Glucose-6-phosphate dehydrogenase (G6PD) Iserlohn and G6PD Regensburg: two new severe enzyme defects in German families

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.     

Thessaly variant of Glucose-6-phosphate Dehydrogenase

Summary A new variant of the erythrocytic enzyme Glucose-6-phosphate Dehydrogenase was detected in two unrelated Greek individuals. The variant was designated G6PD Thessaly. It is characterized by normal levels of G6PD activity in the red cells and electrophoretic migration slower than G6PD B on phosphate and T.E.B. buffers while faster than G6PD B on Tris-HCl buffer. In addition, the Thessaly variant has distinctly decreased affinity for NADP.This study was supported by National Institutes of Health Grant GM 15253.     

2-deoxy-glucose-6-phosphate utilization in the study of glucose-6-phosphate dehydrogenase mosaicism.

The electrophoretic difference between normal glucose-6-phosphate dehydrogenase (G6PD) and two common variants (G6PD A and G6PD A-) has made the G6PD enzyme system very useful for genetic studies and for investigation on the clonal origin of tumors. This approach has not been possible for another common variant, G6PD mediterranean, which has a normal electrophoretic pattern. The different utilization of 2-deoxy-glucose-6-phosphate (2dG6P), an analog of the normal substrate, by the normal enzyme and the Mediterranean variant, allows a convenient determination of the degree of mosaicism in mononuclear cells from heterozygotes.     

Genetic polymorphism of G6PD in a Bulgarian population

Summary Considerable genetic heterogeneity in G6PD was found in the Bulgarian population-14 G6PD variants isolated from 117 hemizygous carriers of G6PD deficiency. Of these, G6PD Mediterranean type was a polymorphic variant and G6PD Corinth occured with high frequency. Two new variants were identified-G6PD Rudosem and G6PD Nedelino. In a selected group of 78 subjects with clinical manifestations, four variants were established: G6PD Mediterranian, G6PD Corinth, G6PD Seattle and G6PD Ohut II.     

G6PD Huntsville: a new glucose-6-phosphate dehydrogenase associated with chronic hemolytic anemia

Summary We describe a previously unreported glucose-6-phosphate dehydrogenase (G6PD) variant. G6PD Huntsville was found in a Caucasian male, resident of Huntsville, Alabama who was investigated for otherwise unexplained chronic hemolytic anemia. An unusual feature of this unique, apparently hemolytic, G6PD mutant is that its red cell enzymatic activity has not been decreased. The mutant enzyme is unstable. Additionally, the enzyme variant is characterized by normal electrophoretic mobility, biphasic and slightly alkaline pH optimum, and abnormal kinetics for the natural substrates G6PD and NADP as well as the artificial substrates deamino NADP. Its activity for another artificial substrate 2-deoxy G6PD is normal. The inhibition constant for NADPH is normal. The subject has had no evidence of episodic jaundice.     

RFLP of the X chromosome-linked glucose-6-phosphate dehydrogenase locus in blacks.

The X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) A(+) variant is found in approximately 20% of blacks. Examination of the structure of the G6PD A(+) gene revealed that AT----GC transition occurred in the variant gene, resulting in the amino acid substitution Asn----Asp at the one hundred forty-second position from the NH2-terminal of the enzyme (Takizawa and Yoshida 1987). The nucleotide change created an additional FokI cleavage site in the variant A(+) gene; thus, the FokI fragment type of the variant A(+) DNA differs from that of the normal B(+) DNA. PvuII fragment type is also found to be polymorphic in blacks, but not in Caucasians. The majority of blacks, as well all nonblacks, have a major hybridization-positive fragment of approximately 4.0 kbp (PvuII type 1), while approximately 20% of blacks have a major fragment of approximately 1.5 kbp (PvuII type 2). The G6PD gene with PvuII type 2 contains an additional PvuII cleavage site approximately 0.7 kbp downstream from the mutation site of the G6PD A(+). Approximately 40% of the G6PD A(+) genes have PvuII type 2, while only approximately 10% of the G6PD B(+) genes are associated with PvuII type 2. The data indicate a statistically significant (X2 = 6.85, P less than .020) linkage disequilibrium between the G6PD types and the PvuII types at the G6PD locus.     

Identification of HeLa cell glucose 6-phosphate dehydrogenase

Although the electrophoretic mobility of HeLa G6PD is similar to that of the common Negro variant G6PD A+, several reports have suggested slight differences between HeLa G6PD and G6PD A+. This study, carried out using the pure homogeneous B+, A+, and HeLa G6PD, showed that (1) the electrophoretic mobility of HeLa G6PD is identical to that of G6PD A+, (2) the enzymatic properties and thermostability of HeLa G6PD are indistinguishable from those of G6PD A+, and (3) the peptide map of the tryptic digest of HeLa G6PD is identical to that of G6PD A+, with one peptide spot of HeLa G6PD different from the corresponding spot of G6PD B+. These results indicate that the structure of HeLa G6PD is identical to that of G6PD A+, and that the amino acid substitution in HeLa G6PD is from one asparagine residue in the wild-type G6PD B+ to an aspartic acid residue in HeLa G6PD.This research was supported by research grant GM 15253 from the National Institutes of Health.     

G6PD lozere and trinacria-like

Summary Two new G6PD variants have been found in red blood cells of the members of a French family originating from Lozere. The father is hemizygous for an electrophoretically fast variant with mild enzyme deficiency (50–60% of normal). The abnormal paternal G6PD gene is segregating in his daughter who is double heterozygous for maternal and paternal variants. This mutant enzyme, different from previously described variants is designated as Gd Lozère. The mother is heterozygous for another G6PD variant. Two sons are hemizygous for this latter mutant enzyme characterized by a moderate deficiency (25–30% of normal) and slower electrophoretic mobility with some slightly altered kinetic properties. This G6PD has been identified as Gd Trinacria like.These two abnormal enzymes are not associated with any hemolytic problem. Case reported is the first showing the segregation of two new mutant enzymes, distinct from common G6PD variants, among the members of the same family.     

G6PD Ube, a glucose-6-phosphate dehydrogenase variant found in four unrelated Japanese families.

A total of 6,120 Japanese males were screened for glucose-6-phosphate dehydrogenase deficiency (G6PD). Five cases with the deficiency were discovered. Two of them and an additional two cases have the same variant, G6PD Ube, characterized by moderate enzyme deficiency, fast moving enzyme activity on electrophoresis, high Ki Nadph, utilization of substrate analogues, kinetics, pH optima, and stability. This variant was distinguished for G6PD A- and from other Oriental variants by biochemical parameters. Differences in the frequency and type of the variants between southern Asia and Japan, suggest that the Japanese who have been isolated on islands where malaria is not endemic, may have developed their own variant traits.     

Glucose 6-Phosphate dehydrogenase variants: A unique variant (G6PD Kobe) showed an extremely increased affinity for galactose 6-phosphate and a new variant (G6PD Sapporo) resembling G6PD Pea Ridge

Summary Two new glucose 6-phosphate dehydrogenase (G6PD) variants associated with chronic nonspherocytic hemolytic anemia were discovered. G6PD Kobe was found in a 16-year-old male associated with hemolytic crisis after upper respiratory infection. The enzyme activity of the variant was about 22% of that of the normal enzyme. The main enzymatic characteristics were slower than normal anodal electrophoretic mobility, high Km G6P, increased thermal-instability, an acidic pH optimum, and an extremely increased affinity for the substrate analogue, galactose 6-phosphate (Gal-6P).G6PD Sapporo was found in a 3-year-old male associated with drug-induced hemolysis. The enzyme activity was extremely low, being 3.6% of normal. In addition, this variant showed high Ki NADPH and thermal-instability.G6PD Kobe utilized the artificial substrate Gal-6P effectively as compared with the common natural substrate, glucose 6-phosphate. In G6PD Sapporo, NADPH could not exert the effect of product inhibition. The structural changes of these variants are expected to occur at the portions inducing conformational changes of the substrate binding site of the enzyme.     

Molecular heterogeneity of the glucose-6-phosphate dehydrogenase deficiency in the Hellenic population

We report results from a systematic study to identify the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on a sample of 299 male subjects from the Hellenic population. Our stepwise approach involved partial biochemical characterization and quantitation of the enzyme's activity, MboII restriction endonuclease digestion to identify the G6PD Mediterranean variant, which represents the most frequent G6PD variant in our population and a nonradioactive polymerase chain reaction-single-strand conformation polymorphism methodology for the detection of the underlying molecular defect(s) in the rest of the non-Mediterranean G6PD-deficient individuals. Through this approach, six different G6PD variants were identified (G6PD Mediterranean, G6PD Hermoupolis, G6PD Cassano, G6PD Seattle, G6PD Ierapetra and G6PD Acrokorinthos), two of which were new (G6PD Hermoupolis, G6PD Acrokorinthos). In essence, this study underlines the remarkable genetic heterogeneity of the G6PD deficiency in the Hellenic population, while the finding of the double mutant, G6PD Hermoupolis, may help to outline the relationship and evolution of mutations in the human G6PD locus.     

Further evidence for heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Papua New Guinea

Summary Four new G6PD variants have been characterized in individuals from Papua New Guinea. This study demonstrates that the previously reported Markham variant and the newly characterized Salata variant may be widely distributed in Papua New Guinea. The data presented here together with those of previously published studies demonstrate a degree of heterogeneity of G6PD deficiency that is much higher than that in other regions of the world where G6PD deficiency is common.     

A new glucose-6-phosphate dehydrogenase variant (G6PD Nagano) associated with congenital hemolytic anemia

Summary A new glucose-6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was reported. The patient, a 6-year-old Japanese male, was noticed to have hemolytic anemia soon after birth, and a diagnosis of G6PD deficiency was made at the age of 2. He had episodes of hemolytic crisis several times after upper respiratory infection. G6PD activity of the patient was 5.5% of normal. The enzymatic characteristics were examined when he was 5 years old, and his G6PD showed faster-than-normal electrophoretic mobility, low Km G6P, high Km NADP, low Ki NADPH, normal utilization of substrate analogues, heat instability, and a normal pH optimum curve. From these results, this was considered to be a new variant and was designated G6PD Nagano. Infection-induced hemolysis and chronic hemolytic anemia seem to be due to markedly impaired enzyme activity and thermal instability.     

Chronic nonspherocytic hemolytic anemia (CNSHA) and glucose 6 phosphate dehydrogenase (G6PD) deficiency in a patient with familial amyloidotic polyneuropathy (FAP)

Summary A new glucose-6-phosphate dehydrogenase (G6PD) variant with severe erythrocytic G6PD deficiency and a unique pH optimum is described in a young patient with chronic nonspherocytic hemolytic anemia (CNSHA) and familial amyloidotic polyneuropathy (FAP). Chronic hemolysis was present in the absence of infections, oxidant drugs or ingestion of faba beans. Residual enzyme activity was about 2.6% and 63% of normal activity in erythrocytes and leucocytes, respectively. A molecular study using standard methods showed G6PD in the patient to have normal electrophoretic mobility (at pH 7.0, 8.0 and 8.8), normal apparent affinity for substrates (K_m, G6P and NADP) and a slightly abnormal utilization of substrate analogues (decreased deamino-NADP and increased 2-deoxyglucose-6-phosphate utilization). Heat stability was found to be markedly decreased (8% of residual activity after 20 min of incubation at 46°C) and a particular characteristic of this enzyme was a biphasic pH curve with a greatly increased activity at low pH. Although molecular characteristics of this variant closely resemble those of G6PD Bangkok and G6PD Duarte, it can be distinguished from these and all other previously reported variants by virtue of its unusual pH curve. Therefore the present variant has been designated G6PD Clinic to distinguish it from other G6PD variants previously described.     

A New Glucose-6-Phosphate Dehydrogenase Variant, G6PD Orissa (44 Ala→Gly), is the Major Polymorphic Variant in Tribal Populations in India

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is usually found at high frequencies in areas of the world where malaria has been endemic. The frequency and genetic basis of G6PD deficiency have been studied in Africa, around the Mediterranean, and in the Far East, but little such information is available about the situation in India. To determine the extent of heterogeneity of G6PD, we have studied several different Indian populations by screening for G6PD deficiency, followed by molecular analysis of deficient alleles. The frequency of G6PD deficiency varies between 3% and 15% in different tribal and urban groups. Remarkably, a previously unreported deficient variant, G6PD Orissa (44 Ala→Gly), is responsible for most of the G6PD deficiency in tribal Indian populations but is not found in urban populations, where most of the G6PD deficiency is due to the G6PD Mediterranean (188 Ser→Phe) variant. The K of G6PD Orissa is fivefold higher than that of the normal enzyme. This may be due to the fact that the alanine residue that is replaced by glycine is part of a putative coenzyme-binding site.     

G6PD Vientiane: A new glucose-6-phosphate dehydrogenase variant with increased stability

Summary A new G6PD variant, called G6PD Vientiane, has been discovered in a patient from Laos.The characteristics of this variant are: mild enzyme deficiency (about 50% of the normal activity) in the granulocytes and the red cells, with normal G6PD-related antigen concentration; increased stability; normal Km glucose 6-phosphate and NADP⁺; increased inhibition constant by NADPH; decreased inhibition by ATP; slightly increased utilization of the substrate analogue; abnormal pH curve, with maximum activity at pH 9.5; slightly reduced starch gel electrophoretic migration. The implications of the molecular stability of a deficient mutant variant are discussed.     

G6PD Sendagi: A new glucose-6-phosphate dehydrogenase variant associated with congenital hemolytic anemia

A new glucose-6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was discovered. It was found in a 2-year-old male who had a hemolytic crisis after an upper respiratory tract infection. The enzyme activity of the variant was 8.4% of that of the normal enzyme. The enzymatic characteristics were slower than normal anodal electrophoretic mobility, low Km G6P, normal Km NADP, increased utilization of substrate analogues, high Ki NADPH, decreased heat stability, and an alkaline pH optimum. From these results, this was considered to be a new variant and was designated G6PD Sendagi.     

A new glucose-6-phosphate dehydrogenase variant with congenital nonspherocytic hemolytic anemia (G6PD Genova)

Summary A new deficient variant of glucose-6-phosphate dehydrogenase (G6PD) causing severe congenital nonspherocytic hemolytic anemia (CNSHA) is described. The variant enzyme, characterized by slow electrophoretic mobility, extreme in vivo and in vitro lability, high K_m for G6P and strongly acidic pH optimum, appears to be unique, and has been designated G6PD Genova. Investigation of an obligate heterozygote using various cytochemical, biochemical and recombinant-DNA techniques showed G6PD mosaicism in the erythrocytes and leukocytes. Therefore, the presence of a disadvantageous mutation at one Gd locus did not determine selection in favor of the normal allele in the heterozygote's hemopoietic cells.     

Altered aggregational properties in a genetic variant of human glucose 6-phosphate dehydrogenase

The Tel Hashomer variant of human G6PD migrates as two prominent components during electrophoresis in several gel systems in which red cell G6PD from other males migrates predominantly as a single band. Since human males normally have but one X-chromosome, the extra band of this variant seemed an exception to earlier biochemical and genetic observations suggesting that human red cell G6PD is determined by a locus on the X chromosome. Results of the present studies indicate that the Tel Hashomer variant is unusually susceptible to the formation of a complex which has a higher molecular weight than normal G6PD and which represents the slow electrophoretic component. The conditions of formation and disruption of this complex in crude and purified Tel Hashomer preparations suggest that it results from the formation of disulfide bridges between molecules of Tel Hashomer G6PD.Supported by U.S. Public Health Service Research Grants AM-11065 and FR-5406 and Research Career Development Award 5 K3 AM 7992.     

Glucose 6-phosphate dehydrogenase in Drosophila: A sex-influenced electrophoretic variant

A variant of glucose 6-phosphate dehydrogenase (G6PD) in Drosophila melanogaster shows different electrophoretic migration in males and females. In heterozygotes, the variant influences the migration of G6PD produced by both chromosomes. Mixing of homogenates of males and females changes migration of the female-produced enzyme, suggesting that a protein produced in males is capable of altering the variant G6PD molecule. The hypothetical protein is also present in pseudomales and intersexes produced by sex transformation genes.This research was supported by NIH grants # 5-T1-GM 216-06 and GM 12768-01 and NSF grants GB 4587 and GB 4824.