Two new variants of glucose-6-phosphate dehydrogenase associated with hereditary non-spherocytic hemolytic anemia: G6PD Wayne and G6PD Huron

Two new deficient variants of glucose-6-phosphate dehydrogenase (G6PD) causing hereditary nonspherocytic hemolytic anemia (HNSHA) are described. Both of these are unique and they have been named G6PD Wayne and G6PD Huron. Patients with G6PD Wayne underwent splenectomy and no objective improvement was noted. The patients with G6PD Huron were under medical observation for a considerable period of time without the diagnosis of G6PD deficiency being entertained because the family was of Northern European origin. Since sporadic variants of G6PD causing HNSHA show no special racial predilection, the diagnosis of G6PD deficiency should always be considered in patients with this syndrome.     

Characterization of a new glucose-6-phosphate dehydrogenase variant: G6PD Central City

Glucose-6-phosphate-dehydrogenase deficiency is the most common disease-producing enzyme deficiency in man. This paper describes a new glucose-6-phosphate-dehydrogenase variant discovered during the evaluation of an episode of acute hemolytic anemia in a 62-year-old black male, which was temporally related to the ingestion of Tolbutamide. The hemolysis resolved within 10 days despite continuation of Tolbutamide. The erythrocyte glucose-6-phosphate-dehydrogenase activity was significantly decreased, and its electrophoretic mobility was indistinguishable from wild type enzyme, though faster on starch gel with tris, borate, and phosphate buffers. The enzyme had a biphasic pH optimum reduced Km for G-6-P and NADP, decreased utilization of deamino-NADP, and reduced Ki for NADPH. Because the kinetic properties of this enzyme were unique, we have designated it as G6PD Central City.     

Two new glucose-6 phosphate dehydrogenase (G6PD) variants associated with hemolytic anemia: G6PD Amman-1 and G6PD Amman-2

Two glucose-6-phosphate dehydrogenase (G6PD) variants were investigated. G6PD Amman-1 was partially purified from the red cells of a patient suffering from recurrent jaundice and spontaneous episodic attacks of severe hemolysis in the absence of oxidant drugs, infection, or fava beans. The enzymatic characteristics of G6PD Amman-1 were markedly reduced activity, fast eletrophoretic mobility, slightly increased km for NADP, normal km for G-6-P, normal heat stability, normal utilization of substrate analogues 2-deoxy G-6-P and deamino-NADP, and a monophasic pH curve with a peak at 8.5 to 9.3. The second variant, G6PD Amman-2, was partially purified from the red cells of a patient suffering from recurrent jaundice with episodic mild hemolysis caused by infection or unknown factors. G6PD Amman-2 characteristics were severely reduced activity, slow electrophoretic mobility, normal km for NADP, decreased km for G-6-P, decreased heat stability, increased utilization of substrate analogues, and a monophasic pH curve with a narrow peak at pH 9.5. The red cell level of reduced glutathione was markedly decreased with twofold increase in the activity of glutathione reductase in the patient with G6PD Amman-2.     

Molecular abnormality of a unique japanese glucose-6-phosphate dehydrogenase variant (G6PD Kobe) with a greatly increased affinity for galactose-6-phosphate

Systematic molecular analysis of a Japanese class 1 glucose-6-phosphate dehydrogenase (GGPD) variant (G6PD Kobe) cONA revealed a unique nucleotide substitution (1318 C to T) in exon 11, which predicts a substitution of leucine for phenylalanine at residue 440. This substitution is located in a region surrounding the putative structural NADP-binding domain. The markedly abnormal kinetics of glucose-6-phosphate (GGP) of GGPD Kobe suggest the interaction between both NADP and G6P binding sites. © 1994 Wiley-Liss, Inc.     

葡萄糖-6-磷酸脱氢酶缺乏症的研究进展

葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症是人类最常见的遗传性细胞酶病，在全世界约有4亿多人受累，在一些国家和地区也已成为一个重要的公共卫生问题。G6PD缺乏症多分布于非洲热带、亚洲热带和亚热带、中东、地中海和巴布亚一新几内亚地区。该文对G6PD缺乏症的流行病学、遗传学、分子生物学、临床表现和诊断等方面的研究进展作一综述。     

Glucose-6-phosphate dehydrogenase (G6PD) iserlohn and G6PD regensburg: Two new severe enzyme defects in german families

Summary Two new inheritable variants of glucose-6-phosphate dehydrogenase have been found in two unrelated German families. Patients with one variant (G6PD Iserlohn, also referred to as G6PD I) suffered from intermittent hemolytic crises caused by fava beans; patients with the other variant (G6PD Regensburg, G6PD II) disclosed chronic nonspherocytic hemolytic anemia aggravated by drug treatment. Due to their unusual biochemical characteristics, the new variants were designated G6PD Iserlohn and G6PD Regensburg. Both variants showed a reduction of enzyme activity to about 6% of the normal in erythrocytes, normal electrophoretic mobility, increased affinity for glucose-6-phosphate, a reduced affinity for NADP and a pH optimum in the neutral region (7.0 and 7.5). G6PD Iserlohn had a decreased affinity for the inhibitor NADPH; G6PD Regensburg had a normal inhibitor constant. Deamino NADP was utilized at an increased rate by G6PD Regensburg. G6PD Iserlohn was thermostable, G6PD Regensburg mildly instable. G6PD activity in leukocytes was normal in G6PD Iserlohn and reduced to the same degree as in erythrocytets in G6PD Regensburg. The cause of the decreased activity of G6PD Iserlohn appears to be in vivo instability; in G6PD Regensburg further mechanisms might include reduced specific activity or reduced synthesis of the variant enzyme.     

Two new glucose 6-phosphate dehydrogenase variants associated with congenital nonspherocytic hemolytic anemia found in Japan: Gd(-) tokushima and gd(-) tokyo

Two new variants of glucose 6-phosphate dehydrogenase (G6PD) deficiency associated with chronic nonspherocytic hemolytic anemia were discovered in Japan. Gd(-) Tokushima was found in a 17-year-old male whose erythrocytes contained 4.4% of normal enzyme activity. Partially purified enzyme revealed a main band of normal electrophoretic mobility with additional two minor bands of different mobility; normal Km G6P, and Km NADP five- to sixfold higher than normal; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; marked thermal instability; a normal pH curve; and normal Ki NADPH. The hemolytic anemia was moderate to severe. Gd(-) Tokyo was characterized from a 15-year-old male who had chronic nonspherocytic hemolytic anemia of mild degree. The erythrocytes contained 3% of normal enzyme activity, and partially purified enzyme revealed slow electrophoretic mobility (90% of normal for both a tris-hydrochloride buffer system and a tris-EDTA-borate buffer system, and 70% of normal for a phosphate buffer system); normal Km G6P and Km NADP; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; greatly increased thermal instability; a normal pH curve; and normal Ki NADPH. These two variants are clearly different from hitherto described G6PD variants, including the Japanese variants Gd(-) Heian and Gd(-) Kyoto. The mothers of both Gd(-) Tokushima and Gd(-) Tokyo were found to be heterozygote by an ascorbate-cyanide test.     

Combined glucose-6-phosphate dehydrogenase and glucosephosphate isomerase deficiency can alter clinical outcome

Glucosephosphate isomerase (GPI) deficiency in humans is an autosomal recessive disorder, which results in nonspherocytic hemolytic anemia of variable clinical expression. A 4-year-old female with severe congenital hemolytic anemia had low red cell GPI activity of 15.5 IU/g Hb (50% of normal mean) indicating GPI deficiency. Subsequent DNA sequence analysis revealed a novel homozygous 921C to G mutation in the GPI gene sequence, predicting a Phe307 to Leu replacement. Strikingly, the red cell GPI activity in this patient was higher than that found in a second patient expressing the same GPI variant, with a more severe clinical phenotype. We propose that the hemolysis in the first patient may be modified by an accompanying deficiency of glucose-6-phosphate dehydrogenase (G6PD). The proband's red cell G6PD activity was reduced at 4.5 IU/g Hb (50% of normal mean) and molecular studies revealed heterozygosity for the G6PD Viangchan mutation and a skewed pattern of X-chromosome inactivation, producing almost exclusive expression of the mutated allele. The G6PD Viangchan variant is characterised by severe enzyme deficiency, but not chronic hemolysis. This study suggests that the metabolic consequences of a combined deficiency of GPI and G6PD might be responsible for a different clinical outcome than predicted for either defect in isolation.     

Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by microarray-based assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy, affecting more than 200 million people worldwide. To date more than 123 mutations in the G6PD gene have been discovered, among which 12 point mutations are found in the Chinese. Setting up a simple and accurate method for detecting these mutations is not only useful for diagnosing G6PD deficiency under some circumstances that it is difficult to measure the activity of the enzyme, but also for studying the frequency of the G6PD genotypes. The purpose of this study was to develop a simple, inexpensive and accurate method for detecting these common mutations. Microarray-based assay was described in this study. Samples from 198 G6PD-deficient persons were investigated. The DNA sequencing data supported the results obtained by microarray-based assay. Thus, we concluded that the microarray-based assay is a rapid, simple, inexpensive, and accurate method for detecting the most common G6PD gene mutations among the Chinese. This method involves the selective amplification of human G6PD gene with specific oligonucleotide primers, fragmentation and labeling of PCR products, followed by hybridization with allele-specific oligonucleotide (ASO) probes on chip.     

Prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Ouest and Sud-Est departments of Haiti

Malaria remains a significant public health issue in Haiti, with chloroquine (CQ) used almost exclusively for the treatment of uncomplicated infections. Recently, single dose primaquine (PQ) was added to the Haitian national malaria treatment policy, despite a lack of information on the prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency within the population. G6PD deficient individuals who take PQ are at risk of developing drug induced hemolysis (DIH). In this first study to examine G6PD deficiency rates in Haiti, 22.8% (range 14.9%–24.7%) of participants were found to be G6PD deficient (class I, II, or III) with 2.0% (16/800) of participants having severe deficiency (class I and II). Differences in deficiency were observed by gender, with males having a much higher prevalence of severe deficiency (4.3% vs. 0.4%) compared to females. Male participants were 1.6 times more likely to be classified as deficient and 10.6 times more likely to be classified as severely deficient compared to females, as expected. Finally, 10.6% (85/800) of the participants were considered to be at risk for DIH. Males also had much higher rates than females (19.3% vs. 4.6%) with 4.9 times greater likelihood (p value 0.000) of having an activity level that could lead to DIH. These findings provide useful information to policymakers and clinicians who are responsible for the implementation of PQ to control and manage malaria in Haiti.     

T-lymphocyte subpopulation changes during hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient children

Lymphocyte subpopulations were studied by means of sheep red blood cell (SRC)-rosetting technique and OKT11, OKT3, OKT4, and OKT8 monoclonal antibodies in ten Sicilian glucose-6-phosphate dehydrogenase-deficient children during the hemolytic crisis due to fava bean ingestion. The number of SRC-rosetting lymphocytes was significantly reduced, while the number of OKT3-positive cells was normal. An inversion of the OKT4/OKT8 ratio was observed, due to a decrease of OKT4-positive cells and an increase of OKT8-positive cells. All these abnormalities reverted to normal in the four children studied after recovery.     

Congenital 6-phosphogluconate dehydrogenase (6PGD) deficiency associated with chronic hemolytic anemia in a Spanish family

Clinical and metabolic studies were performed in four members of a Spanish family with partial (50%) 6 phosphogluconate dehydrogenase (6PGD) deficiency. In all cases the activities of 6 phosphogluconolactone (6PGL) and glutathione reductase (GR) were normal, and the molecular characterization performed in the partially purified 6PGD from the propositus showed normal kinetic and electrophoretic patterns. Two females (the propositus and her sister) suffered from a well-compensated chronic nonspherocytic hemolytic anemia (CNSHA) and exhibited decreased RBC glutathione (GSH) stability with increased oxidative susceptibility, defined by enhanced malonyldialdehyde (MDA) generation “in vitro.” The other two members of the family (the propositus's mother and brother) were clinically asymptomatic. In the propositus and her sister, RBC metabolism exhibited a markedly abnormal concentration of glycolytic intermediates, mainly characterized by striking increases in fructose 1,6 bisphosphate (50-fold), dihydroxiacetone-phosphate (20-fold) and glyceraldehyde 3-phosphate (tenfold). Although the precise mechanism of the hemolysis in the two patients is unknown, the enhanced oxidative threat observed in their RBCs may interfere in some way with the glycolytic pathway function, leading to a marked increase in certain metabolic intermediates located before the glyceraldehyde 3 phosphate dehydrogenase (GA3PD) step. Since it seems that GA3PD half-life is modulated by fluctuations of the cytosolic redox status, an “in situ” approach was simulated by using permeabilized RBCs. In these conditions, GA3PD activity was significantly lower in the propositus and her sister than in the asymptomatic members of the family and the simultaneous normal control. © 1996 Wiley-Liss, Inc.     

G6PD Avenches and G6PD Moosburg: biochemical and erythrocyte membrane characterization

Summary Two new G6PD variants with severe enzyme deficiency in Switzerland (G6PD Avenches, G6PD I) and in Germany (G6PD Moosburg, G6PD II) are described. One patient had suffered from severe postpartal hyperbilirubinemia, the other one presented with chronic hemolysis and remittent hyperbilirubinemia. Both variants showed diminished electrophoretic mobility, both variants were heat labile. The Michaelis-Menten constants K_M for glucose-6-phosphate and for NADP⁺ were normal. 2-Desoxy-glucose-6-phosphate was utilized by G6PD I in a higher and by G6PD II at a lower rate than by the normal enzyme. Desamino-NADP⁺ and galactose-6-phosphate were utilized by both variants at a normal rate. The electrophoretic separation of membrane proteins of G6PD II showed both in the presence and in the absence of 6-mercaptoethanol no difference concerning the formation of membrane protein aggregates between patient and normal control.     

Molecular genetics of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain: identification of two new point mutations in the G6PD gene

Summary .In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain, we have analysed the G6PD gene in 11 unrelated Spanish G6PD-deficient males and their relatives by using the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with a direct PCR-sequencing procedure and PCR-restriction enzyme (RE) analysis. We have identified eight different missense mutations, six of which have been reported in previously described G6PD variants. In nine patients who had presented with acute favism we found the following mutations: G6PD A_^376G–202A (four cases)| G6PD Union^1360T (two cases), G6PD Mediterranean^563T, (one case) and G6PD Aures^143c (one case). In the remaining patient a novel A to G transition was found at nucleotide position 209 which has not been reported in any other ethnic group. This mutation results in a (70) Tyr to Cys substitution and the resulting G6PD variant was biochemically characterized and designated as G6PD Murcia. This new mutation creates a Bsp 12861 recognition site which enabled us to rapidly detect it by PCRRE analysis. In two patients with chronic non-spherocytic haemolytic anaemia (CNSHA) we found the underlying genetic defects, as had been noted previously, to be located within a cluster of mutations in exon 10. One of them had the T to C transition at nucleotide 1153, causing a (3 8 5) Cys to Arg substitution, previously described in G6PD Tomah. The other, previously reported as having a variant called G6PD Clinic, has a G to A transition at nucleotide 1215 that produces a (405) Met to He substitution, thus confirming that G6PD Clinic is a new class I variant.     

Chronic hemolytic anemia is associated with a new glucose-6-phosphate dehydrogenase in-frame deletion in an older woman

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked disorder, is usually observed in hemizygote males and very rarely in females. The G6PD class 1 variants, very uncommon, are associated with chronic hemolytic anemia. Here we report a Portuguese woman who suffered in her sixties from a chronic hemolytic anemia due to G6PD deficiency. Molecular studies revealed heterozygosity for an in-frame 18-bp deletion, mapping to exon 10 leading to a deletion of 6 residues, 362-367 (LNERKA), which is a novel G6PD class 1 variant, G6PD Tondela. Two of her three daughters, asymptomatic, with G6PD activity within the normal range, are heterozygous for the same deletion. The patient's leukocyte and reticulocyte mRNA studies revealed an almost exclusive expression of the mutant allele, explaining the chronic hemolytic anemia. Patient whole blood genomic DNA HUMARA assay showed a balanced pattern of X chromosome inactivation (XCI), but granulocyte DNA showed extensive skewing, harboring the mutated allele, implying that in whole blood, lymphocyte DNA, with a very long lifetime, may cover up the current high XCI skewing. This observation indicates that HUMARA assay in women should be assessed in granulocytes and not in total leukocytes.     

Biochemical and molecular characterization of a new sporadic glucose-6-phosphate dehydrogenase variant described in Italy: G6PD Modena

Summary. A new glucose-6-phosphate dehydrogenase variant detected in an Italian man from the Po delata is described and designated as G6PD Modena. Biochemical characterization of the variant enzyme revealed an activity 21% of normal, a slow electrophoretic mobility, increased K_m value for NADP, decreased K_m value for G6P and a complete absence of NADPH inhibition, which could account for the apparently nonhaemolytic feature of this variant. The cloning and sequencing of the G6PD Modena allele showed a GC transition at nucleotide 844 in exon VIII causing a Asp His amino acid substitution. On the basis of biochemical characterization, G6PD Modena is classified as a genuine variant but it has the same mutation as G6PD Seattle-like.     

Two new mutations of the glucose-6-phospate dehydrogenase (G6PD) gene associated with haemolytic anaemia: clinical, biochemical and molecular relationships

In two unrelated Spanish males with glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemolytic anaemia, and two different novel point mutations in the G6PD gene, have been identified. A C to T transition at nucleotide 406 resulting in a (136) Arg to Cys substitution and a C to G transition at nucleotide 1155 resulting in a (385) Cys to Trp substitution. These two molecular defects have not been described before and are designated G6PD Valladolid^{406 C→T} and G6PD Madrid^{1155 C→G} .
In vitro biochemical characterization of both mutant enzymes showed important differences in their molecular properties according to their different clinical behaviour. In G6PD Valladolid, the mutation of which is located in exon 5, the normal in vitro heat stability may explain its mild clinical expression (low-grade haemolysis interrupted by an acute haemolytic crisis at age 70). In G6PD Madrid, the mutation, located in exon 10, results in a deficient variant associated with neonatal jaundice and life-long chronic nonspherocytic haemolytic anaemia (CNSHA). This finding further emphasizes the importance of this specific region of the G6PD gene in the stabilization of the G6PD molecule. Putative relationships between these single point mutations and the molecular properties of the mutant enzymes are also discussed.     

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

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

Modification to reporting of qualitative fluorescent spot test results improves detection of glucose-6-phosphate dehydrogenase (G6PD)-deficient heterozygote female newborns

Introduction: The glucose‐6‐phosphate dehydrogenase (G6PD) fluorescent spot test (FST) is a useful screening test for G6PD deficiency, but is unable to detect heterozygote G6PD‐deficient females. We sought to identify whether reporting intermediate fluorescence in addition to absent and bright fluorescence on FST would improve identification of mildly deficient female heterozygotes. Methods: A total of 1266 cord blood samples (705 male, 561 female) were screened for G6PD deficiency using FST (in‐house method) and a quantitative enzyme assay. Fluorescence intensity of the FST was graded as either absent, intermediate or normal. Samples identified as showing absent or intermediate fluorescence on FST were analysed for the presence of G6PD mutations using TaqMan@SNP genotyping assays and direct nucleotide sequencing. Results: Of the 1266 samples, 87 samples were found to be intermediate or deficient by FST (49 deficient, 38 intermediate). Of the 49 deficient samples, 48 had G6PD enzyme activity of ≤ 9.5 U/g Hb and one sample had normal enzyme activity. All 38 intermediate samples were from females. Of these, 21 had G6PD activity of between 20% and 60%, and 17 samples showed normal G6PD activity. Twenty‐seven of the 38 samples were available for mutation analysis of which 13 had normal G6PD activity. Eleven of the 13 samples with normal G6PD activity had identifiable G6PD mutations. Conclusion: Glucose‐6‐phosphate dehydrogenase heterozygote females cannot be identified by FST if fluorescence is reported as absent or present. Distinguishing samples with intermediate fluorescence from absent and bright fluorescence improves detection of heterozygote females with mild G6PD deficiency. Mutational studies confirmed that 85% of intermediate samples with normal enzyme activity had identifiable G6PD mutations.     

NBT纸片定性法与G6PD/6PGD比值法在大样本G6PD缺陷症筛查中的联合应用

目的：探讨联合应用葡萄糖-6-磷酸脱氢酶（G6PD）四氮唑蓝（NBT）纸片定性法与G6PD／6PGD比值定量法在大样本筛查中的可行性。方法：用NBT纸片定性法对501例贵州省江口县土家族、525例从江县侗族、586例荔波县瑶族共1612例成人进行定性初筛，再用G6PD／6PGD比值法对初筛阳性样本定量复查。结果：NBT纸片定性法共初筛出G6PD缺陷患者129例，G6PD／G6PD比值法确诊G6PD缺陷123例，2种方法的符合率高达95．35％。结论：对大样本G6PD缺陷症的筛查，先用NBT纸片定性法进行初筛，再用G6PD／6PGD比值法复查确诊，2法联合应用可以提高G6PD缺陷症检出率和节约大量经费和时间。