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.     

Origin and spread of the glucose-6-phosphate dehydrogenase variant (G6PD-Mediterranean) in the Middle East.

A common glucose-6-phosphate dehydrogenase (G6PD) variant characterized by severe enzyme deficiency and B-like electrophoretic mobility is called "G6PD-Mediterranean" because it is found in different populations around the Mediterranean Sea. Sequence analysis of Italian subjects has revealed that the molecular basis of G6PD-Mediterranean is a single C-T transition at nucleotide position 563, causing a serine phenylalanine replacement at amino acid position 188. Most G6PD-Mediterranean subjects also have a silent C-T transition (without amino acid replacement) at nucleotide position 1311. Twenty-one unrelated individuals from Saudi Arabia, Iraq, Iran, Jordan, Lebanon, and Israel with both severe G6PD deficiency and B-like electrophoretic mobility were tested for both mutations by using amplification followed by digestion with appropriate restriction enzymes. All but one had the 563 mutation, and, of these, all but one had the 1311 mutation. Another 24 unrelated Middle Eastern individuals with normal G6PD activity or not known to be G6PD deficient were similarly tested. Four had the silent mutation at position 1311 in the absence of the deficiency mutation at position 563. We conclude that (1) the large majority of Middle Eastern subjects with the G6PD-Mediterranean phenotype have the same mutation found in Italy, (2) the silent mutation is an independent polymorphism in the Middle East, with a frequency of about .13, and (3) the mutation leading to the G6PD-Mediterranean deficiency has probably arisen on a chromosome that already carried the silent mutation.     

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.     

The Thai variant and the distribution of alleles of 6-phosphogluconate dehydrogenase and the distribution of glucose 6-phosphate dehydrogenase deficiency in Thailand

The samples were taken from 3185 subjects from ten provinces throughout Thailand. In 1577 males the frequency of glucose 6-phosphate dehydrogenase deficiency was 11.98%. In the far south the gene frequency was 2.83%; in the remainder of the country the frequency did not vary significantly about a mean of 13.76%. The deficiency is of a severe type. The G6PD of all of the nondeficient individuals had the electrophoretic mobility of type B. The mean frequency of the A/B electrophoretic phenotype of 6-phosphogluconate dehydrogenase is 8.47%. The maximum frequency was in central and southern Thailand with a decline to the north and northeast. A variant form of 6-PGD, referred to as the Thai variant, has been found in which two additional electrophoretic components migrate anodally to the normal A band, confirming that the molecule is at least a dimer. The hypothesis is advanced that erythrocyte 6-PGD is determined by two genetic loci, only one of which is translated in leukocytes.Supported by U.S. Army Contract DA-49-193 MD 2879, U.S.P.H.S. GM 09252, and U.S.P.H.S. 5-K3-GM-15325.     

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.     

At least five polymorphic mutants account for the prevalence of glucose-6-phosphate dehydrogenase deficiency in Algeria

The electrophoretic mobility and level of enzyme activity of glucose-6-phosphate dehydrogenase (G6PD) was established in 100 unrelated Algerian males with G6PD deficiency. DNA from these subjects was analysed for the presence of certain known G6PD mutations by the appropriate restriction enzyme digestion of fragments amplified by the polymerase chain reaction. Where the mutation could not be identified in this way, the samples were subjected to single-strand conformation polymorphism analysis and abnormal fragments were sequenced. In this way, eight different mutations have been identified, of which five are polymorphic and account for 92% of the samples. The most common variants are G6PD A-(46%) and G6PD Mediterranean (23%), both of which were associated with favism. A new polymorphic variant, G6PD Aures, has been identified during the course of this study, whereas another, G6PD Santamaria, has now been established as a polymorphic variant (11%). Thus, G6PD deficiency in Algeria is heterogeneous, suggesting that there has been significant gene flow, both from sub-Saharan Africa and from other parts of the Mediterranean.     

Identification of glucose 6-phosphate dehydrogenase deficiency in a population with a high frequency of thalassemia

High frequencies of both thalassemia trait (5.2%) and glucose 6-phosphate dehydrogenase (G6PD) deficiency for only males (1.3%) have been observed in the Calabrian population. The G6PD activity measurement was carried out on 1239 samples of whole blood from Calabrian subjects of both sexes (age range 10-55) by a differential pH-metry technique which was quite suitable to determine the G6PD deficiency in mass screenings. The analyzed subjects showed: only the thalassemia trait; or only the G6PD deficiency; or only the total iron serum deficiency; or G6PD deficiency associated with the thalassemia trait or with the total iron serum deficiency. The G6PD heterozygous subjects have an enzymatic activity which is masked by both the thalassemia trait and the total iron serum deficiency. In a population showing high frequencies of both thalassemia trait and G6PD deficiency, the comparison of G6PD activity of heterozygous subjects also affected with the thalassemia trait is more reliable if referred to the enzymatic activity of the carriers of the latter inherited anomaly rather than to G6PD activity of normal subjects.     

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.     

G6PD Avenches and G6PD Moosburg: biochemical and erythrocyte membrane characterization

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 KM 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.     

A unique electrophoretic slow-moving glucose 6-phosphate dehydrogenase variant (G6PD Asahikawa) with a markedly acidic pH optimum

Summary A new glucose 6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was discovered in Japan. The patient showed hemolytic crises after upper respiratory infections. The enzyme activity was about 3.8% of the normal. The partially purified enzyme revealed slow anodal electrophoretic mobility, high Km NADP, marked thermal-instability, and increased affinity for a substrate analogue (deamino-NADP). A particular characteristic of this enzyme was a biphasic pH curve with a greatly increased activity at low pH values. From these results, this variant was clearly different from hitherto observed G6PD variants, and was designated G6PD Asahikawa.     

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.     

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.     

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.     

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

Summary A new glucose-6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was found in a 20-year-old Japanese male who showed mild hemolysis after an upper respiratory tract infection. The patient had been noted to have jaundice and reticulocytosis several times before this episode. The enzyme activity of the variant was 1.5% of normal. The enzymatic characteristics were slow anodal electrophoretic mobility, high Km G6P, normal Km NADP, decreased heat stability, and a normal pH optimum. From these results, the enzyme was considered to be a new class 1 variant and was designated G6PD Tsukui.     

G6PD Genotype and Its Associated Enzymatic Activity in a Chinese Population

Knowledge of the G6PD genotype and its associated enzyme activity is significant for population genetics, diagnosis of disease, and management of patients. We tested 2,872 unrelated subjects from a Hakka population in China for G6PD activity by the WHO standard method and for genotype by DHPLC and DNA sequencing. Among female heterozygotes, 78.5% had relatively normal enzyme activity. The phenotype frequency of G6PD deficiency is 0.028, and the causal allele frequency is 0.060 in females. The accuracy, sensitivity, and specificity of DHPLC are more than 98% for detecting G6PD-deficient hemizygotes, heterozygotes, and homozygotes. Measuring enzyme activity alone is not sufficient for the diagnosis of heterozygotes. A combination of enzyme activity and DNA analysis should be used.     

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.     

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.     

G6PD Viangchan: a new glucose 6-phosphate dehydrogenase variant from Laos

Summary We describe a previously unreported glucose-6-phosphate dehydrogenase-(G6PD) variant. G6PD Viangchan was found in a Laotian immigrant to Calgary, Canada, and was characterized by severe enzyme deficiency, normal electrophoretic mobility, increased pH optimum, and abnormal kinetics for the natural substrates G6PD and NADP, as well as the artificial substrates 2-deoxy G6PD and deamino NADP. The inhibition constant for NADPH was decreased. The subject has no evidence suggesting chronic or episodic hemolysis.     

Molecular genetics of the glucose-6-phosphate dehydrogenase (G6PD) Mediterranean variant and description of a new G6PD mutant, G6PD Andalus1361A

Glucose-6-phosphate dehydrogenase (G6PD; E.C.1.1.1.49) deficiency is the most common human enzymopathy; more than 300 different biochemical variants of the enzyme have been described. In many parts of the world the Mediterranean type of G6PD deficiency is prevalent. However, G6PD Mediterranean has come to be regarded as a generic term applied to similar G6PD mutations thought, however, to represent a somewhat heterogeneous group. A C----T mutation at nucleotide 563 of G6PD Mediterranean has been identified by Vulliamy et al., and the same mutation has been found by De Vita et al. in G6PD Mediterranean, G6PD Sassari, and G6PD Cagliari. The latter subjects had an additional mutation, at nucleotide 1311, that did not produce a coding change. We have examined genomic DNA of five patients--four of Spanish origin and one of Jewish origin--having enzymatically documented G6PD Mediterranean. All had both the mutation at nucleotide 563 and that at nucleotide 1311. A sixth sample, resembling G6PD Mediterranean kinetically but with a slightly rapid electrophoretic mobility, was designated G6PD Andalus and was found to have a different mutation, a G----A transition at nucleotide 1361, producing an arginine-to-histidine substitution. These studies suggest that G6PD Mediterranean is, after all, relatively homogeneous.