Glucose-6-phosphate Dehydrogenase Gene Research Articles

Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia.

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway and is highly relevant in the metabolism of Giardia lamblia. Previous reports suggested that the G6PD gene is fused with the 6-phosphogluconolactonase (6PGL) gene (6pgl). Therefore, in this work, we decided to characterize the fused G6PD-6PGL protein in Giardia lamblia. First, the gene of g6pd fused with the 6pgl gene (6gpd::6pgl) was isolated from trophozoites of Giardia lamblia and the corresponding G6PD::6PGL protein was overexpressed and purified in Escherichia coli. Then, we characterized the native oligomeric state of the G6PD::6PGL protein in solution and we found a catalytic dimer with an optimum pH of 8.75. Furthermore, we determined the steady-state kinetic parameters for the G6PD domain and measured the thermal stability of the protein in both the presence and absence of guanidine hydrochloride (Gdn-HCl) and observed that the G6PD::6PGL protein showed alterations in the stability, secondary structure, and tertiary structure in the presence of Gdn-HCl. Finally, computer modeling studies revealed unique structural and functional features, which clearly established the differences between G6PD::6PGL protein from G. lamblia and the human G6PD enzyme, proving that the model can be used for the design of new drugs with antigiardiasic activity. These results broaden the perspective for future studies of the function of the protein and its effect on the metabolism of this parasite as a potential pharmacological target.

Role of genetic factors and ethnicity on the multiplicity of Plasmodium falciparum infection in children with asymptomatic malaria in Yaoundé, Cameroon

In this cross-sectional study, we investigated host genetic factors and ethnic variation in circulating Plasmodium falciparum merozoite surface protein 2 (msp-2) clones among children with asymptomatic malaria.Isolates from seventy two asymptomatic malaria children were used for genotyping block 3 of msp-2 gene by nested polymerase chain reaction (PCR). Sickle cell trait and glucose-6-phosphate dehydrogenase (G6PD) deficiency were analysed by restriction fragment length polymorphism of DNA products from PCR targeting codons 6 and 68 of the beta-globin (HBB) and G6PD genes respectively. ABO blood group was typed by agglutination method.A total of forty two msp-2 genotypes (20 for 3D7 and 22 for FC27) were detected for an average (standard error of mean) multiplicity of infection (MOI) of 2.45 (0.16). The MOI was statistically the same among the five identified ethnic groups (P = 0.83). The overall prevalence of sickle cell trait and G6PD deficiency were 12.50 % and 22.22 % respectively. MOI was similar between children with Hb AA and Hb AS genotypes (P = 0.42). MOI was significantly high among children with a mutant G6PD genotype (P = 0.017). MOI was significantly higher in blood group O than group A (P = 0.03).Our findings show that although ethnicity and sickle cell trait have no association with MOI, the association was observed with G6PD genotype and ABO group. The results suggest the need for extension and expansion of the current study in order to investigate the mechanisms involved.

The gene spectrum of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Guangdong province, China

BackgroundG6PD deficiency presents a higher incidence rate in southern China. Many variants of G6PD resulted from point mutations in the G6PD gene, which lead to decrease of enzyme activity. The objective of this study was to analyze the genotype of G6PD deficiency in four regions of Guangdong province. MethodsGenotype of 1756 cases with G6PD deficiency was identified by reverse dot blotting (RDB). Unidentified Genotype of the samples was further ascertained by direct DNA sequencing. Results34 genotype were found in 1756 cases of G6PD deficiency, Canton (c.1376 G>T) and Kaiping (c.1388 G>A) were the most common variants, accounting for more than 63% of G6PD deficiency individuals, and the following mutations were Gaohe (c.95 A>G), Chinese-5 (c.1024 C>T) and Chinese-4 (c.392 G>T). Two rare mutations Orisa (c.131 C>G) and IVS-5 637/638 T del have been discovered in this study. In addition, c.1311 C>T/IVS-1193 T>C polymorphism had a relatively high frequency in the normal G6PD individuals. ConclusionsThis study provided detailed genotypes of G6PD deficiency in Guangdong, and would be valuable for diagnosis and research of G6PD deficiency in this area.

Loss of cytosolic glucose-6-phosphate dehydrogenase increases the susceptibility of Arabidopsis thaliana to root-knot nematode infection.

Root knot nematodes (RKNs, Meloidogyne spp.) are microscopic roundworms with a wide host range causing great economic losses worldwide. Understanding how metabolic pathways function within the plant upon RKN infection will provide insight into the molecular aspects of plant-RKN interactions. Glucose-6-phosphate dehydrogenase (G6PDH), the key regulatory enzyme of the oxidative pentose phosphate pathway (OPPP), is involved in plant responses to abiotic stresses and pathogenesis. In this study, the roles of Arabidopsis cytosolic G6PDH in plant-RKN interactions were investigated. Enzyme assays and western blotting were used to characterize changes in total G6PDH activity and protein abundance in wild-type Arabidopsis in response to RKN infection. The susceptibility of wild-type plants and the double mutant g6pd5/6 to RKNs was analysed and the expression of genes associated with the basal defence response was tested after RKN infection using quantitative reverse transcription PCR. RKN infection caused a marked increase in total G6PDH activity and protein abundance in wild-type Arabidopsis roots. However, the transcript levels of G6PDH genes except G6PD6 were not significantly induced following RKN infection, suggesting that the increase in G6PDH activity may occur at the post-transcriptional level. The double mutant g6pd5/6 with loss-of-function of the two cytosolic isoforms G6PD5 and G6PD6 displayed enhanced susceptibility to RKNs. Moreover, reactive oxygen species (ROS) production and gene expression involved in the defence response including jasmonic acid and salicylic acid pathways were suppressed in the g6pd5/6 mutant at the early stage of RKN infection when compared to the wild-type plants. The results demonstrated that the G6PDH-mediated OPPP plays an important role in the plant-RKN interaction. In addition, a new aspect of G6PDH activity involving NADPH production by the OPPP in plant basal defence against RKNs is defined, which may be involved in ROS signalling.

Prevalence of G6PD deficiency and associated haematological parameters in children from Botswana

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is commonly seen in malaria endemic areas as it is known to confer a selective advantage against malaria. Recently, we reported a high proportion of asymptomatic reservoir of Plasmodium vivax in Botswana, that calls for intervention with primaquine to achieve radical cure of vivax malaria. Considering that individuals with this enzyme deficiency are at risk of haemolysis following primaquine treatment, assessment of the population for the relative frequency of G6PD deficiency is imperative. Samples from 3019 children from all the districts of Botswana were successfully genotyped for polymorphisms at positions 202 and 376 of the G6PD gene. Haematological parameters were also measured. The overall population allele frequency (based on the hemizygous male frequency) was 2.30% (95% CI, 1.77–2.83), while the overall frequency of G6PD-deficient genotypes A- (hemizygote and homozygote genotypes only) was 1.26% (95% CI, 0.86–1.66). G6PD deficiency is spread in Botswana according to the historical prevalence of malaria with a North-West to South-East decreasing gradient trend. There was no association between G6PD status and P. vivax infection. G6PD A- form was found to be associated with decreased RBC count and haemoglobin levels without a known cause or illness. In conclusion, we report for the first time the prevalence of G6PD deficiency in Botswana which is relevant for strategies in the malaria elimination campaign. Further work to examine the activities of the enzyme in the Botswana population at risk for malaria is warranted.

Validation of the quantitative point-of-care CareStart biosensor for assessment of G6PD activity in venous blood.

IntroductionGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the human population affecting an estimated 8% of the world population, especially those living in areas of past and present malaria endemicity. Decreased G6PD enzymatic activity is associated with drug-induced hemolysis and increased risk of severe neonatal hyperbilirubinemia leading to brain damage. The G6PD gene is on the X chromosome therefore mutations cause enzymatic deficiency in hemizygote males and homozygote females while the majority of heterozygous females have an intermediate activity (between 30–80% of normal) with a large distribution into the range of deficiency and normality. Current G6PD qualitative tests are unable to diagnose G6PD intermediate activities which could hinder wide use of 8-aminoquinolines for Plasmodium vivax elimination. The aim of the study was to assess the diagnostic performances of the new Carestart G6PD quantitative biosensor.MethodsA total of 150 samples of venous blood with G6PD deficient, intermediate and normal phenotypes were collected among healthy volunteers living along the north-western Thailand-Myanmar border. Samples were analyzed by complete blood count, by gold standard spectrophotometric assay using Trinity kits and by the latest model of Carestart G6PD biosensor which analyzes both G6PD and hemoglobin.ResultsBland-Altman comparison of the CareStart normalized G6PD values to that of the gold standard assay showed a strong bias in values resulting in poor area under-the-curve values for both 30% and 80% thresholds. Performing a receiver operator curve identified threshold values for the CareStart product equivalent to the 30% and 80% gold standard values with good sensitivity and specificity values, 100% and 92% (for 30% G6PD activity) and 92% and 94% (for 80% activity) respectively.ConclusionThe Carestart G6PD biosensor represents a significant improvement for quantitative diagnosis of G6PD deficiency over previous versions. Further improvements and validation studies are required to assess its utility for informing radical cure decisions in malaria endemic settings.

Evaluations of newborn screening program performance and enzymatic diagnosis of glucose-6-phosphate dehydrogenase deficiency in Guangzhou

Objective: To reveal the molecular epidemiologic characteristics of glucose-6-phosphate dehydrogenase (G6PD) gene and to evaluate based on the genetic analysis the newborn screening program performance and enzymatic diagnosis of G6PD deficiency in Guangzhou. Methods: G6PD enzyme activities were measured by quantitative fluorescence assay in dry blood spots of 16 319 newborns(8 725 males, 7 594 females) 3-7 days after birth in Guangzhou Newborn Center. They were born in Guangzhou form Oct. 1 to 20, 2016. The cutoff value of G6PD was less than 2.6 U/g Hb in dry blood spots. G6PD deficiency was diagnosed when G6PD<1 700 U/L or G6PD/6PGD<1 in red blood cells. Genetic analysis of G6PD gene was performed on the dry blood spot samples of 823 newborns (including positive 346, negative 477)with various levels of G6PD enzyme activities through fluorescence PCR melting curve analysis(FMCA) to detect 15 kinds of mutations reported to be common among Chinese.G6PD gene Sanger sequency was performed in seven highly suspicious patients with negative results by FMCA. Results: (1) Using the cutoff value of G6PD< 2.6 U/g Hb , a total of 687(4.2%) newborns showed positive screening results, including 560 (6.4%) males and 127(1.7%) females. (2) Among the newborns with positive screening results, 214 males and 122 females were randomly chosen for G6PD gene analysis. The results showed that 197 (92.1%) males were hemizygote and 108(88.6%) females were mutation carriers with one to four alleles. Among the newborns with negative screening results, 41 males with G6PD 2.6-2.8 U/g Hb and 436 females with G6PD 2.6-4.5 U/g Hb were chosen for genetic analysis.Mutations were detected in 5(12.2%)boys, and 226(51.8%) girls were carriers.G6PD gene Sanger sequency of seven highly suspicious patients showed that c.406C>T, c.551C>T, c.835A>T hemizygote were found in 3 male's samples, respectively. (3) The estimated prevalence of harboring mutation was 6.0% in males and 13.5% in females according to rates of mutation in samples with various levels of G6PD enzyme activities. Six common mutations were c.1388G>A、c.1376G>T, c.95A> G, c.871G>A, c.1024C>T, c.392G>T, accounting for 95.5% of detected alleles .(4) based on results of G6PD gene analysis, the newborn scereening of G6PD deficiency with cutoff value G6PD<2.6 U/g Hb yielded a positive predict value(PPV) of 93.5%, a false-positive rate of 0.5%, and a sensitivity of 99.0% for males. A PPV of 88.5%, a false-positive rate of 0.2% . The prevalence of severe type G6PD deficiency in females was about 1.5%. Compared with to genetic analysis, the sensitivity and PPV of G6PD activity assay in red blood cells were 95.5%, 97.2%, respectively. Conclusions: The prevalence of G6PD deficiency in males was 6.0% in Guangzhou. Six mutations c.1388G>A, c.1376G>T, c.95A>G, c.871G>A, c.1024C>T, c.392G>T accounted for 95.5%. The cutoff value of G6PD<2.6 U/g Hb innewborn screening program and the criteria of biochemical diagnosis could accurately identify G6PD deficiency . Combined with biochemical and molecular analysis will improve the accuracy of diagnosis of G6PD deficiency and detect more heterozygous females.

Genetic Polymorphisms of Glucose-6-Phosphate Dehydrogenase in Lagos, Nigeria

Glucose-6-phosphate dehydrogenase (G6PD) is an essential enzyme in the pentose phosphate pathway that prevents oxidative damage to cells. This study determined the genotypic and allelic frequencies of G6PD G202A and A376G and also investigated correlation between G6PD polymorphisms and hemoglobin (Hb) phenotypes in children in Lagos, Nigeria. Seventy-eight children [55 with Hb AA (βΑ/βA) and 23 with Hb AS (βΑ/βS) trait] and 65 Hb SS (βS/βS) (HBB: c.20A>T) subjects in steady state with age range between 5–15 years were recruited for the study. Hemoglobin phenotypes of all study participants were carried out using alkaline electrophoresis and solubility tests. Genomic DNA was extracted from whole blood and restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was used to determine the G202A and the A376G mutations of the G6PD gene. The genotype and allele distributions of G6PD G202A and A376G according to the Hb phenotypes were not statistically significant (p > 0.05). The minor allele frequency 202A was 0.15 (15.0%) and 0.14 (14.0%) in cases and controls, respectively. The overall frequency of 376G allele in the case group was 0.35 (35.0%) and 0.38 (38.0%) in the control group. No statistical significance was observed in the genotype and allele distributions of A376G in both the case and control groups (p > 0.05). The G6PD A− frequency in Hb SS subjects and the control group were 6.2 and 2.6%, respectively. G6PD G202A and A376G polymorphisms were not associated with Hb phenotypes and the allele distributions of 202A and 376G in this study are typical of West African populations.

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.

Glucose-6-Phosphate Dehydrogenase Deficiency in Blood Donors Is Associated with Decreased Post-Transfusion Red Cell Recovery

Background:Pre-transfusion refrigerated storage of red cells (RBCs) induces a series of metabolic and structural changes in stored RBCs (i.e., the “storage lesion”). The resulting, progressive, time-dependent decrease in RBC quality leads to decreased post-transfusion RBC recovery (PTR). Markers of oxidative stress increase during storage, suggesting the possibility that oxidative stress may be a key contributor to the storage lesion. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked condition, decreases the ability of RBCs to withstand oxidative stress by compromising their ability to synthesize NADPH. Therefore, we hypothesized that RBCs from G6PD-deficient donors would have inferior storage quality, as compared to RBCs from G6PD-normal (i.e., control) donors.Aims:This study examined whether G6PD deficiency, the most common enzyme deficiency worldwide, which impairs the RBC response to oxidative stress, also decreases RBC storage quality.Methods:Male volunteers were screened for G6PD deficiency by enzymatic assay, and next generation sequencing was performed to identify the G6PD variant in deficient subjects. In addition, participants were screened for the presence of hemoglobin variants and thalassemia by high performance liquid chromatography and complete blood count. Twenty seven control and 10 G6PD-deficient volunteers each received informed consent, donated 1 unit of leukoreduced RBCs that was stored in AS-3 under standard blood banking conditions, and completed an autologous 51-Chromium-labeled PTR study after 40-42 days of storage. Hemolysis rate in the transfused unit was measured on the day of the PTR study using a Drabkin assay. Results for control and G6PD-deficient PTRs and hemolysis rates were compared using unpaired t-tests.Results:A total of 145 males were screened for G6PD deficiency, yielding 15 deficient (10%) and 130 G6PD normal control (90%) subjects. Twenty seven control (G6PD normal) and 10 deficient participants who did not have hemoglobin variants or microcytosis completed the study. All blood donors met the hemoglobin acceptance criteria for autologous whole blood donation of at least 11.5 g/dL. G6PD gene sequencing of the 10 enrolled G6PD-deficient subjects identified the A- variant in 9 volunteers and the Mediterranean variant in 1. The autologous mean 24-hour PTR for control RBC units was 86.8 ± 2.8% (mean ± SD). The autologous mean 24-hour PTR for G6PD-deficient RBC units was 81.0 ± 7.2% (mean ± SD), which was significantly lower than that for control RBCs (p=0.001). None of the control volunteers (0/27) had PTR results below 75%, one of the key FDA acceptability criteria for stored RBCs. In contrast, two G6PD-deficient volunteers (2/10) had PTR results below 75% (i.e., 68.8% and 70.1%). There was no association between enzymatic G6PD activity and PTR when adjusted for G6PD status (i.e., among control or G6PD-deficient subjects, the enzymatic G6PD activity did not predict PTR). Finally, there was no difference between control and deficient RBC units in mean in vitro hemolysis rates at the end of storage (0.29 ± 0.18% vs. 0.24 ± 0.07%, respectively; p=0.38)Conclusions:This is the first report of the effect of G6PD deficiency on the storage quality at outdate of transfused donor RBCs stored in AS-3 additive solution as assessed by PTR, the FDA gold standard. These results demonstrate that the storage quality of G6PD-deficient RBCs is inferior to G6PD-normal RBCs. In particular, based on the FDA criteria that acceptable RBC units should have a 24-hour PTR of ≥75% at the end of storage, RBC units from G6PD-deficient donors had a 20% failure rate. The clinical consequences of the mean 5.8% decreased PTR of G6PD-defcient RBC units remain to be determined. Patients with sickle cell disease often require lifetime transfusions and up to 12.3% of phenotypically-matched RBC units utilized in their care may be G6PD-deficient. Thus, these data raise the question of whether there is value in screening blood donors for G6PD deficiency, particularly in the setting of chronic transfusion therapy, such as for patients with sickle cell disease and thalassemia. DisclosuresSpitalnik:New Health Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees.

G6PD promotes renal cell carcinoma proliferation through positive feedback regulation of p-STAT3.

Ectopic Glucose 6-phosphate dehydrogenase (G6PD) expression plays important role in tumor cell metabolic reprogramming and results in poor prognosis of multiple malignancies. Our previous study indicated that G6PD is overexpressed in clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC. However, its role in RCC is still unclear. Here, we demonstrate that G6PD is not only up-regulated in all types of RCC specimens but also displays higher activities in RCC cell lines. G6PD overexpression promoted RCC cell proliferation, altered cell cycle distribution, and enhanced xenografted RCC development. G6PD up-regulated ROS generation by facilitating NADPH-dependent NOX4 activation, which led to increased expression of p-STAT3 and CyclinD1. Enhanced ROS generation rescued the p-STAT3 and CyclinD1 expression reduction in G6PD-knockdown cells, while ROS scavengers reversed the up-regulated p-STAT3 and CyclinD1 expression in G6PD-overexpressing cells. Furthermore, p-STAT3 activated G6PD gene expression via binding to the G6PD promoter, demonstrating that p-STAT3 forms a positive feedback regulatory loop for G6PD overexpression. G6PD expression was up or down-regulated in response to the impact of p-STAT3 activators or inhibitors. Therefore, G6PD may be an effective RCC therapeutic target.

Effects of dietary carbohydrate sources on growth, digestive enzyme activity, gene expression of hepatic GLUTs and key enzymes involved in glycolysis-gluconeogenesis of giant grouper Epinephelus lanceolatus larvae

This study aimed to evaluate effects of dietary carbohydrate sources on growth, digestive enzyme activity, gene expression of hepatic glucose transporters (GLUTs) and key enzymes involved in glycolysis-gluconeogenesis in giant grouper Epinephelus lanceolatus larvae through a 21-day growth trial. Four isonitrogenous (50%) and isolipidic (15.4%) experimental diets, including a cellulose control diet and three diets with glucose, maltose or maize starch, were formulated. Dietary CHO level was 18%. The 29 DPH (days post hatching) grouper larvae (initial average weight: 0.078±0.0018g) were cultured in small floating cages (L 120cm×W 70cm×H 50cm) at a density of 80 fish per cage. Experimental cages were labeled and located in three connective 6-m3 indoor concrete tanks (L 3m×W 3m×H 2m) with 4 cages occurring in each tank.Results showed that at 50 DPH, fish fed with maize starch diet had significantly higher weight gain (WG) and thermal growth coefficient (TGC) than fish fed with glucose, maltose or cellulose. Fish fed with glucose had the lowest WG among all experimental treatments. Fish fed with maize starch or cellulose had significantly higher mRNA expression levels of hepatic fructose 1,6 bisphosphatase (FBP) gene compared to fish fed with glucose or maltose at 50 DPH. The highest mRNA expression level of hepatic glucose-6-phosphate dehydrogenase (G6PDH) gene was observed in fish fed with glucose at 50 DPH. Fish fed with maize starch had higher mRNA expression levels of hepatic pyruvate kinase (PK) and hexokinase (HK) genes than fish fed with cellulose, glucose and maltose at 50 DPH. The mRNA expression level of hepatic glucose transporter 4 (GLUT4) gene was significantly lower in fish fed with cellulose compared to that in fish fed with digestible CHOs at 50 DPH. There were no significant differences in mRNA expression levels of hepatic glucose transporter 1 (GLUT1) gene among all experimental fish at 50 DPH. In terms of digestive enzymes, activities of pancreatic trypsin and intestinal lipase increased during the development of fish larvae, but pancreatic α-amylase specific activity showed an opposite trend of variation as those of trypsin and lipase. During the development of experimental fish larvae from 29 DPH to 50 DPH, pancreatic trypsin specific activity in fish fed with glucose was significantly lower than that in fish fed with cellulose or maltose but did not differ from that in fish fed with maize starch. Intestinal lipase specific activity in fish fed with maize starch or maltose were significantly higher than those in fish fed with cellulose but did not differ from those in fish fed with glucose. Generally, giant grouper larvae fed maize starch had better growth than those fed cellulose, glucose or maltose; the mRNA expressions of hepatic HK, PK, FBP and G6PDH genes involved in glycolysis-gluconeogenesis were significantly affected by dietary CHO sources.

Glucose-6-phosphate dehydrogenase (G6PD)-deficient infants: Enzyme activity and gene variants as risk factors for phototherapy in the first week of life.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a recognised cause of severe neonatal hyperbilirubinaemia, and identifying which infants are at risk could optimise care and resources. In this study, we determined if G6PD enzyme activity (EA) and certain gene variants were associated with neonatal hyperbilirubinaemia requiring phototherapy during the first week after birth. Newborn infants with G6PD deficiency and a group with normal results obtained by the fluorescent spot test were selected for analyses of G6PD EA and the 10 commonly encountered G6PD mutations in this region, relating these with whether the infants required phototherapy before discharge from the hospital in the first week. A total of 222 infants with mean gestation and birth weight of 38.3 ± 1.8 weeks and 3.02 ± 0.48 kg, respectively, were enrolled. Of these, n = 121 were deficient with EA ≤6.76 U/g Hb, and approximately half (43%) received phototherapy in the first week after birth. The mean EA level was 3.7 U/g Hb. The EA had good accuracy in predicting phototherapy use, with area under the receiver-operating-characteristic curve of 0.81 ± 0.05. Infants on phototherapy more commonly displayed World Health Organization Class II mutations (<10% residual EA). Logistic regression analysis showed that deficiency in EA and mutation at c.1388G>A (adjusted odds ratio, 1.5 and 5.7; 95% confidence interval: 1.31-1.76 and 1.30-25.0, respectively) were independent risk factors for phototherapy. Low G6PD EA (<6.76 U/g Hb) and the G6PD gene variant, c.1388G>A, are risk factors for the need of phototherapy in newborn infants during the first week after birth.

Pedigree analysis of glucose-6 phosphate dehydrogenase (G6PD) deficiency of a Javanese Chinese family in Indonesia

The molecular and pedigree analyses in a Javanese Chinese family were carried oul on glucose-6-phosphate dehydrogenase deficiencies. By method of MPTP scanning without the sequencing steps, those variants could be confirmed. Two out of three sons were clinically jaundiced at birth due to G6PD deficiency and identified to have a G to T nucleotide change al 1376th nucleotide 01 the G6PD gene (GI376T), corresponding to G6PD Canton. Another son was also identified to have a C to T nucleotide change at 1311st nucleotide 01 the G6PD gene (CI311T), corresponding to a Silent mutation. Their father was normal, but their mother obsorved to have the heleromutation 01 G1376T (G6PD Canton and C1311T (a Silent mutation).

Possible association of 3′ UTR +357 A>G, IVS11-nt 93 T>C, c.1311 C>T polymorphism with G6PD deficiency

ABSTRACTBackground: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked inherited enzymopathic disorder affecting more than 500 million people worldwide. It has so far been linked to 217 distinct genetic variants in the exons and exon–intron boundaries of the G6PD gene, giving rise to a wide range of biochemical heterogeneity and clinical manifestations.Objectives: Reports from different settings suggested the association of intronic and other mutations outside the reading frame of the G6PD gene with reduced enzyme activity and presenting clinical symptoms. The present study aimed to investigate any association of other variations apart of the exonic or exonic intronic boundaries in the development of G6PD deficiency.Methods: Sixty-seven unrelated Palestinian children admitted to the pediatric hospital with hemolytic crises due to G6PD deficiency were studied.Results: In our Palestinian cohort of 67 [59 males (M) and 8 females (F)] G6PD-deficient children, previously hospitalized for acute hemolytic anemia due to favism, molecular sequencing of the G6PD gene revealed four cases (3M and 1F) that did not have any of the variants known to cause G6PD deficiency, but the 3′ UTR c.*+357A>G (rs1050757) polymorphism in association with IVS 11 (c.1365-13T>C; rs2071429), and c.1311C>T (rs2230037).Conclusion: We now provide an additional evidence form Palestinian G6PD-deficient subjects for a possible role of 3′ UTR c.*+357 A>G, c.1365-13T>C, and/or c.1311C>T polymorphism for G6PD deficiency, suggesting that not only a single variation in the exonic or exonic intronic boundaries, but also a haplotype of G6PD should considered as a cause for G6PD deficiency.

Rapid diagnostic test for G6PD deficiency in Plasmodium vivax-infected men: a budget impact analysis based in Brazilian Amazon.

The aim of this study was to estimate the incremental budget impact (IBI) of a rapid diagnostic test to detect G6PDd in male patients infected with Plasmodium vivax in the Brazilian Amazon, as compared with the routine protocol recommended in Brazil which does not include G6PDd testing. The budget impact analysis was performed from the perspective of the Brazilian health system, in the Brazilian Amazon for the years 2013, 2014 and 2015. The analysis used a decision model to compare two scenarios: the first consisting of the routine recommended in Brazil which does not include prior diagnosis of dG6PD, and the second based on the use of RDT CareStart™ G6PD (CS-G6PD) in all male subjects diagnosed with vivax malaria. The expected implementation of the diagnostic test was 30% in the first year, 70% the second year and 100% in the third year. The analysis identified negative IBIs which were progressively smaller in the 3 years evaluated. The sensitivity analysis showed that the uncertainties associated with the analytical model did not significantly affect the results. A strategy based on the use of CS-G6PD would result in better use of public resources in the Brazilian Amazon.

Molecular diagnosis and mutation identification in newborn with glucose-6-phosphate dehydrogenase deficiency by DNA sequencing

Objective To evaluate the clinical significance of molecular diagnosis for detecting neonatal glucose-6-phosphate dehydrogenase (G6PD) deficiency and to identify the novel mutations in G6PD gene. Methods Totally 231 newborn blood samples with decreased G6PD deficiency assessed by first-line newborn screening were recruited from Children's Hospital, Chongqing Medical University. G6PD gene mutation analysis was performed by PCR following by Sanger DNA sequencing, and then hotspot mutation sites were confirmed. The relationship between the enzymatic phenotype and genotype was evaluated by comparing the results between the G6PD activity detection and the molecular diagnosis. Novel variations were mutation predicted to identity the pathogenic mutations. Results Totally 125 G6PD patients were identified in present study group, among which 102 patients were classified as positive by G6PD/6PGD quantitative tests, giving out a detecting rate of 81.6%. The 123 patients were confirmed by molecular diagnosis with a detecting of 98.4%. Within the 16 probably positive cases, 11 definite mutations were confirmed by molecular diagnosis. DNA sequencing also confirmed another group of 12 patients from the 113 negative samples, giving a detecting rate of 10.6%. Two novel mutations of G6PD gene, c. 29C>A and c. 361C>T were found in the study, of which, c. 29C>A was observed in 7.3% (9/123) of the samples and might be a new hotspot site in Chinese population. Conclusions Quantitative ratio testing may be not efficient enough for G6PD deficiency diagnosis in patients carrying heterozygous mutations. Novel mutations can be found in G6PD gene and obviously there are regional diversities in the frequency of hotspot mutations for G6PD deficiency. Therefore, DNA sequencing based molecular testing may serve as the most valuable method for the diagnosis of neonatal G6PD deficiency.(Chin J Lab Med, 2016, 39: 843-847) Key words: Glucosephosphate dehydrogenase deficiency; Glucosephosphate dehydrogenase; Sequence analysis, DNA; Molecular diagnostic techniques; Mutation; Neonatal screening

Optimizing a multiplex high resolution melting curve to diagnose G6PD deficiency based on viangchan and canton mutations

Glucose-6-phosphate dehydrogenase (G6PD) deficiency which is caused by mutation on G6PD gene is the most common enzyme disorder in human. There have been 184 discovered mutations among which Viangchan [Val291Met] and Canton mutation [Arg459Leu] are the most common variants in Vietnamese. Due to the severity of this disease, several methods have been devised for diagnostics. However, time-consuming, low sensitivity and expensiveness are major problems of those techniques. Recently, High Resolution Melting (HRM) has been developed and proven to be an effective method for DNA genotyping, mutation scanning and sequence matching. Hence, in this study a multiplex HRM has been developed aiming at detecting these two mutations concurrently. At first, a singleplex HRM was designed for each mutation. Then, conditions for these singleplex assay were combined and optimized again in order to get the optimal condition for multiplex HRM. This method showed a promising potential with a high accuracy, sensitivity, and specificity, it is impractial to continue developing this method because of the lack of controls. The optimized singleplex PCR-HRM in this study still can be used for single mutation detection and serve as the background to develop PCR-HRM for other G6PD mutations in Vietnamese-Kinh population.

Optimizing amniotic membrane tissue banking protocols for ophthalmic use.

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco's modified eagle's medium (DMEM) and glycerol at -80°C and at -196°C for a period of 6weeks and at 4°C in 70% alcohol for 6weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5×5cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at -196 and -80°C for 6 weeks. Similar sections were also stored at 4°C in 70% alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10% FBS in humidified atmosphere of 5% carbon dioxide at 37°C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8×10(6); 1.59, after 6weeks in DMSO:FBS at -196°C 3.0×10(6); 2.38 and at -80°C 2.1×10(6); 1.60, in DMEM:Glycerol at -196°C 3.6×10(6); 2.33 at -80°C 23×10(6); 1.66 and at 4°C 3.3×10(6); 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at -196°C showed morphology similar to fresh AM in both preservation media and AM stored at -80°C showed disruption of the stromal matrix. At 4°C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at -196°C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.

Abstract 1009: Metabolic reprogramming discriminates aggressive vs. slowly growing preneoplastic lesions at early stages of HCC development

Abstract Introduction and aim: Among the several changes underlying metabolic reprogramming of cancer cells, increased glucose utilization and its uncoupling from oxygen availability is a well-established phenomenon and has been recognized as a hallmark of cancer. To what extent these metabolic changes are important for the progression of slow growing tumors and whether a metabolic rewiring occurs in the very early stages of neoplastic progression represent key questions on the significance of these metabolic alterations in cancer. Here, we compared the metabolic features of preneoplastic hepatic lesions with those of advanced hepatocellular carcinomas (HCCs) and of proliferating liver, following partial hepatectomy (PH). Materials and Methods: Expression levels, activity and modulation of several enzymes with key roles in glycolysis, pentose phosphate pathway (PPP) and oxidative phosphorylation (OXPHOS) were assessed in preneoplastic hepatic lesions and HCC, induced in rats exposed to the Resistant-Hepatocyte (R-H) model. In vitro experiments were performed on HCC cells obtained by perfusion of HCC-bearing rats. Expression of metabolic genes was also investigated in two different cohorts of human patients carrying HCC. Results and discussion: A switch from OXPHOS to PPP was observed in very early preneoplastic lesions generated 10 weeks after the treatment with DENA. Notably, this metabolic reprogramming was observed only in the most aggressive preneoplastic lesions, characterized by positivity for cytokeratin 19 (CK-19+). PPP induction, shown by a strong increase in the expression and activity of glucose 6-phosphate dehydrogenase (G6PD) was supported both by inhibition of pyruvate kinase activity and by TP53-inducible glycolysis and apoptosis regulator (TIGAR) induction. Importantly, such metabolic rewiring was not observed in normal hepatocytes, undergoing proliferation following 2/3 partial hepatectomy (PH). Activation of the NRF2/KEAP1 pathway and down-regulation of miR-1 accompanied the metabolic reprogramming in CK-19+ preneoplastic lesions. Accordingly, NRF2 silencing decreased G6PD and increased miR-1 expression, consequently inhibiting PPP, while forced expression of miR-1 downregulated G6PD expression in HCC cells. Finally, an inverse correlation between miR-1 and its target gene G6PD was found in human HCC patients. Conclusion: These results demonstrate that metabolic reprogramming takes place at early stages of hepatocarcinogenesis and is likely the consequence of the concomitant activation of the NRF2-KEAP1 pathway. Citation Format: Marta A. Kowalik, Giulia Guzzo, Andrea Morandi, Andrea Perra, Silvia Menegon, Ionica Masgras, Elena Trevisan, Maria M. Angioni, Francesca Fornari, Luca Quagliata, Giovanna M. Ledda-Columbano, Laura Gramantieri, Luigi Terracciano, Silvia Giordano, Paola Chiarugi, Andrea Rasola, Amedeo Columbano. Metabolic reprogramming discriminates aggressive vs. slowly growing preneoplastic lesions at early stages of HCC development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1009.