Glucose-6-phosphate Dehydrogenase Mutations Research Articles

The effects of glucose-6-phosphate dehydrogenase deficiency on benzene-induced hematotoxicity in mice

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme deficiency. Our previous study revealed the level of G6PD changed in wild type (WT) mice after benzene exposure. In this study, the pentose phosphate pathway (PPP) in regulation of benzene-induced hematotoxicity was investigated and other potential pathways were discovered in a G6PD deficiency mouse model. WT and G6PD mutation (G6PDmut) mice were exposed to benzene (diluted in corn oil) at doses of 0 and 160 mg/kg by subcutaneous injection for 5 days/week, 4 weeks. Peripheral blood samples and bone marrow cells (BMCs) were obtained and measured. The levels of nicotinamide adenine dinucleotide phosphate (NADPH),reduced glutathione (GSH) and malondialdehyde (MDA) were detected and comet assay was analyzed for DNA damage in BMCs. Finally, RNA sequencing (RNA-seq) of BMCs was performed. The results showed that white blood cells decreased significantly in G6PDmut mice compared with WT mice after benzene treatment. The ratio of hematopoietic stem/progenitor cells significantly decreased in G6PDmut mice exposed to benzene. The reduction of NADPH and GSH revealed the effect on PPP with G6PD deficiency, which then caused the increase of MDA and DNA damage. Finally, RNA-seq results suggested potential genes including SHROOM4, CAMK2B and REN1 played potential roles of G6PD deficiency on benzene-induced hematotoxicity. Renin-angiotensin system and cAMP signaling pathway were potentially involved in the process. Our study provides a better understanding for the effects of G6PD deficiency on benzene-induced hematotoxicity.

Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.

Genotype-Phenotype Correlation of G6PD Mutations among Central Thai Children with G6PD Deficiency.

Background Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked inherited erythroenzymopathy in Thailand. The clinical and hematological manifestations of G6PD deficiency are variable. Objective This study aimed to characterize the genotype-phenotype correlation of G6PD mutations in Thai pediatric patients who were followed-up in Phramongkutklao Hospital, a tertiary center in central Thailand. Material and Method. A total of 102 children including 73 males (71.6%) and 29 females (28.4%) were included in our study. Mutation analysis was performed by direct DNA sequencing of all coding exons of the G6PD gene. Ninety-one patients (89.2%) were presented with neonatal hyperbilirubinemia and 11 patients (10.8%) were presented with acute hemolytic anemia beyond the neonatal period. Results Molecular analysis of the G6PD gene in 102 G6PD-deficient Thai children identified 12 different mutations. G6PD Viangchan (871G > A) and G6PD Canton (1376G > T) were the first (46.2%) and the second (15.4%) most common identified mutations among both male and female G6PD-deficient individuals, respectively. All affected males were hemizygous for G6PD mutations and had an average G6PD level of 16.7 ± 11.5 (3–76) IU/ml.RBC. Majority of female patients (27 in 29, 93.1%) were heterozygous for G6PD mutations and had an average G6PD level of 133.6 ± 43.4 (9–195) IU/ml.RBC. Two female patients (6.9%) were either homozygous or compound heterozygous for the mutations and had G6PD level in the affected male range (35 and 10 IU/ml.RBC). Only 1 in 27 heterozygous females (3.7%) had G6PD level in the affected male range (9 IU/ml.RBC) which is possibly explained by nonrandom X-chromosome inactivation. The correlation of genotypes, G6PD levels, and clinical phenotypes was not demonstrated in our study in which all of the included G6PD-deficient patients were presented with neonatal hyperbilirubinemia and acute hemolytic anemia, since the genotype-phenotype correlation is normally demonstrated in chronic nonspherocytic hemolytic anemia (CNSHA) G6PD-deficient individuals. Conclusion This study characterizes the molecular heterogeneity of G6PD variants causing G6PD deficiency in Thai children. Our study demonstrated the efficiency of direct DNA sequencing which can identify 12 missense mutations in Thai children.

Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C.

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is a hereditary disease caused by pathogenic mutations of G6PD. While most of the pathogenic variants of G6PD have been annotated, hemolysis of unknown etiology but analogous to that in G6PD deficiency persists, implying the existence of undocumented pathogenic variants. In our previous study, we reported four novel G6PD variants in China, for which the pathogenicity remains to be verified.MethodsThe variants were verified by exogenous expression in HEK-293 cells, and their functions were predicted by PolyPhen-2 and SIFT. The CRISPR/Cas9 system was exploited to edit the G6PD c.697G>C variant in HEK-293 cells and K562 cells. The expression of G6PD was detected by quantitative PCR (qPCR) and western blotting. The cell growth capacity was detected by the CCK-8 assay and crystal violet staining. The G6PD enzyme activity was reflected by the G6P/6PG ratio test. The apoptosis of cells was detected by Annexin V-APC/7-AAD staining. The secondary and crystallographic structures were denoted according to the literature and PyMOL software. The G6PD protein was purified from lysis of transformed Escherichia coli (E. coli) cell with Ni-charged Resin Column. The enzymatic activity was detected at different temperatures.ResultsThe G6PD activity of exogenous G6PD c.697G>C in HEK-293 cells was significantly lower than that of wild type (WT) G6PD, a finding that was consistent with the observation in clinical samples. The functional predictions conducted by different algorithms indicated the damage role of the G6PD c.697G>C variant in its enzymatic activity. We recapitulated the G6PD c.697G>C variant both in HEK-293 cells and K562 cells by adapting the CRISPR/Cas9 strategy. Using distinct cell lines expressing the G6PD c.697G>C variant endogenously, we confirmed the deteriorative role of the G6PD c.697G>C variant in its enzymatic activity. Regarding the secondary and crystallographic structure, we found a mutated amino acid approaching the structural NADP+ binding site. Finally, we demonstrated the c.697G>C variant compromised the thermal stability of G6PD protein.ConclusionsOur data delineated the pathogenic role of G6PD c.697G>C variant for G6PD deficiency, implying the wide usage of CRISPR/Cas9 for genetic disease research.

A pilot study of expanded newborn screening for 573 genes related to severe inherited disorders in China: results from 1,127 newborns.

BackgroundNewborn screening (NBS) in China is mainly aimed at detecting biochemical levels of metabolites in the blood, which may generate false-positive/negative results. Current biochemical NBS includes tandem mass spectrometry (MS/MS) screening for metabolites as well as phenylalanine (Phe), thyroid-stimulating hormone (TSH), 17-α-hydroxyprogesterone (17-OHP), and glucose-6-phosphate dehydrogenase (G6PD) test. This study intended to explore whether next-generation sequencing (NGS) for dried blood spots combining with biochemical screening could improve the current screening efficiency and to investigate the carrier frequencies of mutations in causative genes related to amino acid metabolism, organic acid metabolism, and fatty acid oxidation in this cohort.MethodsWe designed a panel of 573 genes related to severe inherited disorders and performed NGS in 1,127 individuals who had undergone biochemical NBS. The NGS screening results of neonates were used to compare with the biochemical results.ResultsNGS screening results revealed that all the four newborns with abnormal G6PD values carried hemizygous G6PD mutations, which were consistent with the decreased G6PD enzymatic activity. The NGS results revealed an individual with compound heterozygous mutations of SLC22A5, who was biochemically negative in 2016. The MS/MS screening results in 2019 showed free carnitine deficiency, which was consistent with the genetic findings. The top five genes with the highest carrier frequencies of mutations in these newborns were PAH (1:56, 1.79%), ETFDH (1:81, 1.23%), MMACHC (1:87, 1.15%), SLC25A13 (1:102, 0.98%), and GCDH (1:125, 0.80%).ConclusionsOur study highlighted that combining NGS screening with biochemical screening could improve the current NBS efficiency. This is the first study to investigate carrier frequencies of mutations in 77 genes causing inherited metabolic diseases (IMDs) in China.

DETERMINATION OF STRUCTURAL STABILITY AND CATALYTIC ACTIVITY OF G6PD VARIANTS USING MOLECULAR DYNAMIC SIMULATION (MDS) APPROACH

BackgroundGlucose‐6‐phosphate dehydrogenase (G6PD) is a known housekeeping enzyme that is important in the pentose phosphate pathway (PPP) and essential for basic cellular function. G6PD also aids in producing compounds to prevent build‐up of reactive oxygen species (ROS) within the red blood cells. Glucose‐6‐phosphate dehydrogenase deficiency (G6PDD) is a known hereditary mutation and X‐linked recessive genetic disorder that have affected over 400 million people worldwide. More than 186 G6PD variants have been characterized that affect enzyme catalytic activities and biochemical reactions leading to various clinical phenotypes.ObjectivesThe intention of this study was to investigate the genotype‐phenotype correlations of selected G6PD mutations via structural analysis and molecular dynamic simulation analysis (MDSA) tools.MethodESPript 3.0 software was used for multiple sequence alignments of G6PD from human and from other organisms. PyMOL molecular visualization tool was also used to shown and evaluate structural changes in ten selected variants of the protein.ResultsMultiple sequence alignment of G6PD from human and three other organisms, namely; Trypanasoma cruzi, Mycobacterium avium and Leuconostoc mesenteroides revealed three conserved regions. The findings of this study suggested that mutation to residues that are unable to adopt a cis conformation would lead to an extremely high activation energy barrier for the enzyme to adopt the correct transition state during the enzymatic reaction. The protein was subjected to several computational approaches as described in previous part. The simulation data from the molecular dynamic simulation helps to determine the protein dynamics through stability and flexibility of G6PD native and mutation.The native G6PD have high RMSD average value than Class I, class II and class III mutation, thus, have low structure stability. The G163D has the lowest RMSD value thus have the most stable structure through simulation. From RMSF analysis, the residue at 310 until 320 (loop 1), 374 until 388 (loop2), 408 until 414 (loop 3) and 424 until 434 (loop 4) responsible for structure flexibility and stability. The loop regions produce larger fluctuation thus, providing the flexibility to the structure.Findings from molecular dynamic analysis reported here will be more valuable if comparison to kinetic data of the mutants are made. Simulation of the enzyme dimer form could stipulate the relationship between structural changes and enzymatic activity of G6PD variants.Support or Funding InformationResearch Center, King Fahad Medical City, Riyadh, KSA

Evaluation of the Functional Effects of an African American Glucose-6-Phosphate Dehydrogenase (G6PD) Polymorphism (Val68Met) on RBC Hemolytic Propensity and Post-Transfusion Recovery in a Humanized Mouse Model

Background: Growing evidence suggest that donor genetic variation is associated with RBCs storage integrity and post-transfusion recovery. In collaboration with the REDS III program, we performed a large-scale genome-wide association study (GWAS) study in ~13,000 healthy human blood donors, which demonstrated that RBCs with an African American G6PD-deficient A- variant (rs1050828, Val68Met) was associated with higher oxidative hemolysis after cold storage in normal volunteers. Despite a high prevalence of X-linked G6PD mutation in African American population (> 10%), blood donors are not routinely checked for G6PD deficiency and its importance in transfusion medicine is relatively understudied. We hypothesize that G6PD V68M SNP is associated with G6PD deficiency and modulates RBCs in vitro hemolytic propensity and in vivo post-transfusion recovery. Methods and Results: CRISPR-Cas9 technology was used to create non-synonymous human G6PD X-linked SNP (Val68Met) in C57B6 mice, and the desired genotypes were isolated by strategic back-crossing and sequential genotyping to ensure passage of SNP. G6PD enzymatic activity in erythrocytes was measured in fresh collected blood using a colorimetric assay kit. The predisposition of fresh and stored RBCs (after 11 days cold storage) to hemolysis was evaluated by subjecting washed mouse RBCs to selected stress assays, including osmotic fragility, mechanical fragility, and oxidative hemolysis using AAPH, diamide or H2O2. Hemolysis was measured by detection of supernatant cell-free hemoglobin using Drabkin's assay. Hematological values were measured using a Hemavet® 950 Hematology Analyzer System. Reticulocyte count was obtained using thiazole orange staining and analyzed by flow cytometry. We found severe disruption of G6PD enzymatic activity in erythrocytes from G6PD V68M SNP mice compared to WT mice (~5% residual activity in hemizygous male and ~60% in heterozygous female mice). Significant increased oxidative hemolysis was observed in both fresh and stored mouse RBCs with G6PD SNP, consistent with the GWAS study in human. G6PD V68M SNP hemizygous male mice had higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) compared to WT mice. However, no difference was observed in storage hemolysis, osmotic fragility, mechanical fragility and reticulocyte counts. Transfusion experiments with stored red blood cells from G6PD hemizygote males into GFP positive recipients will evaluate red blood cell recovery and half-life after standard cold storage and transfusion. Conclusions: We successfully generated a novel mouse strain carrying a "humanized" African American G6PD V68M variant which resembles the phenotype of humans with G6PD deficiency and increased oxidative hemolysis. Studies are undertaken to further investigate the effects of G6PD V68M SNP on RBCs structure, functions and in vivo post-transfusion recovery. Disclosures Gladwin: Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

PB1967 HEREDITARY ELLYPTOCYTOSIS ASSOCIATED WITH HETEROZYGOTE PYRUVATE KINASE AND GLUCOSE‐6‐PHOSPHATE DEHIDROGENASE MUTATIONS. A CASE REPORT AND FAMILY STUDY

Background:Hereditary elliptocytosis (HE) is a red blood cell (RBC) defect characterized by an abnormal shape and mild or moderate chronic haemolytic anaemia. Most cases of HE are due to missense mutations in the RBC membrane skeleton α‐spectrin (SPTA1) gene that encodes α‐spectrin chains, a critical component that maintains cell shape and membrane elasticity. Families carrying α‐HE alleles are in general symptomless or exhibit a mild to moderate haemolysis whereas the more severely affected individuals have been observed in hereditary pyropoikilocytosis (HPP).Aims:The main objective of our task is to provide a fast and efficient diagnosis of Hereditary Haemolytic Anaemia (HHA) using Next Generation Sequencing (NGS).Methods:A NGS analysis including a panel of 35 genes responsible for membranophaty (ANK1, EPB41, EPB42, SLC4A1, SPTB, SPTA1), haemoglobinopathy (HBA1, HBA2, HBB), enzymopathy (ADA, AK1, ALDOA, BPBM, CYB55, G6PD, GCLC, GPI, GSR, GSS, HK1, NT5C3A, PFKM, PGK1,) and CDA (CDAN1, C15orf41, SEC23B, KLF1, GATA1, KIF23) was performed using an Illumina HiSeq2000 device.Results:Two identical twins were prematurely born at 34 weeks of gestation and their parents, a 41‐year‐old women and a 45‐year‐old man were healthy and did not exhibit medical history of interest. After the 10th day of life, an acute severe haemolytic crisis, of unknown origin, appeared in both premature siblings (Hb:70 g/l, reticulocytes:161,6x10^9/l; and Hb 81 g/L, reticulocytes 151x109/L respectively). RBC morphology exhibited unspecific characteristics, and in order to seek for a diagnosis, both parents were evaluated. In the mother, a slight well‐compensated haemolysis (Hb:110 g/l, reticulocytes:102 x109/l) with about 25% of elliptocytes and ovalocytes was found, whereas the father was normal. After NGS study, four different variants were identified in the mother: a) Two α‐spectrin variants in a double heterozygous condition: SPTA1 c.779T>C (p.Leu260Pro), and SPTA1 (c.6531‐12GC>A) α‐LELY), b) A pyruvate kinase gene (PKLR) variant: c.829G>A (p.Glu277Lys); and c) A glucose‐6‐phosphate dehydrogenase (G6PD) A gene variant: c.968T>C (p.Leu323Pro).The SPTA1 (c.779T>C), PKLR (c.829G>A) and G6PD (c.968T>C) mutations, in heterozygous condition, were also found in both children. No gene variants were found in the father.Summary/Conclusion:We present a typical case of symptomless HE in a woman that was studied with NGS due to the severe neonatal haemolytic crisis found in her both premature siblings. Despite this NHC was initially attributed to the mother's disease (HE), the NGS allowed to found two additional mutations of RBC enzyme genes (PK and G6PD) in both siblings but also in the mother that may be the cause of the unexpected crisis. However, despite in the mother the SPTA1 c.779T>C mutation seems to be aggravated by the coexistence, in trans, of the α‐LELY allele (αHE/αLELY diplotype), this was not the case in the siblings because they inherited the α‐LELY allele but not the elliptogenic mutation. Accordingly, after the NGS study it has to be assumed that both proband's sons are at risk allowing to suggest that G6PD deficiency as the mechanism of the NHC observed in both of themThe present report is an example of the utility of NGS to distinguish potential genetic mechanisms of HHA, and to find unexpected genetic variants or mutations in patients with slight or moderate phenotypic expression of the disease. This provides a new challenge to investigate non‐phenotypically suspected complex genetic situations, and likewise to the improvement of the diagnosis and genetic counselling of patients with rare anaemias.image

PB1968 TWO NEW MUTATIONS AND MOLECULAR CHARACTERIZATION OF G6PD GENE AMONG THAI CHILDREN WITH G6PD DEFICIENCY

Background:Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is the most common X‐linked inherited erythroenzymopathy in Thailand. The clinical and hematological manifestations of G6PD deficiency is variable.Aims:This study aimed to characterize the spectrum of G6PD gene mutations in Thai pediatric patients who were followed‐up in Phramongkutklao Hospital, a tertiary center in central Thailand.Methods:A total of 102 children including 73 males (71.6%) and 29 females (28.4%) were included in our study. Mutation analysis was performed by direct DNA sequencing of the G6PD gene. Neonatal hyperbilirubinemia was the most common (87.3%) clinical manifestation in this study.Results:Molecular analysis of G6PD gene in 102 G6PD‐deficient Thai children identified a total of 12 mutational types. G6PD Viangchan (871G>A) was the most common (46.2%) identified mutation. G6PD Canton (1376G > T) was the second most common and was identified in 15.4%, followed by G6PD Kaiping (1388G > A) 14.4% and G6PD Mahidol (487G > A) 8.6%, respectively. Eight uncommon G6PD mutations including G6PD Quing Yan (392G > T) (3.8%), G6PD Aures (143C > T) (1.9%), G6PD Coimbra (592C > T) (1.9%), G6PD Songklanagarind (196T > A) (1.9%), G6PD Union (1360C > T) (1.9%), G6PD Valladolid (406C > T) (1.9%), G6PD Chinese‐5 (1024C > T) (1%) and G6PD Mediterranean (563C > T) (1%) were identified in this study. G6PD Valladolid and G6PD Aures were firstly identified in Thai populations.Summary/Conclusion:This study characterizes the molecular heterogeneity of G6PD variants causing G6PD deficiency in Thai children. Our study demonstrated the efficiency of direct DNA sequencing which can identify mutations in all 102 patients.

Reference levels for glucose-6-phosphate dehydrogenase enzyme activity in infants 7–90 days old in Taiwan

Nationwide newborn screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency has been implemented in Taiwan since 1987 and the G6PD enzyme activity levels were applied for diagnosis confirmation. As the reference value of G6PD enzyme activity was not available for infants aged 7-90 days, this study was performed to determine the enzyme level in different genotypes. Between January 1, 2016 and June 30, 2017, 410 term infants aged 7-90 days old visiting National Taiwan University Hospital Hsinchu branch were enrolled. The comparisons of G6PD enzyme activities among genotype groups were performed. G6PD enzyme activity was negatively correlated with age (R=-0.212, p=0.01). For infants under 30 days of age, the G6PD enzyme activity levels were 1.4±0.9U/g Hb in hemizygotes (n=76), 6.5±2.0U/g Hb in heterozygotes (n=47), and 13.6±3.7U/g Hb in those without G6PD mutations (n=70). Among infants more than 30 days old, G6PD enzyme activity levels were 0.9±0.5U/g Hb in hemizygotes (n=46), 6.0±2.7U/g Hb in heterozygotes (n=23), and 11.7±3.4U/g Hb in those without G6PD mutations (n=148). G6PD levels differed significantly among the groups defined by genotypes. We determined G6PD enzyme activity levels in infants aged between 7 and 90 days in Taiwan. Completing the reference data and determining the cutoff values for different G6PD deficiency disease statuses will help pediatricians to make accurate diagnoses.

Genetic mutation screening of glucose-6-phosphate dehydrogenase deficiency in Dongguan district

To determine the incidence and genotypes of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Dongguan region of Guangdong Province and assess the efficacy and feasibility of flow-through hybridization. Peripheral blood samples were randomly selected and detected by modified G6PD/6PGD ratio method. Flow-through hybridization was used to detect 14 G6PD mutations among all samples. In total 1005 samples were collected, the detection rate for modified G6PD/6PGD ratio method and flow-through hybridization were 2.79% and 20.90%, respectively. The consistency of the two methods was poor(Kappa=0.187). When c.1311C>T mutation is excluded, the consistency of the two methods was good for males (Kappa=0.952) but still poor for females (Kappa=0.194). The most common mutations were c.1376G>T, c.1388G>A and c.95A>G. No G6PD deficiency was found among those only carrying the c.1311C>T mutation. Flow-through hybridization can simultaneously detect 14 loci, covering over 90% of common mutations in Chinese population, and can be easily expanded. The routine method may miss many females carrying homozygous, compound heterozygous and heterozygous mutations, but the detection rate for male hemizygous mutation was much higher.

Increased Methylation of Deamidated Asparagines and Aspartates in Stored Red Blood Cells from Glucose 6-Phosphate Dehydrogenase-Deficient Blood Donors

Blood transfusion is a life-saving intervention for millions of recipients worldwide every year. However, refrigerated storage of red blood cells (RBCs) for up to 42 days promotes impairments in energy and redox homeostasis, which impact RBC hemolytic propensity and post-transfusion performance of the storage-damaged RBC. Since mature RBCs are devoid of de novo protein synthesis - owing to the lack of organelles and nuclei - they evolved metabolic mechanisms to cope with oxidative stress. Two of these involve (i) activation of the pentose phosphate pathway (PPP), which generates reducing equivalents (NADPH) to preserve glutathione homeostasis and recharge NADPH-dependent antioxidant enzymes; and (ii) recycling of oxidatively damaged proteins via methylation of dehydrated and deamidated aspartate and asparagine residues, a process that consumes methionine as the main methyl group donor. Thus, we hypothesize that the latter mechanism is relevant to routine blood bank storage, especially in glucose-6-phosphate dehydrogenase (G6PD)-deficient donors. In this routinely accepted donor population (~10% of donors of African descent), mutations of G6PD, the rate-limiting enzyme of the PPP, result in instability and decreased enzymatic activity (e.g., <1% residual activity in the Mediterranean variant).Within the framework of the REDS-III RBC-Omics study, packed RBC units (n=599) were stored under blood bank conditions for 10, 23 and 42 days and profiled for oxidative hemolysis (following AAPH insult) and time-dependent metabolic dysregulation of the trans-sulfuration pathway. The rate of methionine consumption positively correlated with storage age and oxidative hemolysis. In a separate cohort, RBCs from G6PD-deficient and G6PD-normal donors were also collected and metabolically profiled during storage. The G6PD-deficient donors had higher pre-storage methionine levels as compared against G6PD-sufficient donors, although one week of storage was sufficient to significantly flip this ratio (p<0.01). To ensure that the phenomenon was driven by storage-induced oxidative insults, RBCs were also stored under normoxic, hyperoxic (SO2 >95%), or hypoxic conditions (SO2 = 20%, 10%, 5% of < 3%) for up to 42 days. Of note, hypoxia decreased methionine consumption rates and increased S-adenosylmethionine/S-adenosylhomocysteine ratios in comparison to normoxic control RBCs, while opposite trends were observed in response to oxidative stress. In parallel, RBCs were incubated under normoxic or hypoxic conditions for 16h, in presence or absence of a generic methyltransferase inhibitor, adenosine dialdehyde, or hydrogen peroxide (0.1 or 0.5%). 13C,15N-Methionine uptake and consumption rates were faster in response to pro-oxidant insults and were prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and the formation of methyl-Asp on key structural proteins and enzymes, including band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of GAPDH) and/or residues interacting with the N-terminal cytosolic domain of band 3. Hypoxic incubation with hydrogen peroxide phenocopied normoxic conditions in the presence of pro-oxidant insults, suggesting that the beneficial role of hypoxia in preventing methionine consumption and protein methylation is attributable to decreased oxidative stress. G6PD-deficient donor RBCs had significantly (p<0.0001) higher levels of methylated-deamidated Asn residues in hemoglobin, band 3, and GAPDH, as compared to stored G6PD-normal donor RBCs.Although methylation of basic amino acid residues is an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated Asn residues is a non-epigenetic post-translational sensor of oxidative stress and refrigerated storage in anucleate human RBCs. This damage repair mechanism is exacerbated in RBCs from G6PD-deficient donors, as a compensatory mechanism for the lack of normal PPP function. DisclosuresD'Alessandro:Hemanext inc: Membership on an entity's Board of Directors or advisory committees; Omix Technologies inc: Equity Ownership. Nemkov:Omix Technologies inc: Equity Ownership. Yoshida:Hemanext inc: Employment. Dunham:Hemanext inc: Employment. DuMont:Hemanext inc: Membership on an entity's Board of Directors or advisory committees. Hansen:Omix Technologies inc: Equity Ownership. Spitalnik:Hemanext inc: Membership on an entity's Board of Directors or advisory committees. Zimring:Rubius Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Analysis of Glucose-6-Phosphate Dehydrogenase Genetic Polymorphism in the Hakka Population in Southern China.

BackgroundIn southern China, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a significant health problem. The aim of this study was to investigate the molecular epidemiological characteristic of the G6PD gene among Chinese Hakka in southern Guangdong province.Material/MethodsWe screened 611 unrelated subjects for G6PD genetic polymorphism analyzed by a gene chip analysis for common Chinese G6PD mutations. G-6-PD enzyme activity was determined by use of the G-6-PD quantitative detection kit.ResultsSeven mutation sites were detected from subjects in our study. G6PD Canton (c.1376 G→T)(33.06%), G6PD Kaiping (c.1388 G→A)(30.67%), and polymorphism (c.1311 C→T)(25.89%) account for 89.62% of mutations, followed by G6PD Gaohe (c.95 A→G)(5.97%), G6PD Chinese-5 (c.1024 C→T)(3.58%), G6PD Maewo (c.1360 C→T)(0.39%), and G6PD Viangchan (c.871G→A)(0.39%).ConclusionsWe studied the genetic polymorphisms and frequencies of G6PD gene in the Hakka population of Meizhou. Our results coincide with the results among the Chinese Jiangxi Hakka population. It was consistent with previous research reports on Chinese people. There were differences in the results of reports from some other Asian populations. Our results could be useful for future prevention and control of G6PD deficiency aimed at the Chinese Hakka population.

New cases of Glucose-6-Phosphate Dehydrogenase deficiency in Pulmonary Arterial Hypertension.

Pulmonary Arterial Hypertension (PAH) is a fatal disorder with limited treatment options and reduced life expectancy after diagnosis. Complex genetic backgrounds in PAH complicates identification of causative mutations that is essential for an understanding of the disease diagnostics and etiology especially for idiopathic PAH (iPAH). Hemolysis has been implicated as contributing to the pathobiology of PAH. Glucose-6-Phosphate Dehydrogenase (G6PD) expression and activity define erythrocyte’s antioxidant capacity, and its decrease contributes to erythrocyte fragility. As G6PD deficiency was previously reported in a limited number of PAH cases, we tested whether iPAH patients exhibit underlying G6PD alterations in erythrocytes. A cohort of 22 PAH patients and 8 non-PAH patients were recruited for this study. DNA isolated from Peripheral Blood Mononuclear Cells (PBMC) was used for detection of mutations in the coding region of the G6PD gene. RNA isolated from PBMC was used for determination of G6PD mRNA expression level. G6PD activity was measured in Red Blood Cell (RBC) pellets. Three patients had missense mutations in G6PD (Val291Met, Asn126Asp, Asp194Glu), however, only one mutation (Val291Met) results in a severe G6PD deficiency. A single patient with mutation (Asn126Asp) showed a 21% decrease in G6PD activity, two subjects showed G6PD deficiency without mutations, and one patient had a decreased level of G6PD mRNA and reduced enzyme levels. This study demonstrates that a moderate decrease in G6PD activity is associated with PAH. Screening for G6PD activity and mutations in the G6PD gene may provide early detection of individuals predisposed to PAH.

Mutation of glucose-6-phosphate dehydrogenase deficiency in Chinese Han children in eastern Fujian.

We aim to investigate the mutation types of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Chinese Han children in eastern Fujian Province.A total of 904 Chinese Han neonates (male: 733 with positive G6PD deficiency and 28 with weakly positive deficiency; female: 73 with positive G6PD deficiency and 70 with weakly positive deficiency) received G6PD screening in our center from January 2014 to December 2016 were included in this study. Additionally, 904 age-matched normal Chinese Han individuals (male: 761; female: 143) were selected as control. Neonatal G6PD deficiency screening was performed through blood sample collection from the heels, using the commercial kits. Multicolor melting curve analysis (MMCA) method was used to determine the G6PD mutation type in the 904 cases. If it failed to detect mutations in the cases with abnormal enzyme activity, the polymerase chain reaction (PCR) and gene sequencing were used to determine the mutation sites. PCR and gene sequencing were used to determine the mutation sites in the 904 individuals with normal enzyme activity. Three most common mutation types in Chinese population were compared between Fujian and other provinces.Among the 904 neonates with abnormal G6PD enzyme activity, 17 mutation types were detected including 15 single point mutations and 7 complex mutations. Three most common mutation types were c.1376G > T, c.1388G > A, and c.95A > G accounted for 72.6% of the total mutations in eastern Fujian.The proportion of mutational types in G6PD and the degree of enzyme activity change in various mutational types were found in the neonates of Fujian Province. Our study may enrich the molecular diagnosis of G6PD deficiency meaning Fujian Province.

Diagnostic performances of the fluorescent spot test for G6PD deficiency in newborns along the Thailand-Myanmar border: A cohort study

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an inherited enzymatic disorder associated with severe neonatal hyperbilirubinemia and acute haemolysis after exposure to certain drugs or infections. The disorder can be diagnosed phenotypically with a fluorescent spot test (FST), which is a simple test that requires training and basic laboratory equipment. This study aimed to assess the diagnostic performances of the FST used on umbilical cord blood by locally-trained staff and to compare test results of the neonates at birth with the results after one month of age. Methods: We conducted a cohort study on newborns at the Shoklo Malaria Research Unit, along the Thai-Myanmar border between January 2015 and May 2016. The FST was performed at birth on the umbilical cord blood by locally-trained staff and quality controlled by specialised technicians at the central laboratory. The FST was repeated after one month of age. Genotyping for common local G6PD mutations was carried out for all discrepant results. Results: FST was performed on 1521 umbilical cord blood samples. Quality control and genotyping revealed 10 misdiagnoses. After quality control, 10.7% of the males (84/786) and 1.2% of the females (9/735) were phenotypically G6PD deficient at birth. The FST repeated at one month of age or later diagnosed 8 additional G6PD deficient infants who were phenotypically normal at birth. Conclusions: This study shows the short-comings of the G6PD FST in neonatal routine screening and highlights the importance of training and quality control. A more conservative interpretation of the FST in male newborns could increase the diagnostic performances. Quantitative point-of-care tests might show higher sensitivity and specificity for diagnosis of G6PD deficiency on umbilical cord blood and should be investigated.

Hepatic transcriptional profiling response to fava bean-induced oxidative stress in glucose-6-phosphate dehydrogenase-deficient mice

Favism is an acute hemolytic syndrome caused by the ingestion of fava bean (FB) in glucose 6-phosphate dehydrogenase (G6PD) deficient individuals. However, little is known about the global transcripts alteration in liver tissue after FB ingestion in G6PD-normal and -deficient states. In this study, deep sequencing was used to analyze liver genes expression alterations underlying the effects of FB in C3H (Wild Type, WT) and G6PD-deficient (G6PDx) mice and to evaluate and visualize the collective annotation of a list of genes to Gene Ontology (GO) terms associated with favism. Our results showed that FB resulted in a decrease of glutathione (GSH)-to-oxidized glutathione (GSSG) ratio and an increase of malondialdehyde (MDA) both in the G6PDx and WT-control check (CK) mice plasma. Significantly, liver transcript differences were observed between the control and FB-treated groups of both WT and G6PDx mice. A total of 320 differentially expressed transcripts were identified by comparison of G6PDx-CK with WT-CK and were associated with immune response and oxidation-reduction function. A total of 149 differentially expressed genes were identified by comparison of WT-FB with WT-CK. These genes were associated with immune response, steroid metabolic process, creatine kinase activity, and fatty acid metabolic process. A total of 438 differential genes were identified by comparing G6PDx-FB with G6PD-CK, associated with the negative regulation of fatty acid metabolic process, endoplasmic reticulum, iron binding, and glutathione transferase activity. These findings indicate that G6PD mutations may affect the functional categories such as immune response and oxidation-reduction.

Newborn screening of glucose-6-phosphate dehydrogenase deficiency in Guangxi, China: determination of optimal cutoff value to identify heterozygous female neonates

The aim of this study is to assess the disease incidence and mutation spectrum of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Guangxi, China, and to determine an optimal cutoff value to identify heterozygous female neonates. A total of 130, 635 neonates were screened from the year of 2013 to 2017. Neonates suspected for G6PD deficiency were further analyzed by quantitatively enzymatic assay and G6PD mutation analysis. The overall incidence of G6PD deficiency was 7.28%. A total of 14 G6PD mutations were identified, and different mutations lead to varying levels of G6PD enzymatic activities. The best cut-off value of G6PD activity in male subjects is 2.2 U/g Hb, same as conventional setting. In female population, however, the cut-off value is found to be 2.8 U/g Hb (sensitivity: 97.5%, specificity: 87.7%, AUC: 0.964) to best discriminate between normal and heterozygotes, and 1.6 U/g Hb (sensitivity: 82.2%, specificity: 85.9%, AUC: 0.871) between heterozygotes and deficient subjects. In conclusion, we have conducted a comprehensive newborn screening of G6PD deficiency in a large cohort of population from Guangxi, China, and first established a reliable cut-off value of G6PD activity to distinguish heterozygous females from either normal or deficient subjects.

Diagnostic performances of the fluorescent spot test for G6PD deficiency in newborns along the Thailand-Myanmar border: A cohort study.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an inherited enzymatic disorder associated with severe neonatal hyperbilirubinemia and acute haemolysis after exposure to certain drugs or infections. The disorder can be diagnosed phenotypically with a fluorescent spot test (FST), which is a simple test that requires training and basic laboratory equipment. This study aimed to assess the diagnostic performances of the FST used on umbilical cord blood by locally-trained staff and to compare test results of the neonates at birth with the results after one month of age. Methods: We conducted a cohort study on newborns at the Shoklo Malaria Research Unit, along the Thai-Myanmar border between January 2015 and May 2016. The FST was performed at birth on the umbilical cord blood by locally-trained staff and quality controlled by specialised technicians at the central laboratory. The FST was repeated after one month of age. Genotyping for common local G6PD mutations was carried out for all discrepant results. Results: FST was performed on 1521 umbilical cord blood samples. Quality control and genotyping revealed 10 misdiagnoses. After quality control, 10.7% of the males (84/786) and 1.2% of the females (9/735) were phenotypically G6PD deficient at birth. The FST repeated at one month of age or later diagnosed 8 additional G6PD deficient infants who were phenotypically normal at birth. Conclusions: This study shows the short-comings of the G6PD FST in neonatal routine screening and highlights the importance of training and quality control. A more conservative interpretation of the FST in male newborns could increase the diagnostic performances. Quantitative point-of-care tests might show higher sensitivity and specificity for diagnosis of G6PD deficiency on umbilical cord blood and should be investigated.

Glucose-6-Phosphate Dehydrogenase Deficiency Genetic Variants in Malaria Patients in Southwestern Ethiopia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked erythrocyte enzyme disorder with relevance to malaria treatment policy. Treatment with the antimalarial primaquine can result in hemolytic anemia in G6PD-deficient patients. With increased interest in primaquine use, it is important to identify G6PD variants in Ethiopia to inform malaria treatment policy. In the present study, mutations in the G6PD gene are identified in a sample of patients with malaria in Jimma town in southwest Ethiopia. Plasmodium species of infection were confirmed using polymerase chain reaction (PCR) and gel electrophoresis. PCR and Sanger sequencing were performed to observe a portion of the G6PD gene where the common G6PD mutations (A376G, G202A, and C563T) are found. Molecular analysis revealed that most of the samples were single Plasmodium vivax infections (83.7%). For G6PD genotyping, A376G was detected in 23.26% of individuals, whereas G202A and C563T were absent. Three other uncommon mutations were identified: rs782669677 (535G→A), rs370658483, (485 + 37 G→T), and a new mutation at chrX:154535443(C→T). Bioinformatic analysis of these mutations' potential functional impact suggests minimal effect on protein function. The discovery of both common and uncommon G6PD mutations contributes to the discussion on G6PD deficiency and appropriate primaquine treatment in Ethiopia.