and aggregate analysis of mosaic G6PD expression

Background

Medicines that exert oxidative pressure on red blood cells (RBC) can cause severe hemolysis in patients with glucose‐6‐phosphate dehydrogenase (G6PD) deficiency. Due to X‐chromosome inactivation, females heterozygous for G6PD with 1 allele encoding a G6PD‐deficient protein and the other a normal protein produce 2 RBC populations each expressing exclusively 1 allele. The G6PD mosaic is not captured with routine G6PD tests.

Methods

An open‐source software tool for G6PD cytofluorometric data interpretation is described. The tool interprets data in terms of % bright RBC, or cells with normal G6PD activity in specimens collected from 2 geographically and ethnically distinct populations, an African American cohort (USA) and a Karen and Burman ethnic cohort (Thailand) comprising 242 specimens including 89 heterozygous females.

Results

The tool allowed comparison of data across 2 laboratories and both populations. Hemizygous normal or deficient males and homozygous normal or deficient females cluster at narrow % bright cells with mean values of 96%, or 6% (males) and 97%, or 2% (females), respectively. Heterozygous females show a distribution of 10‐85% bright cells and a mean of 50%. The distributions are associated with the severity of the G6PD mutation.

Conclusions

Consistent cytofluorometric G6PD analysis facilitates interlaboratory comparison of cellular G6PD profiles and contributes to understanding primaquine‐associated hemolytic risk.     

Hemolytic anemia in hereditary pyrimidine 5'-nucleotidase deficiency: nucleotide inhibition of G6PD and the pentose phosphate shunt

We evaluated the erythrocytes of two patients with hereditary pyrimidine 5'-nucleotidase deficiency. Significant findings included an increased reduced glutathione content, increased incubated Heinz body formation, a positive ascorbate cyanide test, and decreased intraerythrocytic pH. The pentose phosphate shunt activity of the patients' red cells as measured by the release of 14CO2 from 14C-1- glucose was decreased compared to high reticulocyte controls. Glucose-6- phosphate dehydrogenase (G6PD) activity in hemolysates from control erythrocytes was inhibited 43% by 5.5 mM cytidine 5'-triphosphate (CTP) and 50% by 5.5 mM in uridine 5'-triphosphate (UTP) at pH 7.1. CTP was a competitive inhibitor for G6P (Ki = 1.7 mM) and a noncompetitive inhibitor for NADP+ (Ki = 7.8 mM). Glutathione peroxidase, glutathione reductase, and 6-phosphogluconate dehydrogenase were not affected by these compounds. Pentose phosphate shunt activity in control red cell hemolysate at pH 7.1 was inhibited to a similar degree by 5.5 mM CTP or UTP. Since the intracellular concentrations of G6P and NADP+ are below their KmS for G6PD, these data suggest that high concentrations of pyrimidine 5'-nucleotides depress pentose phosphate shunt activity in pyrimidin 5'-nucleotidase deficiency. Thus, this impairment of the pentose phosphate pathway appears to contribute to the pathogenesis of hemolysis in pyrimidine 5'-nucleotidase deficiency hemolytic anemia.     

Molecular characterization of glucose-6-phosphate dehydrogenase deficient variants in Baghdad city - Iraq

Background  

Although G6PD deficiency is the most common genetically determined blood disorder among Iraqis, its molecular basis has only recently been studied among the Kurds in North Iraq, while studies focusing on Arabs in other parts of Iraq are still absent.     

Loss of glucose 6-phosphate dehydrogenase function increases oxidative stress and glutaminolysis in metastasizing melanoma cells

The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To address this, we mutated the substrate binding site of glucose 6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors, but when these tumors spontaneously metastasized, the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species, decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing malic enzyme activity and glutamine consumption. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutaminase than control melanomas, both for oxidative stress management and anaplerosis. The oxidative pentose phosphate pathway, malic enzyme, and glutaminolysis thus confer layered protection against oxidative stress during metastasis.

The pentose phosphate pathway is an important source of NADPH for oxidative stress resistance (1–5). The oxidative branch of the pentose phosphate pathway contains two enzymes that generate NADPH from NADP⁺, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) (SI Appendix, Fig. S1). NADPH is an important source of reducing equivalents for oxidative stress resistance because it is used by cells to convert oxidized glutathione (GSSG) to glutathione (GSH), an abundant redox buffer. Complete deficiency for G6PD is embryonic-lethal in mice (2, 6, 7) but hypomorphic G6PD mutations are common in certain human populations, perhaps because they protect against malaria (8, 9). These partial loss-of-function G6PD mutations are well tolerated in adults, though they sensitize red blood cells to hemolysis from oxidative stress under certain circumstances (10).Several studies have reported a lower incidence and mortality for certain cancers in people with hypomorphic mutations in G6PD (11–14), suggesting that cancer cells depend upon G6PD to manage oxidative stress. Cells experience high levels of oxidative stress during certain phases of cancer development and progression, including during metastasis (15–17). Antioxidant mechanisms thus promote the survival of cells during oncogenic transformation (18, 19) as well as during metastasis (15, 16). For example, relative to primary cutaneous tumors, metastasizing melanoma cells exhibit increased dependence upon the folate pathway (15), monocarboxylate transporter-1 (MCT1) (20), and glutathione peroxidase-4 (GPX4) (21), each of which directly or indirectly attenuate oxidative stress. By better understanding the mechanisms that confer oxidative stress resistance in cancer cells, it may be possible to develop pro-oxidant therapies that inhibit cancer progression by exacerbating the oxidative stress experienced by cancer cells.G6PD (22) or PGD deficiency (23–25) reduce the growth of some cancers, including melanoma, but G6PD deficiency has little effect on primary tumor formation by K-Ras–driven epithelial cancers (26). This is at least partly because loss of G6PD in these cancers leads to compensatory increases in the function of other NADPH-generating enzymes, including malic enzyme and isocitrate dehydrogenase (1, 27). Nonetheless, pentose phosphate pathway function may increase during metastasis (20, 28–30) and higher G6PD expression is associated with worse outcomes in several cancers (31–33), raising the question of whether metastasizing cells are particularly dependent upon G6PD. G6PD is not essential for metastasis in a breast cancer cell line but it reduces their capacity to form metastatic tumors (26).Melanoma cells show little evidence of oxidative stress in established primary tumors but exhibit increased levels of reactive oxygen species (ROS) and dependence upon antioxidant mechanisms during metastasis (15, 20, 21). To test if these cells are more dependent upon the pentose phosphate pathway during metastasis, we generated three G6PD mutant melanomas, including two patient-derived xenografts and one human melanoma cell line. Reduced G6PD function had little effect on the formation or growth of primary subcutaneous tumors but significantly increased ROS levels and reduced spontaneous metastasis. G6PD mutant melanomas compensated by increasing malic enzyme activity and glutamine consumption, both to increase oxidative stress resistance and to replenish tricarboxylic acid (TCA) cycle intermediates through anaplerosis. Melanoma cells thus have redundant layers of protection against oxidative stress during metastasis, including the abilities to alter fuel consumption and antioxidant pathway utilization.     

Inhibition of the pentose phosphate shunt by lead: a potential mechanism for hemolysis in lead poisoning

Recent investigations have disclosed a decrease in pentose phosphate shunt activity in hereditary pyrimidine 5'-nucleotidase deficiency. Clinical lead poisoning is associated with an acquired decrease in pyrimidine 5'-nucleotidase activity. The current investigations were undertaken (1) to determine if pentose shunt activity was decreased in erythrocytes exposed to lead, and (2) to compare the mechanism of inhibition to that seen in hereditary pyrimidine 5'-nucleotidase deficiency. Normal erythrocytes incubated with lead acetate in vitro demonstrated increased Heinz body formation, decreased reduced glutathione, a positive ascorbate cyanide test, and a reversible suppression of pentose shunt activity in the intact erythrocyte. Lead acetate added to normal red cell hemolysates markedly inhibited the activities of glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase. The mean Kis of lead for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) for G6PD were 1.5 microM and 2.1 microM, respectively, which is within the range of intraerythrocytic lead concentrations found in clinical lead poisoning. Magnesium enhanced the ability of lead to inhibit G6PD. Thus, the shortened erythrocyte survival in lead poisoning appears to be due, in part, to increased oxidant sensitivity secondary to inhibition of G6PD and the pentose shunt. The mechanism of shunt inhibition is, in part, similar to that seen in hereditary pyrimidine 5'-nucleotidase deficiency.     

脂联素球状结构域对3T3-L1脂肪细胞磷酸戊糖途径关键酶表达的影响

目的:通过探讨脂联素球状结构域(gAd)对3T3-L1脂肪细胞磷酸戊糖途径关键酶表达的影响,进而探讨gAd促进脂肪细胞摄取的葡萄糖是否经磷酸戊糖途径代谢。方法:用gAd干预分化成熟的3T3-L1脂肪细胞,干预结束后测定细胞残液的葡萄糖浓度,并以实时荧光定量PCR(RT-PCR)法检测各组细胞磷酸戊糖途径关键酶葡萄糖-6-磷酸酶(G6PD)转录水平的表达情况,进行统计学分析。结果:各实验组细胞残液中葡萄糖浓度均显著低于对照组(均P     

Red cell and platelet‐derived microparticles are increased in G6PD‐deficient subjects

In response to oxidative stress and during apoptosis, cells often shed microparticles (MPs), submicron elements carrying phosphatidylserine and protein antigens. Glucose‐6‐phosphate dehydrogenase (G6PD)‐deficient cells are extremely sensitive to oxidative damage that may lead to the formation of MPs. To determine whether G6PD deficiency alters membrane phospholipid asymmetry and increases MPs production, we determined the concentrations and cellular origins of MPs in G6PD‐deficient individuals using flow cytometry. G6PD‐deficient individuals showed an increase in circulating MPs concentrations as compared with G6PD‐normal individuals [1051/μL (865–2532/μL) vs. 258/μL (235–575/μL), P < 0.01]. MPs concentrations were significantly increased with the severity of G6PD deficiency. Median MPs concentrations from individuals with severe G6PD deficiency, and individuals with moderate G6PD deficiency were 2567/μL (1216–2532/μL) and 984/μL (685–2107/μL), respectively (P < 0.01). Importantly, G6PD enzymatic activity was significantly correlated with MPs concentrations with r² = 0.731. MPs found in G6PD deficiency individuals were largely derived from red blood cells (RBCs) (45%) and platelets (30%). Additionally, Atomic Force Microscopy was used to study the morphology and measures the diameter of MPs found in G6PD‐deficient individuals. The mean (SD) width and height of RMPs were 0. 41 (0.18) and 2.04 (0.14) μm, respectively. Together, these results indicate that MP concentration is significantly correlated with G6PD enzymatic activity and is increased in G6PD‐deficient as compared with G6PD‐normal individuals. Our data also provide an evidence for an alteration in cell membrane associated with a decreased in G6PD activity. However, the significance of MPs in G6PD deficiency needs further clarification.     

Glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) is expressed in all tissues, where it catalyses the first step in the pentose phosphate pathway. G6PD deficiency is prevalent throughout tropical and subtropical regions of the world because of the protection it affords during malaria infection. Although most affected individuals are asymptomatic, there is a risk of neonatal jaundice and acute haemolytic anaemia, triggered by infection and the ingestion of certain drugs and broad beans (favism). A rare but more severe form of G6PD deficiency is found throughout the world and is associated with chronic non-spherocytic haemolytic anaemia. Many deficient variants of G6PD have been described. DNA sequence analysis has shown that the vast majority of these are caused by single amino acid substitutions. The three-dimensional structure of G6PD shows a classical dinucleotide binding domain and a novel beta + alpha domain involved in dimerization.     

Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation drives part of the excessive superoxide production implicated in the pathogenesis of heart failure. Pacing-induced heart failure was performed in eight chronically instrumented dogs. Seven normal dogs served as control. End-stage failure occurred after 28 +/- 1 days of pacing, when left ventricular end-diastolic pressure reached 25 mm Hg. In left ventricular tissue homogenates, spontaneous superoxide generation measured by lucigenin (5 microM) chemiluminescence was markedly increased in heart failure (1338 +/- 419 vs. 419 +/- 102 AU/mg protein, P < 0.05), as were NADPH levels (15.4 +/- 1.5 vs. 7.5 +/- 1.5 micromol/gww, P < 0.05). Superoxide production was further stimulated by the addition of NADPH. The NADPH oxidase inhibitor gp91(ds-tat) (50 microM) and the NO synthase inhibitor L-NAME (1 mM) both significantly lowered superoxide generation in failing heart homogenates by 80% and 76%, respectively. G6PD was upregulated and its activity higher in heart failure compared to control (0.61 +/- 0.10 vs. 0.24 +/- 0.03 nmol/min/mg protein, P < 0.05), while superoxide production decreased to normal levels in the presence of the G6PD inhibitor 6-aminonicotinamide. We conclude that the activation of myocardial G6PD is a novel mechanism that enhances NADPH availability and fuels superoxide-generating enzymes in heart failure.     

Favism: Erythrocyte Metabolism during Haemolysis and Reticulocytosis

The reduced activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate; NADP+ 1-oxidoreductase; G6PF) in Mediterranean erythrocytes explains the precarious equilibrium of the hexose monophosphate pathway (HMP) and the susceptibility of these cells to haemolytic agents. G6PD-deficient erythrocytes, in steady-state conditions, have a low NADPH/NADP+ ratio, thus allowing the HMP to operate at its maximal intracellular rate and to compensate the intrinsic erythrocyte enzyme deficiency. Studies started soon after accidental intake of fava beans by sensitive G6PD-deficient subjects demonstrate a decrease of both NADPH/NADP+ ratio and reduced glutathione. The metabolic effects induced by fava beans may be attributed to oxidative stress probably associated with an inhibitor effect of some unknown metabolite on the HMP. The availability of erythrocytes from subjects recovering from haemolysis with high reticulocyte counts and increased G6PD activity, provides new information on the rate of synthesis as well as on the in vivo decay of the mutant enzyme. Correlation of G6PD activity to reticulocyte count and extrapolation to an ideally homogenous population of reticulocytes reveal that the mutant enzyme is synthesized at a nearly normal rate. Furthermore, the present correlation allows an estimate of the in vivo half-life of Mediterranean G6PD. The rate of decline of about 8 d observed in this study well correlates to the intracellular metabolic aspects of G6PD Mediterranean erythrocytes.     

Mechanism of inhibition of growth of 3T3-L1 fibroblasts and their differentiation to adipocytes by dehydroepiandrosterone and related steroids: role of glucose-6-phosphate dehydrogenase.

Dehydroepiandrosterone (DHEA) and certain structural analogues block the differentiation of 3T3-L1 mouse embryo fibroblasts to adipocytes. These steroids also are potent uncompetitive inhibitors of mammalian glucose-6-phosphate dehydrogenases (G6PDs). We provide direct evidence that treatment of the 3T3-L1 cells with DHEA and its analogues results in intracellular inhibition of G6PD, which is associated with the block of differentiation: (i) Levels of 6-phosphogluconate and other products of the pentose phosphate pathway are decreased; (ii) the magnitude of these decreases depends on the potency of steroids as inhibitors of G6PD and on concentration and duration of exposure, and it is accompanied by a proportionate block of differentiation; (iii) in cells exposed to 16 alpha-bromoepiandrosterone (a more potent inhibitor of G6PD than DHEA) at concentrations that block differentiation, introduction of exogenous 6-phosphogluconate in liposomes raises the levels of 6-phosphogluconate and other products of the pentose phosphate pathway and partially relieves the steroid block of cell growth and differentiation.     

Iron deficiency in chronic kidney disease patients with diabetes mellitus

Backgrounds

Iron deficiency has been studied extensively in patients with chronic kidney disease on hemodialysis therapy. However, few studies looked at iron treatment in the non-dialysis chronic kidney disease population.

Platelet-Function Studies in Patients with Glucose-6-Phosphate Dehydrogenase Deficiency

S ummary . Tests of platelet function were carried out in 17 patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Despite the clinical absence of defective haemostasis impaired platelet function was observed in all patients studied. This consisted of reduced platelet-factor 3 (PF 3) availability, a reduced prothrombin-consumption time, and/or reduced platelet retention in a glass bead column. PF3 assay of freeze-thawed platelets demonstrated that this reduction of PF3 availability represents a true deficiency rather than defective release. Platelet aggregation with ADP, collagen and adrenaline was normal. These findings suggest that G6PD deficiency, which leads to impaired NADPH production, may cause reduced fatty-acid and phospholipid synthesis by the platelets resulting in PF3 deficiency. The normal platelet aggregation suggests that energy derived from the hexose-moaophospliatc shunt is not essential for this function.     

Two new class III G6PD variants [G6PD Tunis (c.920A > C: p.307Gln > Pro) and G6PD Nefza (c.968T > C: p.323 Leu > Pro)] and overview of the spectrum of mutations in Tunisia

We screened 423 patients referred to our laboratory after hemolysis triggered by fava beans ingestion, neonatal jaundice or drug hemolysis. Others were asymptomatic but belonged to a family with a history of G6PD deficiency. The determination of enzymatic activity using spectrophotometric method, revealed 293 deficient (143 males and 150 females). The molecular analysis was performed by a combination of PCR-RFLP and DNA sequencing to characterize the mutations causing G6PD deficiency. 14 different genotypes have been identified : G6PD A^? (376A > G;202G > A) (46.07%) and G6PD Med (33.10%) were the most common variants followed by G6PD Santamaria (5.80%), G6PD Kaiping (3.75%), the association [c.1311T and IVS11 93c] (3.75%), G6PD Chatham (2.04%), G6PD Aures (1.70%), G6PD A^? Betica (0.68%), the association [ 376G;c.1311T;IVS11 93c] (0.68%), G6PD Malaga, G6PD Canton and G6PD Abeno respectively (0.34%). Two novel missense mutations were identified (c.920A > C: p.307Gln > Pro and c.968T > C: p.323 Leu > Pro). We designated these two class III variants as G6PD Tunis and G6PD Nefza. A mechanism which could account for the defective activity is discussed.     

Decreased blood activity of glucose-6-phosphate dehydrogenase associates with increased risk for diabetes mellitus

Glucose-6-phosphate dehydrogenase (G6PD) deficiency predisposes affected individuals highly susceptible to oxidative stress, which is one of the risk factors for diabetes. To evaluate the relationship between blood level of G6PD activity and diabetes in Taiwan, blood G6PD activity was analyzed among 237 patients with diabetes and 656 healthy subjects. A significant difference in the distribution of G6PD activities as grouped by an increment of 100 U/10¹² red blood cells (RBCs) was observed between diabetic patients and healthy subjects. The odds ratio for diabetes was 1.46 (95% confidence interval=1.11−1.92) for every decrement of 100 U/10¹² RBC G6PD activities in these subjects. The data indicate that low G6PD activity is another risk factor for diabetes.     

Regulation of the pentose phosphate pathway in human astrocytes and gliomas

Several aspects of the regulation of the pentose phosphate pathway were examined in cultured normal human cortical astrocytes and gliomas of pathological grades I-IV. The generation of radiolabeled CO₂ from [l-¹⁴C]glucose by the oxidative arm of the pentose phosphate pathway is a saturable process and has a maximum flux rate of 8–9 nmol/hr/mg cell protein. The flux can be blocked by the glycolytic inhibitor iodoacetamide but is unaffected by agents which inhibit oxidative phosphorylation. The magnitude of the pentose phosphate flux is directly related to the glioma grade. Grade IV gliomas (glioblastoma) show a pentose phosphate flux rate of approximately 4% of the total glucose flux. The flux rate can be increased by pharmacological agents which decrease the NADPH/NADP⁺ ratio. Both the activity and the regulation of glioma glucose-6-phosphate dehydrogenase (G6PDH) are altered in high-grade gliomas. While the affinity constants for cofactors in whole homogenates were not significantly different in glioma or normal astrocyte homogenates, normal astrocytes have a lower K_m for glucose-6-phosphate and a G6PDH activity which is 10-fold greater than that of gliomas. NADPH is a powerful regulator of G6PDH activity in the normal astrocytes and in gliomas. At a NADPH/NADP⁺ ratio of 7:1 the normal astrocyte G6PDH is entirely inhibited, while the glioma enzyme is only 70% inhibited even at a ratio of20: 1. Increased metabolic flux through the oxidative arm of the pentose phosphate pathway is apparently due to an altered form of G6PDH.     

A Contradiction Between in vivo and in vitro Activities of Normal and Variant Glucose 6-Phosphate Dehydrogenase

The presumed “physiologic activity” of normal glucose 6-phos-phate dehydrogenase (G6PD), i.e. activity assayed in the presence of physiologic concentrations of ATP, 2,3-diphosphoglycerate, glucose 6-phosphate, NADP and NADPH in the normal red cells, is comparable to shunt pathway activity of intact normal red cells. In contrast, the presumed “physiologic activity” of G6PD variants associated with enzyme deficiency (Gd A® and Gd Mediterranean) is one order of magnitude higher than actual shunt pathway activity of the variant red cells. The difference of kinetic parameters of the enzyme in artificial buffer media and presumed physiologic medium in red cells cannot explain the discrepancy. The apparent discrepancy could be attributed either to (a) over-estimation of NADP and under-estimation of NADPH concentrations in the G6PD deficient red cells due to rapid oxidation of NADPH to NADP in preparation of hemolysate, or to (b) a large portion of NADP which accumulated in the G6PD deficient red cells binds with cellular components of intact red cells and is not available as substrate for G6PD.

Whatever the mechanisms are, the balance of NADP and NADPH is a major factor for regulating shunt pathway activity, and the sensitivity of NADPH inhibition (i.e. K_m for NADP versus K_i for NADPH) can be considered as an important factor in differentiating hemolytic severity of several Gd variants.