Characterization of glucose-6-phosphate dehydrogenase variants. II. G6PD Kephalonia, G6PD Attica, and G6PD "Seattle-like" found in Greece.
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Chromatography↗
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A glucose-6-phosphate dehydrogenase (G6PD) splice site consensus sequence mutation associated with G6PD enzyme deficiency.
A glucose-6-phosphate dehydrogenase (G6PD) deficient strain of mouse (GPDX) which was developed using the ethylating agent ethylnitrosourea (ENU) has been used to study clonality in epithelial tissues. While the biochemical defect has been quantified, the genetic basis of the deficiency is unknown. The G6PD gene is composed of 13 exons. Exon 1 is not translated, and the ATG start site is near the 5' end of exon 2. Direct sequencing of the exonic regions of the gene from GPDX, C3H, 101, C57BL/6 and BALB/c mice was carried out. The coding region, in which (with a single exception) all mutations found to cause G6PD deficiency in man are situated, showed identical sequences in three of the four strains studied (101 coding region sequence was not examined). However, the G6PD gene in the GPDX mouse showed a single base difference from the other four strains and from the published mouse G6PD sequence (BALB/c) in the 5' splice site consensus sequence at the 3' end of exon 1, part of the untranslated region. The difference was confirmed in four different GPDX mice. This mutation was of the type (A to T transversion) that is known to be induced by ENU; its effect is likely to be exerted through a defect in transcription, splicing or translation, leading to a reduction in protein levels. By Western blot we have found a marked decrease in the G6PD protein levels in the GPDX mouse, with the C3H X GPDX heterozygote showing a lesser decrease. Recently, an increasing number of mutations in the untranslated regions of genes have been found which have effects on protein levels. We believe that the reduced enzyme activity in the GPDX mouse is due to the mutation in the 5' untranslated region (UTR), and that similar mutations may be relevant in other inherited conditions.
Alternative Splicing↗
Investigation of glucose 6-phosphate dehydrogenase (G6PD) kinetics for normal and G6PD-deficient persons and the effects of some drugs.
In the present study, blood samples from 1183 children aged 0.5-6 years were taken. Three children were found with G6PD deficiency by examining the enzyme activity and hemoglobin ratio. Some kinetic properties of glucose 6-phosphate dehydrogenase enzyme (G6PD) were studied after the purification of the enzyme with ammonium fractionation, dialysis and 2',5' ADP-Sepharose 4B affinity chromatography from a healthy person and from three G6PD-deficient people. The purity of the enzymes was confirmed by SDS-PAGE electrophoresis. The effects of some drugs which are known inhibitors of G6PD activity were studied. Some of the drugs stimulated the activity of the enzyme in two of the three cases with G6PD deficiency. KM values, Vmax values for G6P and NADP+, optimum pH and optimum temperature for the enzyme from the healthy person and the three G6PD-defficient people are reported.
Child↗
Molecular abnormality of G6PD Konan and G6PD Ube, the most common glucose-6-phosphate dehydrogenase variants in Japan.
G6PD Konan and G6PD Ube are the most common glucose-6-phosphate dehydrogenase (G6PD) variants found in Japan. To clarify the molecular abnormality of these two variants, the entire coding region was amplified by polymerase chain reaction from genomic DNA (G6PD Konan) or cDNA (G6PD Ube). Direct sequencing revealed that both variants have the same nucleotide substitution (241 C to T) in exon 4, which predicts an Arg to Cys substitution at amino acid 81.
Arginine↗
Linkage between a PvuII restriction fragment length polymorphism and G6PD A-202A/376G: evidence for a single origin of the common G6PD A- mutation.
DNA samples from 29 males with the G6PD A- phenotype and 14 males with a G6PD B phenotype were studied for the presence of each of four polymorphic restriction sites in the glucose-6-phosphate dehydrogenase locus. All G6PD A- subjects with the G6PD A-202A/376G genotype, regardless of population origin, shared identical haplotypes. In view of the fact that at least one of the restriction sites, the PvuII site in the intron between exon 5 and 6, has thus far been uncommon in the populations studied, it seems likely that the G6PD A- mutation at nucleotide 202 arose relatively recently and in a single individual.
Base Sequence↗
Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency by natural and amplification created restriction sites: five mutations account for most G6PD deficiency cases in Taiwan.
We have developed a rapid and simple method to diagnose the molecular defects of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Chinese in Taiwan. This method involves the selective amplification of a DNA fragment from human G6PD gene with specific oligonucleotide primers followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. Ninety-four Chinese males with G6PD deficiency were studied. The results show that 50% (47 of 94) were G to T mutation at nucleotide (nt) 1376, 21.3% (20 of 94) were G to A mutation at nt 1388, 7.4% (7 of 94) were A to G mutation at nt 493, 7.4% (7 of 94) were A to G mutation at nt 95, 4.2% (4 of 94) were C to T mutation at nt 1024, 1.1% (1 of 94) was G to T mutation at nt 392, and 1.1% (1 of 94) was G to A mutation at nt 487. These results show that the former five mutations account for more than 90% of G6PD deficiency cases in Taiwan. Aside from showing that G to T change at nt 1376 is the most common mutation, our research indicates that nt 493 mutation is a frequent mutation among Chinese in Taiwan. We compared G6PD activity among different mutations, without discovering significant differences between them.
Base Sequence↗
Linkage disequilibrium of polymorphic sites in the G6PD gene in African populations and the origin of G6PD A-.
So far, four polymorphic sites are known to exist in the glucose 6-phosphate dehydrogenase (G6PD) gene of people of African origin: the mutation in G6PD A (which creates a FokI restriction enzyme site); the additional mutation in G6PD A- (which creates an NlaIII site); and two restriction fragment length polymorphisms (PvuII and PstI). We have investigated the status of these four sites in 78 men of African descent and found them to exist in linkage disequilibrium--only five of the ten haplotypes expected at least twice under a random assortment regimen were observed. The mutation of G6PD A- is found only in the presence of the PvuII and PstI sites and we therefore suggest that it has arisen only once. We propose a likely sequence for the evolution of these different mutations in the G6PD gene.
Africa↗
Glucose-6-phosphate dehydrogenase (G6PD) deficiency in southern Italy: a case of G6PD A(-) associated with favism.
During a routine screening for G6PD deficiency in the Province of Matera (Southern Italy), an eleven-year-old boy was brought to our attention who had fever obviously caused by a viral infection, but who also had hepatosplenomegaly and haemoglobinuria. The boy had previously experienced two severe haemolytic attacks. At the age of six months severe haemolysis occurred after the ingestion of cooked fava beans. At the age of seven years, the haemolytic episode was very likely triggered by oral administration of co-trimoxazole. The G6PD activity level in erythrocyte lysate was clearly defective (25% of normal). The electrophoretic mobility of G6PD was 110% of normal. These data together with those obtained from biochemical and molecular characterisation allowed the variant to be identified as G6PD A(-). This is the first report of an association between the African type G6PD deficiency variant and favism.
Adolescent↗
Detection of point mutations in exon 2 of the G6PD gene in Chinese G6PD variants.
In the past few years, a total of 6 different mutations of the G6PD gene have been reported in China. One of these, the C6 mutation (A95-->G), accounted for about 15.4% of the Chinese G6PD variants. In order to develop a strategy for rapid detection of mutation-containing exons of the G6PD gene, we applied the single-strand conformation polymorphism (SSCP) technique to the detection of mutations in exon 2 of this gene. We observed four patients with abnormal migration patterns of the exon 2 band among 20 cases of G6PD variants. Direct PCR sequencing confirmed a T to C substitution in exon 2 that has previously been reported. This procedure is therefore of particular importance for the rapid detection of mutation-containing exons in the G6PD gene.
Base Sequence↗
Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency.
In population-based studies it has been established that inherited deficiency of erythrocyte (E) glucose-6-phosphate dehydrogenase (G6PD) confers protection against severe Plasmodium falciparum (P falciparum) malaria. Impaired growth of parasites in G6PD-deficient E in vitro has been reported in some studies, but not in others. In a systematic analysis, we have found that with five different strains of P falciparum (FCR-3, KI, C10, HB3B, and T9/96), there was no significant difference in either invasion or maturation when the parasites were grown in either normal or G6PD-deficient (Mediterranean variant) E. With all of these strains and at different maturation stages, we were unable to detect any difference in the amount of P falciparum-specific G6PD mRNA in normal versus deficient parasitized E. The rate of 14C-CO2 production from D-[1-14C] glucose (which closely reflects intracellular activity of G6PD) contributed by the parasite was very similar in intact normal and deficient E. By contrast, in studies of phagocytosis of parasitized E by human adherent monocytes, we found that when the parasites were at the ring stage (ring-stage parasitized E [RPE]), deficient RPE were phagocytosed 2.3 times more intensely than normal RPE (P = .001), whereas there was no difference when the parasites were at the more mature trophozoite stage (trophozoite-stage parasitized E [TPE]). Phagocytic removal markers (autologous IgG and complement C3 fragments) were significantly higher in deficient RPE than in normal RPE, while they were very similar in normal and deficient TPE. The level of reduced glutathione was remarkably lower in deficient RPE compared with normal RPE. We conclude that impaired antioxidant defense in deficient RPE may be responsible for membrane damage followed by phagocytosis. Because RPE, unlike TPE, are nontoxic to phagocytes, the increased removal by phagocytosis of RPE would reduce maturation to TPE and to schizonts and may be a highly efficient mechanism of malaria resistance in deficient subjects.
Animals↗
Neutrophils and monocytes from subjects with the Mediterranean G6PD variant: effect of Plasmodium falciparum hemozoin on G6PD activity, oxidative burst and cytokine production.
Glucose 6-phosphate dehydrogenase (G6PD) activity and oxidative burst were measured in neutrophils and monocytes from five, hemizygous, G6PD-deficient (Mediterranean variant) individuals and five normal controls. Additionally, tumor necrosis factor (TNF), interleukin-10 (IL-10), interleukin-12 (IL-12) release and phagocytosis of the malarial pigment hemozoin or opsonized erythrocytes (RBC) were measured in monocytes recovered from G6PD-deficient and normal individuals. G6PD activity was significantly lower in "deficient monocytes" (38% residual activity, p = 0.01) and not significantly different in "deficient neutrophils" (79% residual activity, p = 0.83) compared to homologous leukocytes recovered from normal controls. Oxidative burst was not significantly different in "deficient" versus "normal" neutrophils and monocytes. Previous phagocytosis of hemozoin decreased the phorbol ester induced oxidative burst in "deficient" and "normal" monocytes but not in neutrophils. Phagocytosis of hemozoin and RBC strongly stimulated cytokine production. With the exception of IL-10, the cytokine production pattern was comparable in "deficient" versus "normal" cells. Incubation with high concentrations of hemozoin (equivalent to 300 RBC per monocyte) strongly stimulated TNF production. Lipopolysaccharide (LPS) had an additive effect on TNF production induced by hemozoin or opsonized RBC. IL-12 production was induced only by the presence of large amounts of hemozoin. IL-10 production was increased in normal monocytes incubated with RBC or hemozoin. LPS increased IL-10 production significantly in monocytes incubated with RBC or low amounts of hemozoin (equivalent to 30 RBC per monocyte), but had no effect when given alone or in conjunction with high concentrations of hemozoin. Interestingly, deficient monocytes produced less IL-10 than normal cells under these conditions. In conclusion, except for IL-10 production, we did not find major functional differences between neutrophils and monocytes from individuals with or without the Mediterranean G6PD mutation.
Animals↗
Marked decrease in specific activity contributes to disease phenotype in two human glucose 6-phosphate dehydrogenase mutants, G6PD(Union) and G6PD(Andalus).
Clones overexpressing clinical glucose 6-phosphate dehydrogenase (G6PD) mutants Union (c.1360C>T/p.Arg454Cys) and Andalus (c.1361G>A/p.Arg454His), have been constructed. These abolish a salt bridge between Arg454 and Asp 286. One mutant is reportedly a Class II clinical variant and the other a Class I. Kinetic studies of the purified proteins reveal that, for both mutants, kcat is about 10-fold decreased, thus giving a 90% decrease in the WHO assay, and also presumably under physiological conditions. In contrast with unfavourable changes in Vmax for both mutants, Km values for both G6P and NADP+ are decreased approximately 5-fold. Measurements with alternative substrates confirm that G6PD Union, like the wild-type enzyme, follows a rapid-equilibrium random-order mechanism, allowing calculation of enzyme-substrate dissociation constants from initial-rate parameters. The mutations result in several-fold tighter binding of glucose 6-phosphate to the free enzyme. Binding, however, is clearly less productive than with normal enzyme. G6PD mutations are thought to cause haemolytic anaemia by compromising enzyme stability. Both these mutants indeed show somewhat decreased thermostability. However, at 37 degrees C and with NADP+, the stability differences are only moderate. Decreased catalytic efficiency clearly contributes to the disease phenotype of these two mutants, entirely accounting for reported decrease in leukocyte G6PD levels, though not for still lower levels in erythrocytes. Neither the kinetic nor the stability effects appear to justify the different clinical classification of these mutations.
Anemia, Hemolytic↗
G6PD Ferrara I has the same two mutations as G6PD A(-) but a distinct biochemical phenotype.
The cloning and sequencing of the normal glucose-6-phosphate dehydrogenase (G6PD) gene has led to the study of the molecular defects that determine enzymatic variants. In this paper, we describe the mutations responsible for the Ferrara I variant in an Italian man with a family history of favism, from the Po delta. Nucleotide sequencing of this variant showed a G-->A mutation at nucleotide 202 in exon IV causing a Val-->Met amino acid exchange, and a second A-->G mutation at nucleotide 376 in exon V causing an Asn-->Asp amino acid substitution. Although on the basis of its biochemical properties this variant was classified as G6PD Ferrara I, it has the same two mutations as G6PD A(-), which is common in American and African blacks, and as the sporadic Italian G6PD Matera. The mutation at nucleotide 202 was confirmed by NlaIII digestion of a polymerase chain reaction amplified DNA fragment spanning 109 bp of exon IV. The 109-bp mutated amplified sequence is not distinguishable from the normal sequence in single strand conformation polymorphism analysis.
Cloning, Molecular↗
Glucose-6-phosphate dehydrogenase (G6PD) activity in tumoral tissues of G6PD-deficient subjects affected by larynx carcinoma.
The activity of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the hexose monophosphate (HMP) shunt pathway, was measured in both normal and tumoral larynx tissues from normal and G6PD deficient subjects. Significant increases of this enzymatic activity were found in tumoral tissues of both normal and G6PD deficient subjects, who were characterized by very low levels of G6PD activity in erythrocytes as well as in larynx tissue.
Aged↗
Glucose-6-phosphate dehydrogenase (G6PD) mutations and UDP-glucuronosyltransferase promoter polymorphism among G6PD deficient Kuwaitis.
Screening of 1,080 Kuwaiti male blood donors for glucose-6-phosphate dehydrogenase (G6PD) deficiency revealed this condition in 70 (6.5%) individuals. Mutation analysis of all 70 G6PD deficient samples performed by PCR/RFLP and direct sequencing identified the 563C-->T (Mediterranean) in 72.9%, 202G-->A (A(-)) in 14.3%, 1003G-->A (Chatham) in 7.1%, and 143T-->C (Aures) in 1.4%. In 3 cases (4.3%) mutations remain unknown. Genotyping of all G6PD deficient samples for UDP-glucuronosyltransferase 1 (UDPGT1) gene promoter polymorphism revealed (ta)6/(ta)6 in 38.6%, (ta)7/(ta)7 in 15.7%, (ta)6/(ta)7 in 44.3%, and (ta)7/(ta)8 allele in 1.4% of cases. Thus, 4% of males in the Kuwaiti population have G6PD deficiency coexisting with low activity of the UDPGT1 promoter.
Gene Frequency↗
Quantitative expression of G6PD activity of different phenotypes of G6PD and haemoglobin in a Sudanese population.
597 unrelated persons, comprising of 401 males and 196 females, were investigated for glucose-6-phosphate dehydrogenase (G6PD) and haemoglobin phenotypes by starch gel electrophoresis. The levels of G6PD activity were assayed in order to study the quantitative expression of G6PD phenotypes and the influence of haemoglobin phenotypes on such expression. There was no significant different in the levels of G6PD activity in subjects with GdA or GdB. The mean levels of the enzyme activity were 165.5 +/- 33.7 and 164.8 +/- 33.8 IU/10(12) red cells in males and 159.3 +/- 27.8 and 163.4 +/- 33.5 IU/10(12) red cells in females, respectively. 14 subjects with Gd(+) "Khartoum" had significantly (p less than 0.001) higher level of enzyme activity with a mean above 200 IU/10(12) red cells. On the other hand, 20 subjects with GdB(int) (demonstrated by visual comparison of starch gel) showed significantly (p less than 0.001) lower levels of enzyme activity (107.6 +/- 23.5 IU/10(12) red cells). The heterozygotes GdAB also had slightly, but not significantly lower levels of enzyme activity than either GdA or GdB. The mean level of activity for GdAB was 140.1 +/- 29.4 IU/10(12) red cells.
Adolescent↗
Congenital dyserythropoietic anaemia type II associated with a new type of G6PD deficiency (G6PD Gabrovizza).
A 6-year-old boy with chronic haemolytic anaemia was found to have glucose 6-phosphate dehydrogenase (G6PD) deficiency and the morphological, ultrastructural and serological features of congenital dyserythropoietic anaemia (CDA) type II. The patient's mother was heterozygous for G6PD deficiency. G6PD from the patient's red cells, upon partial purification and full characterization, was found to be a new variant designated G6PD Gabrovizza. We conclude that two distinct genetic abnormalities coexisted in this patient. We suggest that CDA type II may become clinically more expressed when another abnormality of the erythrocytes coexists.
Anemia, Dyserythropoietic, Congenital↗
Entrapment of normal and mutant glucose 6-phosphate dehydrogenase (G6PD) within G6PD deficient erythrocytes.
G6PD-deficient erythrocytes (Mediterranean type of this enzyme disorder) were loaded with a) normal G6PD purified to homogeneity from human erythrocytes, b) G6PD Mediterranean purified from deficient granulocytes. The first set of experiments led to complete normalization of biochemical properties of erythrocytes, as assessed by evaluating their metabolic competence under steady state conditions and under oxidative stress as well. The second type of experiment allowed us to conclude that mutant G6PD is not appreciably destroyed within the affected erythrocytes.
Erythrocytes↗