Molecular analysis of glucose-6-phosphate dehydrogenase variants in the Solomon Islands.
Explore the source record for details and available documents.
Black People↗
Biomedical subjects
A Hirono
Publications and source records attributed to A Hirono.
Red cell enzymopathies as a model of inborn errors of metabolism.
The molecular abnormalities of erythroenzymopathies associated with hereditary hemolytic anemia have been determined using molecular techniques. Pyruvate kinase (PK) deficiency is the most common and well-characterized enzyme deficiency involving the glycolytic pathway and causing hereditary hemolytic anemia. We have identified six distinct missense mutations and a form of splicing mutation in 11 unrelated families with homozygous PK deficiency. Mutations located near the substrate binding site may change the conformation of the active site, resulting in a drastic loss of activity and severe clinical symptoms. Up to now, including these genetic defects, 21 missense, 1 nonsense and 2 splicing mutations, 2 insertions, and 3 deletions have been determined. G6PD deficiency is the most common metabolic disorder, and is associated with chronic and drug- or infection-induced hemolytic anemia. To date, sixty different mutations have now been identified. Except for three kinds of variants with small gene deletions or three nucleotide substitutions, all of those were found to be produced by one or two nucleotide substitutions. Molecular studies disclosed that all the class 1 variants associated with chronic hemolysis have the mutations surrounding either the substrate or the NADP binding site. Among rare enzymopathies, missense mutations have been determined in glucosephosphate isomerase deficiency, aldolase deficiency, triosephosphate isomerase (TPI) deficiency, phosphoglycerate kinase deficiency, and adenylate kinase deficiency. Compound heterozygous cases with missense mutation/nonsense mutation and missense mutation/decreased mRNA have been reported in TPI deficiency and diphosphoglyceromutase deficiency, respectively. In phosphofructokinase (PFK) deficiency, three kinds of 5'-splice junction mutations resulting in abnormally spliced PFK-M mRNA were identified. An exception is a hemolytic anemia due to increased adenosine deaminase activity. The basic abnormality appears to result from overproduction of structurally normal enzyme.
Anemia, Hemolytic↗
Molecular study of eight Japanese cases of glucose-6-phosphate dehydrogenase deficiency by nonradioisotopic single-strand conformation polymorphism analysis.
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.
Base Sequence↗
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 (G6PD) variant (G6PD Kobe) cDNA 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 (G6P) of G6PD Kobe suggest the interaction between both NADP and G6P binding sites.
Base Sequence↗
Cellulose-treated blood is a good source of DNA for molecular analysis.
Explore the source record for details and available documents.
Base Sequence↗
G6PD Nara: a new class 1 glucose-6-phosphate dehydrogenase variant with an eight amino acid deletion.
In the course of molecular studies on Japanese glucose-6-phosphate dehydrogenase (G6PD) variants using single-strand conformation polymorphisms (SSCP) analysis, we found an unusual class 1 G6PD variant that had nucleotide deletion in exon 9. The patient showed chronic nonspherocytic hemolytic anemia associated with frequent episodes of severe hemolytic attack. The hemolysate exhibited no measurable activity. Although the partially purified enzyme had detectable activity, we could not perform kinetic studies because of its extreme instability. Nucleotide sequencing showed a unique 24 bp deletion at nucleotide 953-976 that predicts an eight amino acid deletion of TKGYLDDP at residue 319-326. While this is one of the most drastic structural alterations found in G6PD variants, the region with the amino acid deletion was distant from both the G6P and NADP+ binding sites and was located in a domain with low sequence homology among species. The comparatively low functional importance of the deleted region may have saved the patient from lethal tissue dysfunction.
Amino Acid Sequence↗
Hereditary triosephosphate isomerase (TPI) deficiency: two severely affected brothers one with and one without neurological symptoms.
A 13-year-old Hungarian boy (B.J.Jr.) with congenital haemolytic anaemia (CHA) and hyperkinetic torsion dyskinesia was found to have severe triose-phosphate isomerase (TPI) deficiency. One of his two brothers (A.J.), a 23-year-old amateur wrestler, has CHA as well, but no neurological symptoms. Both have less than 10% TPI activity and a highly increased dihydroxyacetone phosphate (DHAP) level in their red blood cells. Their TPI had a slow electrophoretic mobility and was heat unstable. Both parents and a third brother are healthy heterozygous carriers of the defect. A.J. represents a unique phenotype from the point of view that all published "homozygotes" had severe neurological alterations from infancy or early childhood except one infant who died at 11 months, probably too young for neurological symptoms to be noted. In contrast to the two affected Hungarian brothers all but one "homozygote" has died before the age of 6 years. The striking difference in the clinical course of the defect between the two brothers with the same severe red blood cell enzyme deficiency may originate from unusual differences between two double heterozygous brothers resulting inter alia in different levels of TPI expression in various tissues. Significantly lower TPI activities were found in both the T- and B-cells of the propositus as compared to the respective cells of the neurologically symptom-free brother.
Adult↗
Molecular abnormality of G6PD Konan and G6PD Ube, the most common glucose-6-phosphate dehydrogenase variants in Japan.
G6PD Konan and G6PD Ube are the most common glucose-6-phosphate dehydrogenase (G6PD) variants found in Japan. To clarify the molecular abnormality of these two variants, the entire coding region was amplified by polymerase chain reaction from genomic DNA (G6PD Konan) or cDNA (G6PD Ube). Direct sequencing revealed that both variants have the same nucleotide substitution (241 C to T) in exon 4, which predicts an Arg to Cys substitution at amino acid 81.
Arginine↗
Human glucose-6-phosphate dehydrogenase: structure and function of normal and variant enzymes.
Glucose-6-phosphate dehydrogenase (G6PD) is a unique enzyme with many genetic variants. Recent progress in molecular biological techniques have provided several important findings about the structure-function relationship of human G6PD. A putative substrate glucose-6-phosphate binding (around Lys 205) and a putative 'structural' NADP binding site (around Lys 386) have been identified. A conservative segment (amino acid 38-44) near the N-terminus was proposed to be the possible second NADP binding region (amino acid 38-44). Analysis of amino acid substitutions of variants revealed that there might be some relationship between the position of substitution and the properties of variants.
Amino Acid Sequence↗
A single amino acid substitution (157 Gly----Val) in a phosphoglycerate kinase variant (PGK Shizuoka) associated with chronic hemolysis and myoglobinuria.
We have determined a single amino acid substitution in a new phosphoglycerate kinase (PGK) variant, PGK Shizuoka, associated with chronic hemolysis and myoglobinuria. PGK Shizuoka had an extremely low enzyme activity with normal kinetic properties and normal electrophoretic mobility. Total blood cell RNA of the patient was reverse-transcribed and amplified by the polymerase chain reaction. A single nucleotide substitution from guanine to thymine at position 473 of PGK messenger RNA was found. This nucleotide change causes a single amino acid substitution from Gly to Val at the 157th position, which is located in the NH2-terminal domain of the enzyme. This mutation creates a new Bst XI cleavage site in exon 5, and we thus confirmed the mutation in the variant gene. The replacement of Gly by Val is considered to affect enzyme catalysis.
Adult↗
Identical point mutations of the R-type pyruvate kinase (PK) cDNA found in unrelated PK variants associated with hereditary hemolytic anemia.
We cloned and sequenced the cDNAs for R-type pyruvate kinase (R-PK) from three homozygous PK patients. A point mutation (1151ACG to ATG) identified in the R-PK cDNA of PK Nagasaki was identical with that previously identified in an unrelated family, PK Tokyo. The mutation has also been identified in the PK L gene of a Lebanese family, PK Beirut, by Neubauer et al (Blood 77:1871, 1991). These results suggest either that the point mutation is very old or that the same mutation occurs sporadically in the same hot spot in unrelated families. A point mutation, 1261CAG to AAG, was detected in both PK Fukushima and PK Maebashi; this mutation causes a single amino acid substitution, from Gln421 to Lys, in R-PK. Consequently, the hydrophobicity properties near the mutated site are drastically changed. A silent mutation (1705AGG to CGG) was also identified in those variants. The screening of these three point mutations showed only the silent mutation in a normal individual as well as in individuals with PK variants. These results indicate that the multiplicity of the mutant PK allele is smaller than expected, and that the silent mutation is a polymorphic change commonly distributed in the Japanese population.
Anemia, Hemolytic, Congenital↗
Molecular abnormality of a Japanese glucose-6-phosphate dehydrogenase variant (G6PD Tokyo) associated with hereditary non-spherocytic hemolytic anemia.
The entire coding sequence of a Japanese class 1 variant (G6PD Tokyo) was amplified by the polymerase chain reaction from genomic DNA. Nucleotide analysis by a direct sequencing technique revealed a unique nucleotide substitution (1246 G to A) in exon 10, which predicts a Glu to Lys substitution at the 416th amino acid. This is another member of a conspicuous mutation cluster surrounding the putative NADP-binding domain.
Anemia, Hemolytic↗
A single nucleotide substitution in the phosphoglycerate kinase (PGK)-1 gene occurred after the separation of PGK-1 and PGK-2.
The mammalian genome contains two functional loci for the production of phosphoglycerate kinase, PGK-1, an X-linked gene expressed in all somatic cells, and PGK-2, an autosomal intron-less gene expressed exclusively in late spermatogenesis. A nucleotide substitution from guanine to thymine was recently found at position 473 of PGK-1 mRNA in PGK Shizuoka. The mutation was not found in the PGK-2 gene and might have occurred after separation of PGK-1 and PGK-2.
Animals↗
cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia.
cDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-specific primer. The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino acids, the same number as that by rat R-type PK. Compared with human L-type PK, R-type PK was 31 amino acids longer at the amino terminus. We also cloned and characterized R-type PK cDNA clones from patients with hereditary hemolytic anemia from a PK deficiency, PK Tokyo. A single nucleotide substitution (ACG to ATG) was found at nucleotide 1151 of the coding sequence of the R-type PK, which caused an amino acid substitution, Thr384----Met. Dot blot hybridization of PCR-amplified genomic DNA from patients and their parents by allele-specific oligonucleotide probes showed that the parents, who were second cousins, were heterozygous. To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coli and characterized the enzyme. The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris.HCl, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK. From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene.
Amino Acid Sequence↗
Identification of the binding domain for NADP+ of human glucose-6-phosphate dehydrogenase by sequence analysis of mutants.
Human erythrocyte glucose-6-phosphate dehydrogenase is normally quite stable in the presence of 10 microM NADP+. Certain glucose-6-phosphate dehydrogenase variants lose virtually all their activity at this concentration of NADP+ but are reactivated by 200 microM NADP+. Such variants presumably have a defect in their NADP+-binding site. We analyzed the sequence of cDNA or genomic DNA from seven unrelated patients with hemolytic anemia due to the inheritance of variants that are reactivated by NADP+. Six patients had substitutions of one of three adjacent amino acids, and the seventh patient had another amino acid substitution 23 residues downstream. These amino acids are highly conserved, all being present in rat and all but one being found also in Drosophila. The anomalous electrophoretic behavior of some of the variants can be explained by their loss of ability to bind NADP+. We conclude that the region in which these mutations occur defines the binding domain for NADP+ and that binding NADP+ that has been designated as "structural" and as "catalytic" probably occurs at the same site.
Amino Acid Sequence↗
Alternative splicing of human glucose-6-phosphate dehydrogenase messenger RNA in different tissues.
Different forms of glucose-6-phosphate dehydrogenase (G-6-PD) have been described in different tissues. Moreover, the directly determined amino acid sequence amino end of the red cell enzyme does not exactly match the sequence deduced from cDNA isolated from HeLa cells or lymphoblasts. We have therefore investigated the sequence of cDNA from sperm, granulocytes, reticulocytes, brain, placenta, liver, lymphoblastoid cells, and cultured fibroblasts. A novel human cDNA, which has extra 138 bases coding 46 amino acids, was isolated from a lymphoblastoid cell library. Sequencing of genomic DNA amplified by the polymerase chain reaction (PCR) revealed that the extra sequence was derived from the 3'-end of intron 7 by alternative splicing. This longer form of mRNA was also detected in sperm and granulocytes. Sequence analysis using PCR-amplified cDNA revealed that the 5'-end of the coding sequence of G6PD mRNA in reticulocytes is identical to those in other tissues.
Amino Acid Sequence↗
The molecular biology of variation in glucose-6-phosphate dehydrogenase.
The complete cDNA sequence of G-6-PD is now known, and the application of modern technology has made it possible to rapidly accumulate sequence data regarding G-6-PD variants. The information that has become available demonstrates that there are several different types of G-6-PD A(-) but all have in common the mutation that is characteristic of G-6-PD A(+), viz. a substitution of G for A at nucleotide 376. Moreover, the same nucleotide substitutions have been found in Spanish and Italian subjects, indicating that the G-6-PD A(-) mutation is much more common in non-African populations than had been thought. G-6-PD Mediterranean, on the other hand, seems to be more uniform than had been thought previously. A wealth of information regarding structure/function relationship of enzymes is potentially available through the study of G-6-PD variants.
Amino Acid Sequence↗
Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-).
Glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) A(-) is a common variant in Blacks that causes sensitivity to drug-and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3' end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C33----G, G202----A, and A376----G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The base substitution at position 376, identical to the substitution that has been reported in G6PD A(+), was present in all G6PD A(-) samples and none of the control G6PD B(+) samples examined. The substitution at position 202 was found in four of the five G6PD A(-) samples and no normal control sample. At position 33 guanine was found in all G6PD A(-) samples and seven G6PD B(+) control samples and is, presumably, the usual nucleotide found at this position. The finding of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein.
Amino Acid Sequence↗