Diagnosis of glucose-6-phosphate dehydrogenase (G6PD) mutations by DNA amplification and allele-specific oligonucleotide probes.

We have recently identified that at least four types of mutation are responsible for the glucose-6-phosphate dehydrogenase (G6PD) polymorphism in the Chinese of Taiwan. Two mutations (487 G-->A and 493 A-->G) occurring at nucleotide position 487 and 493, respectively, create Alu I and Ava II recognition sites which enabled us to directly examine these two mutations by PCR/restriction enzyme (RE) digestion. However, the other two mutations (1376 G-->T and 1388 G-->A), which do not generate any recognizable restriction sites, were detected by DNA sequencing method which is not suitable for rapid diagnosis. Using the PCR technique, we have successfully developed a simple and rapid method for the detection of 1376 and 1388 mutations. This method involves the selective amplification of a DNA fragment from human G6PD gene with specific oligonucleotide primers, followed by hybridization with allele-specific oligonucleotide (ASO) probes. Using the PCR/ASO and PCR/RE methods, we have successfully examined two families and 20 unrelated subjects with G6PD deficiency. Our results indicate that the PCR/ASO method is suitable for the rapid determination of 1376 and 1388 mutations. The combined use of PCR/ASO and PCR/RE methods will be suitable for rapid diagnosis of four known G6PD mutations in Chinese.     

Molecular heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Malays in Malaysia

Multiplex polymerase chain reaction (PCR) using multiple tandem forward primers and a common reverse primer (MPTP) was recently established as a comprehensive screening method for mutations in X-linked recessive diseases. In the work reported here, MPTP was used to scan for mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene. Mutations in exons 3,4,5,6,7,9, 11, and 12 of the G6PD gene were screened by MPTP in 93 unrelated Malaysian patients with G6PD deficiency. Of the 93 patients, 80 (86%) had identified mutations. Although all of these were missense mutations, identified nucleotide changes were heterogeneous, with 9 mutations involving various parts of the exons. These 9 mutations were G-to-A nucleotide changes at nucleotide 871 of the G6PD gene (G871A), corresponding to G6PD Viangchan, G6PD Mediterranean (C563T), G6PD Vanua Lava (T383C), G6PD Coimbra (C592T), G6PD Kaiping (G1388A), G6PD Orissa (C131G), G6PD Mahidol (G487A), G6PD Canton (G1376T), and G6PD Chatham (G1003A). Our results document heterogeneous mutations of the G6PD gene in the Malaysian population.     

G6PD San Francisco: a new variant of glucose-6-phosphate dehydrogenase associated with congenital nonspherocytic hemolytic anemia

Congenital nonspherocytic hemolytic anemia in an adult male of Scandinavian ancestry was associated with virtual absence of G6PD activity in red cells. Characterization of G6PD purified from leukocytes using standard WHO techniques revealed diminished electrophoretic mobility, marked lability on heating at 46 degrees C, normal pH optimum and utilization of alternate substrates (2-deoxy G6P, D-amino NADP), elevated Km NADP, and striking susceptibility to NADPH inhibition. The variant G6PD, which appears to be unique, has been designated G6PD San Francisco. An unusual feature of the variant enzyme, susceptibility to inactivation by brief periods of dialysis, could be prevented by addition of 200 microM NADP to the dialysis solution. In red cells, where G6PD activity was essentially absent, regeneration of reduced glutathione was totally curtailed in vitro, while in leukocytes, where residual G6PD activity was approximately 60% of normal, hexose monophosphate shunt activity, oxygen consumption during phagocytosis, and bacterial killing were unimpaired. Thus, instability of the variant enzyme rather than its unfavorable kinetics appeared to be an important determinant of abnormal cell function.     

Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in Chinese infants with or without severe neonatal hyperbilirubinaemia

Summary. To characterize mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene in Chinese infants, we studied 213 G6PD-deficient infants without blood exchange transfusion (BET) therapy, and 34 patients who required BET therapy for their severe hyperbilirubinaemia after birth. Nine different point mutations were characterized in all infants. Of these mutations, the G to T substitution at cDNA nucleotide (nt) 1376, which accounts for the mutations in 131 (53.0%) neonates, followed by G to A substitution at nt 1388 in 18 (10.5%) infants, A to G substitution at nt 493 in 17 (6.9%) infants, A to G substitution at nt 95 in 10 (4.1%) infants, C to T substitution at nt 1024 in six (2.4%) infants, and G to T substitution at nt 392 in three (1.2%) infants, G to A substitution at nt 487 in two (0.8%) infants, C to T substitution at nt 1360 in two (0.8%) infants and C to T substitution at nt 592 in two (0.8%) infants. Mutations in 48 (19.5%) G6PD-deficient infants were not characterized. Most (64.7%) mutations in the G6PD-deficient infants who required BET therapy after birth result from a G to T substitution at nt 1376. The enzyme activity of G6PD deficient infants who required BET therapy is significantly lower than for those who did not, even in a group with the same variant (as in 1376 mutation). Severe neonatal jaundice requiring BET therapy can take place with the majority of variants encountered in this area.     

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.     

Diverse point mutations result in glucose-6-phosphate dehydrogenase (G6PD) polymorphism in Taiwan.

Glucose-6-PHOSPHATE dehydrogenase (G6PD; EC 1.1.1.49) deficiency is the most common human enzymopathy, affecting more than 200 million people worldwide. Although greater than 400 variants have been described based on clinical and biochemical criteria, little is known about the molecular basis of these G6PD deficiencies. Recently, the gene that encodes human G6PD has been cloned and sequenced, which enables us to examine directly the heterogeneity of G6PD at the DNA level. During the past 10 years, we examined the G6PD activity in 21,271 newborn Chinese infants (11,400 males and 9,871 females) and identified 314 (2.8%) males and 246 (2.5%) females having low G6PD activity. The G6PD gene from 10 randomly selected affected individuals and their relatives was polymerase chain reaction (PCR) amplified, subcloned, and sequenced. Our results indicate that at least four types of mutation are responsible for the G6PD polymorphism in Taiwan. The first type of mutation (487 G----A) was found in an affected Chinese with a G to A change at nucleotide 487, which results in a (163)Gly to Ser substitution. The second type of mutation (493 A----G) is a novel mutation that has not been reported in any other ethnic group and was identified in two affected Chinese. This mutation causes an A to G change at nucleotide position 493, producing an (165)Asn to Asp substitution. Interestingly, the 487 G----A and 493 A----G mutations create Alu I and Ava II recognition sites, respectively, which enabled us to rapidly detect these two mutations by PCR/restriction enzyme (RE) digestion method. The third mutation (1376 G----T) was found in four affected Chinese. This mutation causes a G to T change at nucleotide position 1376 that results in an (459)Arg to Leu substitution. The 1376 G----T mutation seems to be the dominant allele that causes G6PD deficiency in Taiwan. Finally, two affected Chinese were identified as having the fourth mutation (1388 G----A). This mutation causes a G to A change at nucleotide 1388 that produces an (463)Arg to His substitution. Our studies provide the direct proof of the genetic heterogeneity of G6PD deficiency in the Chinese populations of Taiwan and the PCR/RE digestion method is suitable for simultaneous detection of the 487 G----A and 493 A----G mutations.     

Neonatal jaundice and molecular mutations in glucose-6-phosphate dehydrogenase deficient newborn infants

Molecular mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene and clinical manifestations of neonatal jaundice in 112 male and 50 female Chinese neonates with G6PD deficiency were studied. In the 112 males, the nucleotide (nt) 1376 (G→T) mutation was the dominant type (50.0%), followed by nt 1388 (G→A) (16.1%), nt 493 (A→G) (8.0%), nt 1024 (C→T) (6.2%), nt 95 (A→G) (5.4%), nt 392 (G→T) (1.8%), nt 487 (G→A) (1.8%), nt 871 (G→A) (0.9%), and nt 1360 (C→T) (0.9%). The nt 871 variant has not been reported in Taiwan before. The occurrence rates for nt 1376, nt 1388, nt 493, nt 95, and nt 1024 mutations in the 50 females were 44.0%, 18.0%, 12.0%, 6.0%, and 6.0%, respectively. The type of G6PD mutation in 10 male and 7 female neonates has not been identified yet. Although G6PD deficient neonates had higher frequency of phototherapy than G6PD normal neonates in both sexes, a significant difference in the prevalence of hyperbilirubinemia (peak bilirubin ≥ 15.0 mg/dl) between G6PD deficient and normal neonates was found only in males. Further analysis showed that duration of phototherapy was longer in G6PD deficient male neonates than in the control group, while the outcome of phototherapy was better in subjects with non-nt 1376 mutations than subjects with the nt 1376 mutation. Most (78.3%) of the 23 G6PD deficient neonates who subsequently suffered from neonatal hyperbilirubinemia carried the nt 1376 mutation. The results of this study indicate that the nucleotide substitution at 1376 is the most common and important mutation for G6PD deficiency in Chinese neonates in Taiwan. © 1996 Wiley-Liss, Inc.     

A New Variant of Glucose-6-Phosphate Dehydrogenase Deficiency Hereditary Hemolytic Anemia, G6PD Cornell: Erythrocyte, Leukocyte, and Platelet Studies

A variant of glucose-6-phosphate-dehydrogenase deficiency associated withchronic hereditary hemolytic anemia wasdiscovered in a 9-yr-old white male. Theerythrocytes contained 5% of normalenzyme activity, the Km NADP was twoto three times normal, the pH optimumwas decreased, and the heat stabilitywas markedly decreased. The Km G6PD,electrophoretic mobility (B), and utilization of substrate analogues 2-deoxy-G6Pand deamino-NADP were normal. Theactivity of G6PD in the leukocytes andplatelets was 15% and 28% of normalvalues, respectively, but bactericidal activity and platelet function were unaffected by the deficiency of G6PD.

Submitted on September 10, 1973 Accepted on March 8, 1974     

Haemolytic Anaemia due to Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency: Demonstration of Two New Biochemical Variants, G6PD Hamm and G6PD Tarsus

Two new variants of erythrocytic glucose-6-phosphate dehydrogenase have been found in one German patient and in another patient of Turkish origin. The enzymes were partially purified 165-fold and 111-fold respectively. Both revealed reduced activity, increased thermolability and a pH-optimum in the alkaline region (8.5 and 9.0). One variant (G6PD Hamm) had a low Km-value for glucose-6-phosphate, the other (G6PD Tarsus) exhibited an increased affinity for glucose-6-phosphate and a reduced affinity for NADP+. This enzyme showed an increased inhibitor constant for NADPH with respect to NADP+. Electrophoretic mobility was normal in both cases. 2-Desoxy glucose-6-phosphate was utilized to an increased rate by both variant enzymes (46% and 33%). Also galactose-6-phosphate (29% and 25%) and deamino-NADP+ (230% and 261%) gave increased utilization rates. The mothers of both patients could be identified as heterozygous for this enzyme deficiency.     

A novel mutation in the glucose-6-phosphate dehydrogenase gene in a subject with chronic nonspherocytic hemolytic anemia--characterization of enzyme using yeast expression system and molecular modeling

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. Human G6PD gene is highly polymorphic, with over 130 mutations identified, many of which cause hemolytic anemia. We studied a novel point mutation in the G6PD gene 1226 C-->G, predicting the proline 409 to arginine substitution (G6PD Suwalki). We expressed the human wild-type and mutated G6PD gene in yeast Saccharomyces cerevisiae which allowed the characterization of the Suwalki variant. We showed that human wild-type, as well as the mutated (1226 C-->G) G6PD gene, functionally complemented the phenotype displayed by the yeast strain with disruption of the ZWF1 gene (homologue of the human G6PD gene). Comparison of wild-type (wt) human G6PD purified from yeast and from blood shows no significant differences in the Km values for G6P and in the utilization rate for the substrate analogue, 2-deoxyG6P. The P409R substitution leads to drastic changes in G6PD kinetics. The specific activity as well as stability of mutated G6PD is also significantly reduced. Besides this, the effect of this mutation was analyzed using a model of the tertiary structure of the human enzyme. The localization of the P409R mutation suggests that it may influence the stability of the whole protein by changing tetramer interactions and disturbing the binding of structural NADP+.     

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.     

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.     

A glucose-6-phosphate dehydrogenase variant, Gd(-) Santamaria found in Costa Rica

Red cell glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-chromosomal-linked abnormality often associated with hemolytic anemia. The G6PD variants obtained from 2 unrelated males, one associated with enzyme deficiency and history of hemolytic jaundice, and the other associated with enzyme deficiency but no hemolytic problems, were examined. Although the 2 subjects have no known consanguinity, the two enzymes could not be distinguished from each other in respect to their electrophoretic mobilities and kinetic properties, both exhibiting slower than normal anodal electrophoretic mobility, lower Km for G6P and NADP and higher rate of utilization of 2-deoxy-G6P and deamino-NADP. An unique double-banded pattern was observed in starch gel electrophoresis at pH 7.0 and pH 8.6. The variant is distinguished from all reported Gd variants, and it is designated Gd(-) Santamaria.     

Glucose-6-phosphate dehydrogenase of malaria parasite Plasmodium falciparum

Plasmodium falciparum growth is impaired in glucose-6-phosphate dehydrogenase (G6PD)-deficient red blood cells (RBCs), and malaria has been implicated in the spreading of deficient variants in malaria- endemic areas. Recent reports suggest that the malaria parasite can adapt itself to grow in these variant RBCs by producing its own G6PD, but studies on parasite G6PD are very limited. In this report, we define the properties of the parasite G6PD. G6PD was partially purified from infected and uninfected variant RBCs associated with severe G6PD deficiency. G6PD from infected RBCs contained two components separable by starch gel electrophoresis: a major component (approximately 90% activity) with a very slow anodal electrophoretic mobility and a minor component (approximately 10% activity) with the same mobility as the host G6PD. Parasite G6PD exhibited much higher affinity (low Km) to G6P and nicotinamide-adenine dinucleotide phosphate (NADP) than did human G6PD. Southern blot hybridization indicated that the parasite genome contained nucleotide sequences that were hybridizable with the human G6PD cDNA. These data indicate that the parasite is capable of adapting to G6PD-deficient RBCs by producing its own G6PD.     

Clonal origin of cells restricted to monocytic differentiation in acute nonlymphocytic leukemia

Two patients with acute monocytic leukemia and heterozygous for the Mediterranean variant of the X-linked enzyme, glucose-6-phosphate dehydrogenase (G6PD), were investigated to determine the number and type of progenitor cells involved. Mosaicism for Mediterranean G6PD was assessed by the different rate of utilization of 2-deoxy glucose-6- phosphate (2dG6P) by normal and Mediterranean variants of G6PD. The monocytoid blasts were found to express one type of G6PD only, indicating their clonal origin from a common progenitor cell, whereas all other hemopoietic cell populations tested expressed the heterozygous phenotype. The finding of a unique involvement of the monocytic line in two cases of acute nonlymphocytic leukemia (ANLL) represents further evidence of heterogeneity of stem cell involvement in ANLL.     

Glucose-6 phosphate dehydrogenase mutations and haplotypes in various ethnic groups

Mutations that produce glucose-6-phosphate dehydrogenase (G6PD) deficiency have been identified in samples from patients with hemolytic disease in the United States, and in G6PD-deficient samples from Greece, the Canary Islands, the Czech and Slovak Republics, South China, and in samples from the Coriell Cell Repository. Eight new mutations are described. Particularly unusual were a nonsense mutation ("G6PD Georgia"1284A), a deletion of six bases ("G6PD Stony Brook" 724- 729 del) coding for two amino acids, and a deletion of the invariant dinucleotide ApG at the 3' acceptor splice site in the highly conserved sequence between intron 10 and exon 11 ("G6PD Varnsdorf"). In addition, five new missense point mutations were identified: "G6PD Cleveland"820A creates a deduced AA 274 Glu-->Lys; "G6PD West Virginia"910T AA 303 Val- ->Phe; "G6PD Fushan"1004A, AA 335 Ala-->Asp; "G6PD Olomouc"1141C AA 381 Leu-->Phe; and "G6PD Praha"1166G AA 389 Glu-->Gly. All of the new mutations except for "G6PD Fushan"1004A were found in patients with hereditary nonspherocytic hemolytic anemia. A coincidental finding in the case of G6PD "West Virginia" was a C-->T transition at nucleotide 1,191. This silent mutation, Asn-->Asn, appears to be rare. Haplotype analysis of mutations in samples from the Canary Islands and South China agreed with previous findings.     

G-6-PD Long Prairie: a new glucose-6-phosphate dehydrogenase mutant exhibiting normal sensitivity to inhibition by NADPH and accompanied by nonspherocytic hemolytic anemia.

The enzymatic properties of a new glucose-6-phosphate dehydrogenase (G-6-PD) variant (G-6-PD Long Prairie) were studied in a white patient with chronic nonspherocytic hemolysis. The red cells were found to have 2.3%-7.7% normal enzymatic activity. The mutant enzyme exhibited marked heat instability, an increased pH optimum, a moderately decreased Km for G-6-P, and increased utilization of 2-deoxyglucose-6-phosphate and deamino NADP. The Km for NADP and Ki for NADPH were both normal. G-6-PD Long Prairie is an interesting new G-6-PD variant that demonstrates that chronic hemolysis can be associated with modestly decreased G-6-PD activity despite normal sensitivity to inhibition by NADPH. Although increased sensitivity to inhibition by NADPH has been postulated to decrease intracellular enzyme activity, resulting in enhanced susceptibility to hemolysis in certain G-6-PD variants with only moderately decreased enzymatic activity, an alternative mechanism of hemolysis, possibly enzyme thermolability, exists in G-6-PD Long Prairie.     

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.     

Molecular study of eight Japanese cases of glucose-6-phosphate dehydrogenase deficiency by nonradioisotopic single-strand conformation polymorphism analysis [see comments]

Using a newly developed nonradioisotopic method of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis combined with the direct sequencing using the fluorescence-labeled terminator, we identified seven missense mutations, 527 A-->G, 1003 G-- >A, 1159 C-->T, 1160 G-->A, 1229 G-->A, 1246 G-->A, and 1361 G-->A, in eight Japanese patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Except for the 527 A-->G, each mutation has been reported to cause variants G6PD Chatham, G6PD Guadalajara, G6PD Beverly Hills, G6PD "Japan", G6PD Tokyo, and G6PD Andalus, respectively. In addition, a single base deletion in intron 5 was found in the patients with G6PD Guadalajara or G6PD Andalus. The variant with unique 527 A-->G was characterized and designated as G6PD Shinshu. We also characterized G6PD "Japan" and found that the variant had the striking resemblance with G6PD Riverside, bearing a missense mutation in the same codon, but causing a different amino acid substitution. Our modified PCR-SSCP analysis using minigel and ethidium bromide staining could detect six of the eight diverse mutations in the G6PD gene. Because it is easy and requires no special apparatus, this modified method will be useful for screening mutations in the G6PD gene.     

Four new electrophoretically slow-moving glucose 6-phosphate dehydrogenase variants associated with congenital nonspherocytic hemolytic anemia found in Japan: Gd(-) Kurume,Gd(-) Fukushima,Gd(-) Yamaguchi and Gd(-) Wakayama

Four new glucose 6-phosphate dehydrogenase (G6PD) variants which had electrophoretically slow-moving characteristics associated with congenital nonspherocytic hemolytic anemia were discovered in Japan. Gd(-) Kurume was found in a 17-year-old male whose red cells contained only 0.8% of normal enzyme activity. Gd(-) Kurume had a normal Km G6P, normal Km NADP, low Ki NADPH, normal utilization of 2-deoxy-G6P and deamino-NADP, very low heat stability and a biphasic pH curve. The hemolytic anemia was mild. Gd(-) Fukushima was characterized from a 33-year-old male and had 2.8% of normal enzyme activity, normal Km G6P, normal Km NADP, low Ki NADPH, normal utilization of 2-dcoxy-G6P, low utilization of deamino-NADP, low heat stability and a normal pH curve. The hemolytic anemia was mild. Gd(-) Yamaguchi was characterized from an 8-year-old boy and had 3.5% of normal enzyme.