Spontaneous and induced activation of genes affecting the phenotypic expression of glucose 6-phosphate dehydrogenase in Daphnia pulex

Summary Evidence has previously been presented on the occurrence spontaneous variations in the electrophoretic mobility (EM) of glucose-6-phosphate dehydrogenase (G6PD E.C. 1.1.1.49) in laboratory clones of parthenogenetically reproducing Daphnia (Ruvinsky et al. 1983). The present study is concerned with a natural population of Daphnia living under the extreme conditions of shallow, dessicating pond. The number of individuals having the slow (S) variant of the EM of G6PD increased sharply during their 1.5 month life span. This increase is suggested to result from alternational variability related to activation of latent genetic material.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

Glucose 6-phosphate dehydrogenase inDrosophila melanogaster: Starch gel electrophoretic variation due to molecular instability

Two starch gel electrophoretic variants of glucose 6-phosphate dehydrogenase (G6PD) in Drosophila melanogasterwere partially purified and characterized biochemically. The difference in their migration on starch gel is not due to a charge difference, but rather to a difference in molecular size due to instability of one of the variants. Crosses between the two variants did not produce offspring showing an intermediate electrophoretic band in the heterozygous female. That an hybrid molecule does exist in the heterozygous female, however, was demonstrated by taking advantage of differences in heat lability and structural stability of the two variants. Hence, we conclude that the G6PD locus in both X-chromosomes is active in each cell in female Drosophila.Our findings with DrosophilaG6PD emphasize the importance to protein variation and enzyme function of mutations leading to amino acid substitutions which do not produce a change in net charge. Aided by National Institutes of Health grants HD 00486, HD 00004, and GM 14155.     

The extent of linkage disequilibrium caused by selection on G6PD in humans

The gene coding for glucose-6-phosphate dehydrogenase (G6PD) is subject to positive selection by malaria in some human populations. The G6PD A- allele, which is common in sub-Saharan Africa, is associated with deficient enzyme activity and protection from severe malaria. To delimit the impact of selection on patterns of linkage disequilibrium (LD) and nucleotide diversity, we resequenced 5.1 kb at G6PD and approximately 2-3 kb at each of eight loci in a 2.5-Mb region roughly centered on G6PD in a diverse sub-Saharan African panel of 51 unrelated men (including 20 G6PD A-, 11 G6PD A+, and 20 G6PD B chromosomes). The signature of selection is evident in the absence of genetic variation at G6PD and at three neighboring loci within 0.9 Mb from G6PD among all individuals bearing G6PD A- alleles. A genomic region of approximately 1.6 Mb around G6PD was characterized by long-range LD associated with the A- alleles. These patterns of nucleotide variability and LD suggest that G6PD A- is younger than previous age estimates and has increased in frequency in sub-Saharan Africa due to strong selection (0.1 < s < 0.2). These results also show that selection can lead to nonrandom associations among SNPs over great physical and genetic distances, even in African populations.     

IN VIVO Function of Rare G6pd Variants from Natural Populations of DROSOPHILA MELANOGASTER

From 1981 to 1983, 15,097 X-chromosomes were genetically extracted from a number of North American populations of D. melanogaster and were electrophoretically screened for rare mobility and activity variants of glucose-6-phosphate dehydrogenase (G6PD). Overall, 13 rare variants were recovered for a frequency of about 10^-3. Eleven variants affect electrophoretic mobility and are apparently structural, and two variants exhibit low G6PD activity. One low activity variant is closely associated with a P-element insertion at 18D12-13—all of the variants were subjected to the previously described genetic scheme used to identify relative in vivo activity differences between the two common electrophoretic variants associated with the global polymorphism. Most of the rare variants exhibit apparent in vivo activities that are similar to one or the other of the common variants, and these specific rare variants appear to be geographically widespread. Several variants have significantly reduced function. All of the variants were measured for larval specific activity for G6PD as a first measure of in vitro activity. It appears that specific activity alone is not a sufficient predictor for G6PD in vivo function.     

A new improved electrophoretic method of glucose-6-phosphate dehydrogenase with discontinuous buffer and gel system

A new improved electrophoretic method of glucose-6-phosphate dehydrogenase (G6PD) was devised by using a slab disc electrophoresis with a discontinuous gel of 1-mm thickness. This was suitable for electrophoresis of thermolabile G6PD variants with low activities. Using this system, sharp G6PD bands and acceptable separations of G6PD Konan, G6PD Tokyo, and G6PD Yokohama, were obtained and compared with those of starch gel electrophoresis.     

Clonal evolution in human lymphoblast cultures.

We established lymphoblast cultures from normal females heterozygous for electrophoretic variants of glucose-6-phosphate dehydrogenase (G6PD), and the X-linked markers have permitted us to look at evolution of these cell populations in culture. The established cultures were phenotypically heterozygous at onset, having both of the mosaic cell populations resulting from X chromosome inactivation. However, by the tenth subculture, the population of cells no longer reflected the heterozygous genotype in 50% of the cultures, as only a single G6PD isozyme was expressed. The ultimate cell composition seems to be influenced by the initial composition, by the nature of alleles at heterozygous X-linked loci that may provide a growth advantage (or disadvantage), as well as by stochastic events. Our results show that lymphoblast cultures may not reflect the X-linked phenotype of the cells from which they were derived. The fate of such cultures seems to be evolution toward clonal cell populations.     

Gd(-) Muret and Gd(-) Colomiers,two new variants of glucose-6-phosphate dehydrogenase associated with favism

Summary Two males subjects are described with hitherto undescribed glucose-6-phosphate dehydrogenase (G6PD) variants. The first is of French ancestry, the second of Sicilian extraction. Each subject suffered from acute hemolytic anemia following ingestion of broad beans (Vicia fava). In both cases the hemolytic crisis occurred in a late period of life (29 and 58 years). No previous hemolytic crisis was recorded. The electrophoretic and kinetic properties of the mutant enzymes examined after purification from the red cells allowed each to be distinguished from other G6PD variants reported until now. The first variant was named Gd(-) Muret, the other Gd(-) Colomiers.     

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.     

Glucose-6-phosphate dehydrogenase (G6PD) Guantánamo and G6PD Caujerí: Two new glucose-6-phosphate dehydrogenase-deficient variants found in Cuba

Glucose-6-phosphate dehydrogenase (G6PD) deficiency was identified in two children who were studied because of hemolytic episodes. The electrophoretic and kinetic properties of the mutant enzymes allowed us to conclude that both of them were new variants. They were named G6PD Guantánamo and G6PD Caujerí.     

Stability of the "two active X" phenotype in triploid somatic cells.

We examined triploid cells of XXY karyotype heterozygous for glucose 6 phosphate dehydrogenase (G6PD) electrophoretic variants with regard to the stability of their X chromosome phenotype. Clonal populations of cells derived from these human fibroblasts maintained a precise 1:2:1 ratio of A:heteropolymer:B isozymes throughout their life span, indicating stability of the two active X chromosomes in these cells. To determine the influence of the autosomal complement on X chromosome expression, we attempted to perturb the relationship. Fusion of these triploid cells with human diploid fibroblasts carrying a novel G6PD variant (B') resulted in heterokaryons exprssing a novel heteropolymer, presumably indicating that all three parental X chromosomes were active. However, no derepression of the inactive X chromosome was observed. Analysis of interspecific hybrids derived from triploid cells and mouse fibroblasts confirmed that activity of parental X chromosomes is maintained. Some human mouse hybrid clones, however, expressed only a single human G6PD isozyme, probably attributable to segregation of the pertinent X chromosome, but elimination of a relevant autosome cannot be excluded. The triploid cells transformed by SV40 showed alterations in LDH pattern and an approximately 10-20% decrease in chromosome number, but maintained the precise G6PD phenotype of the untransformed cell. These studies provide evidence for the stability of the X chromosome phenotype in triploid cells.     

Genetic heterogeneity at the glucose-6-phosphate dehydrogenase locus in southern Italy: a study on a population from the Matera district

Summary Glucose-6-phosphate dehydrogenase (G6PD) has been analyzed by gel electrophoresis and by quantitative assay in an unselected sample of 1524 schoolboys from the province of Matera (Lucania) in southern Italy. We have identified 43 subjects with a G6PD variant. Of these, 31 had severe G6PD deficiency, nine had mild to moderate deficiency, and three had a non-deficient electrophoretic variant. The overall rate of G6PD deficiency was 2.6%. The frequency of G6PD deficiency, ranging from 7.2% on the Ionian Coast to zero on the eastern side of the Lucanian Apennines, appears to be inversely related to the distance of each town examined from the Ionian Coast, suggesting that this geographic distribution may reflect, at least in part, gene flow from Greek settlers. Biochemical characterization has shown that most of the G6PD deficiency in this population is accounted for by G6PD Mediterranean. In addition, we have found several examples of two other known polymorphic variants (G6PD Cagliari and G6PD A^–): three new polymorphic variants. G6PD Metaponto (class III), G6PD Montalbano (class III), and G6PD Pisticci (class IV); and two sporadic variants, G6PD Tursi (class III) and G6PD Ferrandina (class II). These data provide further evidence for the marked genetic heterogeneity of G6PD deficiency within a relatively narrow geographic-area and they prove the presence in the Italian peninsula of a sene (Gd ^A–) regarded as characteristically African.     

Cycloheximide-resistance in Chinese hamster ovary cells and human fibroblast cells

Summary A total of 3000 men living in Yamaguchi were screened for glucose-6-phosphate dehydrogenase (G6PD) deficiency using Beutler's spot test and three types of starch gel electrophoresis. These electrophoresis used a phosphate buffer system at pH 7.0, a TRIS-EDTA-borate buffer system at pH 8.6, and a TRIS-hydrochloride buffer system at pH 8.8. Fifteen G6PD-deficient variants were found at the rate of 0.5% and classified into four groups. As new variants, G6PD Konan, Kamiube, and Kiwa were identified. These three variants had a mild to moderate G6PD deficiency and were not associated with any clinical signs. G6PD Konan had fast electrophoretic mobility as compared with normal levels, G6PD Kiwa had slightly elevated electrophoretic mobility, and G6PD Kamiube had normal electrophoretic mobility. These three variants had normal levels of Km G6P, Km NADP, and Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NAPD, normal heat stability, and a normal pH curve. The other variant was G6PD Ube, which we had previously found in Yamaguchi (Nakashima et al., 1977). One boy with G6PD Ube was Korean.     

Alcohol dehydrogenase of Biomphalaria glabrata (Mollusca: Pulmonata): Polymorphism,genetic analysis,and interspecific variations

Alcohol dehydrogenase of Biomphalaria glabrata has been characterized by electrophoresis, substrate specificities, and other physicochemical means. It exists as a multiple molecular form possessing a minimum number of three bands in ovotestis, five in digestive gland, and six in albumen gland. Each organ shows characteristic electrophoretic forms which differ in substrate specificities and the response to the organomercurial inhibitor p-hydroxymercuribenzoate. Mercaptoethanol treatment has no effect on any electrophoretic form. Genetic analyses of the electrophoretic variants show that three different loci are responsible for the synthesis of the various electrophoretic forms observed in this species. Different species vary in their electrophoretic patterns. A possible role of alcohol dehydrogenase isozymes in the phylogenetic relationship among three species, B. glabrata, B. tenagophila, and B. straminea, has been discussed.This work was supported by a grant from the Conselho Nacional de Pesquisas, Brazil.     

Polymorphism at the G6pd and 6Pgd loci in Drosophila melanogaster. IV. Genetic factors modifying enzyme activity

Different homozygous lines of similar genotype with respect to G6pd and 6Pgd were shown to have different enzyme activities for G6PD and 6PGD. Crosses between high and low lines suggested that there were modifying genes present on the autosomes, while others were probably located on the X chromosome. Allelic variation within each electrophoretic class of G6pd and 6Pgd might, however, also have contributed to this variation. An experiment on adaptation to sodium octanoate demonstrated that in adapted flies selection for lower enzyme activity had occurred, which provided further evidence for the existence of genetic differences in activity. Furthermore, a strong positive correlation between the activities of G6PD and 6PGD was found for each genotype. Since no correlation was found between MDH and the two enzymes G6PD and 6PGD, it could be concluded that this correlation was probably rather specific for G6PD and 6PGD. Interaction between genotypes with respect to activity was also found. It was shown that the variation at 6Pgd influenced the activity of G6PD within a genotype. The data are discussed in relation to fitness differences presented in foregoing articles.     

Gd- allele distribution patterns in Azerbaijan. III. The identification of mutant forms of glucose-6-phosphate dehydrogenase

In 28 families with G6PD deficiency living in 3 settlements of Shekii district of Azerbaijan 11 G6PD variants of II and III classes differing by kinetic properties were identified according WHO program. 9 of them are characterized with the same electrophoretic mobility. Comparison of G6PD spectra in two subpopulations and in a mixed group permits to make a conclusion about existence of common and rare G6PD alleles in examined population. They distribute by gene drift supported by natural selection. Among 7 samples of G6PD with normal and increased activity two new variants of IV class -- Nukha and Bash-Kungut -- were found.     

Congenital Non-Spherocytic Hemolytic Anemia

A family with congenital non-spherocytic hemolytic anemia associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency was studied. Two females, heterozygous for the enzyme deficency, had evidence of a hemolytic anemia. The results of chromium-51 erythrocyte life span studies prior to, during, and after periods of primaquine administration suggested that the hemolytic anemia in these women was due to the presence of two populations of red blood cells in their circulation. One population had normal G6PD levels and a normal life span, whereas the other had diminished enzyme activity and a shortened life span.In vitro metabolic studies of the erythrocytes of a heterozygous female and a hemizygous male suggested that, in spite of G6PD deficiency, the synthesis and breakdown of adenosine triphosphate and 2,3-diphosphoglyceric acid was similar to that in normal erythrocytes.     

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.     

Description of new mutant forms of erythrocyte glucose-6-phosphate dehydrogenase in man]

9 variants of human erythrocyte glucose-6-phosphate dehydrogenase (G6PD) were isolated from erythrocytes of patients with G6PD deficiency and partially purified according to WHO program for stanartization of methods for studying G6PD. The results of physico-chemical study of these enzymes (determination of electrophoretic mobility, kappaM for G6P and NADP, pH optimum and thermostability) permit tu consider 5 of them to be new mutations of G6PD previously not described in literature. The observed high geterogeneity of variants of G6PD in Azerbaijan is discussed.     

Genetic heterogeneity at the glucose-6-phosphate dehydrogenase locus in southern Italy: a study on the population of Naples

Summary Glucose-6-phosphate dehydrogenase (G6PD) electrophoretic phenotype was determined in red cells from 979 male subjects born in Naples (Southern Italy). In 0.7% of the cases no activity could be detected in haemolysates, while in 1.3% of the cases G6PD activity was approximately 20% of normal and electrophoretic mobility was altered. Moveover in two subjects a G6PD with altered mobility and normal activity was shown. G6PD was characterized in 10 subjects with variant phenotype. We conclude that the G6PD(-) phenotype in the population of Naples consists of at least six different G6PD variants associated with mild deficiency and at least one, G6PD Mediterranean, associated with severe deficiency.