Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency

Objective:Glucose-6-phosphate isomerase(GPI)deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants.This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics,often posing challenges for precise diagnoses using conventional methods.To this end,this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family.Methods:The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis.Novel compound heterozygous variants of the GPI gene,c.174C>A(p.Asn58Lys)and c.1538G>T(p.Trp513Leu),were identified using whole-exome and Sanger sequencing.The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure.Results:By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study,we found that most variants were located in exons 3,4,12,and 18,with a few localized in exons 8,9,and 14.This study identified novel compound heterozygous variants associated with GPI deficiency.These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids.Conclusion:Early family-based sequencing analyses,especially for patients with congenital anemia,can help increase diagnostic accuracy for GPI deficiency,improve child healthcare,and enable genetic counseling.

Genome-wide Analysis of Glucose-6-phosphate Dehydrogenase(G6PDH) and Its Evolution in Eucalyptus grandsis

[Objective] The aim of this study was to perform genome-wide analysis of glucose-6-phosphate dehydrogenase（G6PDH） and reveal its evolution in Eucalyptus grandsis.[Method] The gene character,protein sequence and phylogenetic tree of G6PDH gene were analyzed by BLAST and other bioinformatics software within Eucalyptus grandsis whole genome database.[Result] Six G6PDH genes,including one cytomic type and five plastids,were detected in the E.grandsis genome.All the G6PDHs have conserved motifs of motif 1,motif 2,motif 3,motif 7,motif 9 and motif 11.Furthermore,promoter sequences of all E.grandsis G6PDH contain TATA box,enhancer,light-responsive,hormone-responsive and stress-responsive regulatory elements.[Conclusion] This study provided reference for the further revealing molecular function of E.grandsis G6PDH gene family

Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freez- ing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydro- genase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana ta- bacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.

Involvement of the circular RNA/microRNA/glucose-6-phosphate dehydrogenase axis in the pathological mechanism of hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is the third most common cause of cancer-related death worldwide with high mortality.The incidence of HCC is increasing in China.Abnormal activation of glucose-6-phosphate dehydrogenase(G6 PD)exists in all malignant tumors,including HCC,and is closely related to the development of HCC.In addition,the differential expression of non-coding RNAs is closely related to the development of HCC.This systematic review focuses on the relationship between G6 PD,HCC,and noncoding RNA,which form the basis for the circ RNA/mi RNA/G6 PD axis in HCC.The circular RNA(circ RNA)/micro RNA(mi RNA)/G6 PD axis is involved in development of HCC.We proposed that non-coding RNA molecules of the circ RNA/mi RNA/G6 PD axis may be novel biomarkers for the pathological diagnosis,prognosis,and targeted therapy of HCC.

Is glucose-6-phosphate dehydrogenase deficiency more prevalent in Carrion's disease endemic areas in Latin America?

Glucose-6-phosphate dehydrogenase(G6PD) is a cytoplasmic enzyme with an important function in cell oxidative damage prevention.Erythrocytes have a predisposition towards oxidized environments due to their lack of mitochondria,giving G6 PD a major role in its stability.G6 PD deficiency(G6PDd) is the most common enzyme deficiency in humans:it affects approximately 400 million individuals worldwide.The overall G6 PDd allele frequency across malaria endemic countries is estimated to be 8%.corresponding to approximately 220 million males and 133 million females.However,there are no reports on the prevalence of G6 PDd in Andean communities where bartonellosis is prevalent.

Prevalence of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in India: A Systematic Review

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the most common enzyme deficiency of human erythrocyte affecting more than 400 million people worldwide. In India, G6PD deficiency was first reported in 1963 and since then various investigations have been conducted across country. The objective of this work was to study the prevalence of G6PD deficiency in different ethnic, caste and linguistic groups of Indian population. A systematic search of published literature was undertaken and the wide variability of G6PD deficiency has been observed ranging from 0% - 30.7% among the different caste, ethnic, and linguistic groups of India. It was observed that the incidence of G6PD deficiency was found to be considerably higher among the tribes (9.86%) as compared to other ethnic groups (7.34%) and significantly higher in males as compared to females.

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Pigeon Pea (Cajanus cajan) Seeds

Glucose-6-phosphate dehydrogenase has been purified from pigeon pea (Cajanus cajan) seeds and subjected to characterization. The enzyme was purified 123.69 fold with a yield of 21.37% by ammonium sulphate fractionation, PEG-4000 precipitation, CM cellulose column chromatography and DEAE cellulose column chromatography. The catalytically active enzyme is a dimer of 113 KDa with a subunit molecular weight of 55 KDa. Thermal inactivation of enzyme follows first order kinetics at 30&#176C and 40&#176C with half life of 6 and 1.5 min respectively. Km value for glucose-6-phosphate and NADP+ was found to be 2.68 mM and 0.75 mM respectively whereas Vmax value was found to be 0.11 U/mL and 0.13 U/mL respectively. The enzyme shows more affinity towards NADP+ than glucose-6-phosphate. The pKa value was found to be 10.41 indicating that the amino acid residue at active site might be lysine. The enzyme exhibited maximum catalytic activity at pH 8.2. The enzyme was found to be highly thermosensitive with gradual loss of activity above 30&#176C temperature.

Sapium ellipticum(Hochst) Pax ethanol leaf extract modulates glucokinase and glucose-6-phosphatase activities in streptozotocin induced diabetic rats

Objective:To examine the effects of Sapium ellipticum(SE) leaf extract on the hepatic activities of glucokinase and glucose-6-phosphatase in streptozotocin-induced diabetic Wistar rats.Methods:STZ-induced diabetic Wistar rats(four groups,n = 8) were used in this study.SE was assessed at two different doses,400 and 800 mg/kg BW,in comparison with metformin(METF)(12 mg/kg BW) as a reference antidiabetic drug.All treatments were done orally(p.o),twice daily at 8 h interval for a period of 21 days.Glucokinase and glucose-6-phosphatase activities were respectively determined using standard protocols.Hepatic and muscle glycogen contents were estimated as well.Results:STZ caused significant decrease in glucose-6-phosphatase activity and concomitant increase in glucokinase activity.SE extract especially at 400 mg dosage significantly reversed the alterations by increasing glucokinase activity by 40.31% and inhibiting glucose-6-phosphatase activity by 37.29% compared to diabetic control animals.However,the effects were significantly lower than that of METF which enhanced glucokinase activity by94.76% and simultaneously inhibited glucose-6-phosphatase activity by 49.15%.The extract also improved hepatic glycogen level by 32.37 and 27.06% at 400 and 800 mg dosage respectively.HPLC-MS analysis of some SE fractions in dynamic MRM mode(using the optimized compound-specific parameters) revealed among other active compounds,the presence of amentoflavone,which has been associated with antidiabetic function.Conclusions:The ability of SE extract to concurrently inhibit glucose-6-phosphatase and activate glucokinase in this study suggests that it may be a treatment option for type 2 diabetes patients,and the presence of amentoflavone in the plant extract may account for its anti-diabetic potential.

Glucose-6-phosphate dehydrogenase(G6PD) deficiency is associated with asymptomatic malaria in a rural community in Burkina Faso

Objective:To investigate 4 combinations of mutations responsible for glucose-6—phosphate dehydrogenase(G6PD) deficiency in a rural community of Burkina Faso,a malaria endemic country.Methods:Two hundred individuals in a rural community were genotyped for the mutations A376 G.G202A,A542 T,G680T and T968 C using TaqMan single nucleotide polymorphism assays and polymerase chain reaction followed by restriction fragment length polymorphism.Results:The prevalence of the G6 PD deficiency was 9.5%,in the study population.It was significantly higher in men compared to women(14.23%vs 6.0%,P=0.049).The 202A/376 G G6PD Awas the only deficient variant detected.Plasmodium falciparum asymptomatic parasitemia was significantly higher among the C6PD-non—deficient persons compared to the G6PD-deficient(P<0.001).The asymptomatic parasitemia was also significantly higher among G(SPI) nondeficient compared to C6PD—heterozygous females(P<0.001).Conclusions:This study showed that the G6 PD A- variant associated with protection against asymptomatic malaria in Burkina Faso is probably the most common deficient variant.

Is there any role of glucose-6-phosphate dehydrogenase in obesity induced metabolic disorder

The present study was designed to explore the possible mechanism of obesity associated metabolic syndrome. 150 subjects (120 men and 30 women) in the age-group of 17 - 26 years were studied. Body Mass Index and Waist-to-Hip Ratio were taken as a measure of generalized obesity and abdominal adiposity. The serum concentration of glucose-6-phosphate dehydrogenase increased with increasing levels of Body Mass Index and was found to be significant in obese subjects (Body Mass Index ≥ 30.0 kg/m2) and more so in the obese subjects with abdominal adiposity (p = 0.002) as compared to normal-weight subjects. Karl Pearson coefficient of correlation revealed a significant positive correlation of glucose-6-phosphate dehydrogenase with Body Mass Index (r = 0.499;p < 0.001) and malondialdehyde (a biomarker of oxidative stress) (r = 0.736;p < 0.001) but inverse correlation with adiponectin (r = -0.524;p < 0.001). Thus, we conclude that increased expression of glucose-6-phosphate dehydrogenase in obese subjects (more if it is associated with abdominal adiposity) might mediate the onset of obesity associated metabolic disorders by increasing oxidative stress.

The diagnostic significance of glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibody in rheumatoid arthritis patients

Objective: To investigate whether glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibodies could be applied for the clinical diagnostic markers of rheumatoid arthritis (RA) and its associations with RA activity states. Methods: The levels of G6PI antigens and anti-G6PI Abs in sera from 176 RA patients in different states, 35 non-RA patients and 100 healthy donors and in synovia fluids from 33 patients and 11 non-RA patients were measured by ELISA. Results: The sensitivity and specificity of G6PI antigens in the RA patients were 75.0% and 93.3%, respectively. The levels of serum G6PI antigens in 176 RA patients were significantly higher than non-RA patients and the health controls. Especially, there was a significant difference between the active phase and the inactive phase in G6PI antigens levels. The levels of G6PI antigens in synovia fluid were also significantly higher in RA groups than in non-RA patients. With the values of the anti-G6PI Abs in sera, there were no marked differences among RA, non-RA patients and health controls. Also, there was no significant difference between the active phase and the inactive phase in RA patients. However, there were no significant differences of G6PI and anti-G6PI between RA patients and health controls in synovial fluid. Conclusions: G6PI is highly correlated with the activity states of RA, and could be applied for a clinical biomarker with high sensitivity and specificity for the diagnosis of RA.

HEXOKINASE, GLUCOSE-6-PHOSPHATASE DEHYDROGENASE AND ALEOSE REDUCTASE IN HUMAN FETAL LENSES

The lens HK, G6PD, AR activity and its relationship with fetal age was determined.There is a positive correlation between the age of fetus and the activity(IU/mg pro.) of HK and G6PD(r=0.8069, 0.8204, P<0.01) and a negetive correlation between the age of fetus and activity of AR(r=-0.810 1,0.05>P>0.01).

G6PT-H6PDH-11βHSD1 triad in the liver and its implication in the pathomechanism of the metabolic syndrome

The metabolic syndrome, one of the most common clinical conditions in recent times, represents a combination of cardiometabolic risk determinants, including central obesity, glucose intolerance, insulin resistance, dyslipidemia, non-alcoholic fatty liver disease and hypertension. Prevalence of the metabolic syndrome is rapidly increasing worldwide as a consequence of common overnutrition and consequent obesity. Although a unifying picture of the pathomechanism is still missing, the key role of the pre-receptor glucocorticoid activation has emerged recently. Local glucocorticoid activation is catalyzed by a triad composed of glucose-6-phosphate-transporter, hexose-6-phosphate dehydrogenase and 11β-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum. The elements of this system can be found in various cell types, including adipocytes and hepatocytes. While the contribution of glucocorticoid activation in adipose tissue to the pathomechanism of the metabolic syndrome has been well established, the relative importance of the hepatic process is less understood. This review summarizes the available data on the role of the hepatic triad and its role in the metabolic syndrome, by confronting experimental findings with clinical observations.

Rice Glucose 6-Phosphate/Phosphate Translocator 1 is required for tapetum function and pollen development

In plants, non-green plastids in heterotrophic tissues are sites for starch and fatty acids biosynthesis,which are essential for plant development and reproduction. Distinct from chloroplasts, the metabolites for these processes in non-green plastids have to be imported through specific transporters. Glucose 6-Phosphate/Phosphate Translocator 1 is required for the uptake of cytosolic Glucose 6-Phosphate into non-green plastids. In Arabidopsis, GPT1 has been demonstrated to play essential roles in male, female gametophyte and embryo development. However, the roles of GPTs in other species are yet largely unknown. Here, we reported that rice OsGPT1 is indispensable for normal tapetal degeneration and pollen exine formation during anther and pollen development. OsGPT1 is localized in the plastid and distributed in the anther wall layers and late-stage pollen grains. Different from the gametic defects caused by mutation in At GPT1, disruption of OsGPT1 does not affect male and female gamete transmission as well as embryo development. On the contrary, osgpt1 mutant exhibits delayed tapetum degeneration,decreased Ubisch bodies formation and thinner pollen exine, leading to pollen abortion at the mature stage. Furthermore, the expression of several genes involved in tapetal programmed cell death(PCD)and sporopollenin formation is decreased in osgpt1. Our study suggests that OsGPT1 coordinates the development of anther sporophytic tissues and the male gametophyte by integrating carbohydrate and fatty acid metabolism in the plastid.

Molecular Cloning of a Cytosolic G6PDH Gene from Populus Suaveolens and Its Expression to Improve the Cold Resistance of Tobacco Plants

Glucose-6-phosphate dehydrogenase (G6PDH,EC 1.1.1.49) is the first and main regulated enzyme of oxidative pentose phosphate pathway (OPPP),catalyzing the conversion of glucose-6-phosphate to 6-phospho-gluconolactone and playing important roles in the growth and development of plants. It is preciously reported that the enhancement of freezing resistance of Populus suaveolenscuttings is clear related to the distinct increase in cytosolic G6PDH activity. Here,a 1697 bp cDNA fragment (PsG6PDH) is amplified by RT-PCR from cold-induced total RNA of the freezing-tolerant P. suaveolens. A sequence analysis showed that PsG6PDH coding region had 1 530 bp and encoded 510 predicted amino acid residues. Genomic Southern analysis revealed that the isoform is encoded by a few copies of the gene in the poplar genome. The cloned gene PsG6PDHis cloned into binary vector pBI121 and used to transform tobacco. PCR and Southern blotting results verified integration of this gene into the genome of tobacco. Moreover,cold treatment experiments and membrane defense enzymeactivity analysis confirmed that overexpression of the PsG6PDHgene could enhance the tolerance to cold or frigid stresses in transgenic plants.

Etiology analysis for term newborns with severe hyperbilirubinemia in eastern Guangdong of China

BACKGROUND Neonatal hyperbilirubinemia is one of the common diseases of newborns that typically presents with yellow staining of skin,resulting in sequelaes such as hearing loss,motor and intellectual development disorders,and even death.The pathogenic factors of neonatal hyperbilirubinemia are complex.Different cases of hyperbilirubinemia may have a single or mixed etiology.AIM To explore the etiological characteristics of severe hyperbilirubinemia in term newborns of eastern Guangdong of China.METHODS Term newborns with severe hyperbilirubinemia in one hospital from January 2012 to December 2021 were retrospectively analyzed.The etiology was determined according to the laboratory results and clinical manifestations.RESULTS Among 1602 term newborns with hyperbilirubinemia in eastern Guangdong of China,32.20%(580/1602)was severe hyperbilirubinemia.Among the causes of severe hyperbilirubinemia,neonatal hemolysis accounted for 15.17%,breast milk jaundice accounted for 12.09%,infection accounted for 10.17%,glucose-6-phosphate dehydrogenase(G6PD)deficiency accounted for 9.14%,and the coexistence of multiple etiologies accounted for 6.55%,unknown etiology accounted for 41.72%.ABO hemolysis and G6PD deficiency were the most common causes in the 20 cases with bilirubin encephalopathy.94 severe hyperbilirubinemia newborns were tested for uridine diphosphate glucuronosyl transferase 1A1(UGT1A1)*6 variant(rs4148323,c.211G>A,p.Arg71Gly),9 cases were 211 G to A homozygous variant,37 cases were 211 G to A heterozygous variant,and 48 cases were wild genotypes.CONCLUSION The main cause for severe hyperbilirubinemia and bilirubin encephalopathy in eastern Guangdong of China were the hemolytic disease of the newborns,G6PD deficiency and infection.UGT1A1 gene variant was also a high-risk factor for neonatal hyperbilirubinemia.Targeted prevention and treatment according to the etiology may reduce the occurrence of bilirubin encephalopathy and kernicterus.

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.

Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of southwestern China

The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31% (19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2―13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen inde-pendent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping (1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.

Alleviation of PEGylated Puerarin on Erythrocyte Hemolysis Induced by Puerarin in Glucose-6-phosphate Dehydrogenase-deficient Rats

Objective To explore and analyze the reducing hemolytic effects of PEGylated puerarin (PEG-PUE) on erythrocytes induced by PUE in glucose-6-phosphate dehydrogenase (G6PD)-deficient rats. Methods The rat model with G6PD-deficiency was established via sc injecting 1% acetylphenyl-hydrazine. Then the G6PD-deficient erythrocyte suspension obtained from this rat model was used to evaluate the hemolytic effects of PUE and the reducing hemolytic effects of PEG-PUE via hemolytic activity and erythrocyte osmotic fragility assay. Results It was found that PUE could cause a serious hemolysis to the erythrocyte suspension with the increase of drug concentration and the prolongation of drug incubation time, the hemolytic rate of PUE was up to 40%, while the addition of PEG-PUE to the erythrocyte suspension revealed no significant hemolysis. Additionally, the result of erythrocyte osmotic fragility indicated that PEG-PUE exerted a slight effect on the erythrocyte membranes, and the NaCl concentration that induced 50% hemolysis (32 mmol/L) was about one-third PUE. Conclusion These results demonstrate that PEG-PUE could play a significant role in reducing the side effect of hemolysis induced by PUE. The low hemolytic activity of PEG-PUE makes it a favorable candidate for in vivo tests and PEG-PUE could also provide the useful insight for the further formulation development as an innovative drug.

Glucose-6-phosphate isomerase is associated with disease activity and declines in response to infliximab treatment in rheumatoid arthritis

Background:Rheumatoid arthritis (RA), a systemic autoimmune disease characterized by synovial inflammation, can cause cartilage and bone damage as well as disability. The aim of this study was to explore whether serum glucose-6-phosphate isomerase (GPI) is correlated with disease activity and the value of GPI in the evaluation of infliximab treatment in patients with RA.Methods:Sixty-two patients with RA who had an inadequate response to methotrexate (MTX) were enrolled in Peking University People’s Hospital from July 1, 2016 to July 31, 2018. Infliximab (3 mg/kg, intravenous at weeks 0, 2, and 6 and then every 8 weeks) was administered to patients with stable background MTX therapy. Serum samples were obtained at baseline and week 18. Serum GPI levels were determined using enzyme-linked immunosorbent assay. The associations between serum GPI levels and clinical features were analyzed.Results:Serum GPI was positively correlated with Disease Activity Score in 28 joints (DAS28), swollen joint count, tender joint count and C-reactive protein level ( P < 0.001, P < 0.001, P < 0.001, and P = 0.033, respectively). The change of DAS28 in GPI-positive patients was greater than that in GPI-negative patients ( P < 0.001). Compared with those for patients receiving MTX monotherapy at baseline, the GPI levels were significantly declined when MTX was combined with infliximab ( P < 0.001). Conclusion:Serum GPI is related to disease activity and clinical response to infliximab treatment.