Mutant Erythrocytes Research Articles

Hereditary spherocytosis in zebrafish riesling illustrates evolution of erythroid beta-spectrin structure, and function in red cell morphogenesis and membrane stability.

Spectrins are key cytoskeleton proteins with roles in membrane integrity, cell morphology, organelle transport and cell polarity of varied cell types during development. Defects in erythroid spectrins in humans result in congenital hemolytic anemias with altered red cell morphology. Although well characterized in mammals and invertebrates, analysis of the structure and function of non-mammalian vertebrate spectrins has been lacking. The zebrafish riesling (ris) suffers from profound anemia, where the developing red cells fail to assume terminally differentiated erythroid morphology. Using comparative genomics, erythroid beta-spectrin (sptb) was identified as the gene mutated in ris. Zebrafish Sptb shares 62.3% overall identity with the human ortholog and phylogenetic comparisons suggest intragenic duplication and divergence during evolution. Unlike the human and murine orthologs, the pleckstrin homology domain of zebrafish Sptb is not removed in red cells by alternative splicing. In addition, apoptosis and abnormal microtubule marginal band aggregation contribute to hemolysis of mutant erythrocytes, which are features not present in mammalian red cells with sptb defects. This study presents the first genetic characterization of a non-mammalian vertebrate sptb and demonstrates novel features of red cell hemolysis in non-mammalian red cells. Further, we propose that the distinct mammalian erythroid morphology may have evolved from specific modifications of Sptb structure and function.

Somatic Cell Mutations at the Glycophorin A Locus in Erythrocytes of Atomic Bomb Survivors: Implications for Radiation Carcinogenesis

To clarify the relationship between somatic cell mutations and radiation exposure, the frequency of hemizygous mutant erythrocytes at the glycophorin A (GPA) locus was measured by flow cytometry for 1,226 heterozygous atomic bomb (A-bomb) survivors in Hiroshima and Nagasaki. For statistical analysis, both GPA mutant frequency and radiation dose were log-transformed to normalize skewed distributions of these variables. The GPA mutant frequency increased slightly but significantly with age at testing and with the number of cigarettes smoked. Also, mutant frequency was significantly higher in males than in females even with adjustment for smoking and was higher in Hiroshima than in Nagasaki. These characteristics of background GPA mutant frequency are qualitatively similar to those of background solid cancer incidence or mortality obtained from previous epidemiological studies of survivors. An analysis of the mutant frequency dose response using a descriptive model showed that the doubling dose is about 1.20 Sv [95% confidence interval (CI): 0.95-1.56], whereas the minimum dose for detecting a significant increase in mutant frequency is about 0.24 Sv (95% CI: 0.041-0.51). No significant effects of sex, city or age at the time of exposure on the dose response were detected. Interestingly, the doubling dose of the GPA mutant frequency was similar to that of solid cancer incidence in A-bomb survivors. This observation is in line with the hypothesis that radiation-induced somatic cell mutations are the major cause of excess cancer risk after radiation exposure. Furthermore, the dose response was significantly higher in persons previously or subsequently diagnosed with cancer than in cancer-free individuals. This may suggest an earlier onset of cancer due to elevated mutant frequency or a higher radiation sensitivity in the cancer group, although the possibility of dosimetry errors should be considered. The findings obtained in the present study suggest that the GPA mutant frequency may reflect the cancer risk among people exposed to radiation.

Do mutations at the glycophorin A locus i patients treated for childhood Hodgkin's disease predict secondary leukaemia?

Childhood Hodgkin's disease has a high cure rate but second cancers are common, related to treatment and perhaps genetic predisposition. The glycophorin A (GPA) mutation assay measures the frequency of NO and NN mutant erythrocytes of MN blood-group heterozygous individuals. Mutant frequencies in Hodgkin's disease patients were compared with controls. No significant difference from controls was found pretreatment or in patients treated with radiotherapy only. Patients who received chemotherapy had a significantly increased frequency (total mutation frequency per 106 cells: 31 vs 11, p<0·001), which may be relevant to their known increased risk of secondary leukaemia.

The micronucleus assay with 1,3-propane sultone

The Pig-a assay is a useful in vivo mutation detecting test and is easier to perform than the in vivo transgenic mutation assay. This assay is now recognized to be able to detect a number of mutagenic chemicals administered to rats in sub-acute or sub-chronic dose studies. The present investigation was conducted to evaluate the usefulness of peripheral blood Pig-a assays with total red blood cells (RBC Pig-a assay) and with reticulocytes (PIGRET assay) using two genotoxic rodent carcinogens, 1,3-propane sultone (1,3-PS) and 2-acetylaminofluorene (2-AAF). Male rats were orally administered a single dose of each test compound, and both the RBC Pig-a and PIGRET assays were performed using flow cytometry to measure the Pig-a mutant frequency (MF) before and after dosing on Days 8, 15 and 29. In the experiment with 1,3-PS, significant increases in Pig-a MF were observed from Day 15 and Day 8 in the RBC Pig-a and PIGRET assays, respectively. The results of both assays demonstrated that the increases in Pig-a MF were detectable after a single treatment with 1,3-PS. Furthermore, the difference in the kinetics of the increase in Pig-a MF between the RBC Pig-a and PIGRET assays with 1,3-PS suggests that the PIGRET assay has an advantage in detecting the mutant erythrocytes earlier than the RBC Pig-a assay. In contrast, no significant increases were observed in the Pig-a assays using either RBC or reticulocytes with 2-AAF. The negative results in both assays with 2-AAF may indicate the limitation of the single dose method; however, further investigation at higher doses is necessary to determine limitation of the single dose method.

Glucose-6-phosphate isomerase deficiency associated with nonspherocytic hemolytic anemia in the mouse: an animal model for the human disease

The first two mutations causing hereditary glucose-6-phosphate isomerase (GPI) deficiency associated with chronic nonspherocytic hemolytic anemia in nonhuman mammals are described in the mouse. As in humans, the hemolytic syndrome, which is characterized by a diminished erythrocyte number, lower hematocrit, lower hemoglobin, higher number of reticulocytes and plasma bilirubin concentration, as well as increased liver- and spleen-somatic indices, was exclusively manifested in homozygous mutants. In comparison with wild type, heterozygous individuals exhibited neither hematologic differences nor alterations of other physiologic parameters, including plasma concentration of glucose, pyruvate and lactate, body weight, organo-somatic indices of liver, lung, kidney, spleen, and heart, as well as viability. Glycolytic intermediates, adenine nucleotides, and metabolic rate were not significantly altered in erythrocytes from heterozygotes. On the contrary, if allowance is made for the young erythrocyte population, homozygous mutant erythrocytes showed an increased concentration of glucose-6-phosphate and normal or decreased concentrations of glycolytic metabolites following the enzymatic block. The concentration of adenosine triphosphate and the glycolytic rate also appeared to be reduced. Homozygous anemic mice showed hepatosplenomegaly and typical adaptations to hypoxia, such as an elevated heart-somatic index and, for one mutant line, an enhanced lung-somatic index. Further, these animals were characterized by a marked reduction of body weight and an increase of lethality both correlated with the degree of enzyme deficiency in tissues. The latter findings were attributed to a reduced glycolytic capability of the whole organism caused by the enzyme defect in tissues, rather than representing secondary consequences of GPI deficiency in erythrocytes. The similarity in physicochemical and kinetic properties of the mutant murine proteins reported earlier with those of allozymes found in human GPI deficiency, as well as the comparable metabolic and physiologic consequences of this enzyme defect in mice and humans support that these murine mutants are excellent animal models for the human disease.

Glucose-6-Phosphate Isomerase Deficiency Associated With Nonspherocytic Hemolytic Anemia in the Mouse: An Animal Model for the Human Disease

The first two mutations causing hereditary glucose-6-phosphate isomerase (GPI) deficiency associated with chronic nonspherocytic hemolytic anemia in nonhuman mammals are described in the mouse. As in humans, the hemolytic syndrome, which is characterized by a diminished erythrocyte number, lower hematocrit, lower hemoglobin, higher number of reticulocytes and plasma bilirubin concentration, as well as increased liver- and spleen-somatic indices, was exclusively manifested in homozygous mutants. In comparison with wild type, heterozygous individuals exhibited neither hematologic differences nor alterations of other physiologic parameters, including plasma concentration of glucose, pyruvate and lactate, body weight, organo-somatic indices of liver, lung, kidney, spleen, and heart, as well as viability. Glycolytic intermediates, adenine nucleotides, and metabolic rate were not significantly altered in erythrocytes from heterozygotes. On the contrary, if allowance is made for the young erythrocyte population, homozygous mutant erythrocytes showed an increased concentration of glucose-6-phosphate and normal or decreased concentrations of glycolytic metabolites following the enzymatic block. The concentration of adenosine triphosphate and the glycolytic rate also appeared to be reduced. Homozygous anemic mice showed hepatosplenomegaly and typical adaptations to hypoxia, such as an elevated heart-somatic index and, for one mutant line, an enhanced lung-somatic index. Further, these animals were characterized by a marked reduction of body weight and an increase of lethality both correlated with the degree of enzyme deficiency in tissues. The latter findings were attributed to a reduced glycolytic capability of the whole organism caused by the enzyme defect in tissues, rather than representing secondary consequences of GPI deficiency in erythrocytes. The similarity in physicochemical and kinetic properties of the mutant murine proteins reported earlier with those of allozymes found in human GPI deficiency, as well as the comparable metabolic and physiologic consequences of this enzyme defect in mice and humans support that these murine mutants are excellent animal models for the human disease.

A new mutant erythrocyte pyrimidine 5'-nucleotidase characterized by fast electrophoretic mobility in a Chinese boy with chronic hemolytic anemia

A new mutant erythrocyte pyrimidine 5'-nucleotidase characterized by fast electrophoretic mobility in a Chinese boy with chronic hemolytic anemia

Ankyrin and the hemolytic anemia mutation, nb, map to mouse chromosome 8: presence of the nb allele is associated with a truncated erythrocyte ankyrin.

Mice with normoblastosis, nb/nb, have a severe hemolytic anemia. The extreme fragility and shortened lifespan of the mutant erythrocytes result from a defective membrane skeleton. Previous studies in our laboratory indicated a 50% deficiency of spectrin and an absence of normal ankyrin in erythrocyte membranes of nb/nb mice. We now report genetic mapping data that localize both the nb and erythroid ankyrin (Ank-1) loci to the centromeric end of mouse chromosome 8. Using immunological and biochemical methods, we have further characterized the nature of the ankyrin defect in mutant erythrocytes. We do not detect normal sized (210 kDa) erythroid ankyrin by immunoblot analysis in nb/nb reticulocytes. However, nb/nb reticulocytes do contain a 150-kDa ankyrin immunoreactive protein. The 150-kDa protein is present with normal-sized ankyrin in nb/+ reticulocytes but is not found in +/+ reticulocytes. Our genetic and biochemical data indicate that the nb mutation results from a defect in the erythroid ankyrin gene. A human hereditary spherocytosis putatively resulting from an ankyrin defect maps to a segment of human chromosome 8 that is homologous to the nb-ankyrin region of mouse chromosome 8. The linkage data suggest that the mouse and human diseases result from mutations in homologous loci.

Search for a relationship between molecular anomalies of the mutant erythrocyte pyruvate kinase variants and their pathological expression

Anomalies of the mutant pyruvate kinase variants and clinical symptoms have been compared in 22 unrelated patients with congenital red cell pyruvate kinase deficiency. This study suggests that some characteristics of the mutant enzymes could play a role in the intensity of haemolysis, namely residual activity, affinity for the substrate phosphoenolpyruvate and for the allosteric activator fructose 1,6 diphosphate and inhibition by ATP.

Purine Base Transport in Normal and Mutant Erythrocytes

The uptake of adenine and hypoxanthine in HGPRT-deficient and normal human erythrocytes was measured using a rapid filtering centrifugation technique. The transport of hypoxanthine as well as of adenine is impaired in the mutant cells. The transport of hypoxanthine into HGPRT-deficient erythrocytes differs from that into normal cells with respect to a higher accumulation capacity, to lower initial velocities and to the kinetic properties of the translocator. In addition, a higher accumulation capacity and lower initial velocities of adenine uptake could be demonstrated in mutant cells. A linkage of the purine translocator with purine phosphoribosyltransferases associated with the erythrocyte membrane is discussed.

Hereditary erythrocyte pyruvate kinase deficiency: molecular and functional studies of four mutant PK variants detected in Spain

The mechanisms of the defect in enzyme activity and the functional anomalies have been studied for four different mutant erythrocyte pyruvate kinase (PK) variants found in Spain. In two cases the PK variants were heat stable, and their antigenic concentration was nearly normal in the patients' red cells: the defect in activity was due to decreased catalytic activity of the mutant molecules. In the two other observations the mutant PK variants were heat-labile, and their antigenic concentration was reduced in the red cells: the defect in activity was entirely due to this decrease of the enzyme molecule concentration in one case, and to decrease of the enzyme molecule concentration plus decrease of catalytic activity of these mutant molecules in the other case. Kinetic properties were normal for one variant and drastically modified in the three other variants. These latter variants showed decreased apparent affinity for PEP, which cannot be simply explained by a shift of the R α T allosteric equilibrium towards the T form: the results of the study of ATP inhibition and FDP activation, indeed, did not correspond to the expected results for a PK enzyme present in a predominant T form. We point out the heterogeneity and the complexity of the anomalies of the mutant PK variant, most likely reflecting the diversity of the possible punctual mutations of the PK structural gene.

Biphasic reaction kinetics in an anomalous isozyme of erythrocyte pyruvate kinase

A mutant erythrocyte isozyme of pyruvate kinase (PK) (ATP:pyruvate phosphotransferase, EC 2.7.1.40) has been found in association with chronic hemolytic anemia in two siblings who were doubly heterozygous for the isozyme and for quantitative PK deficiency of the usual form. The isozyme was characterized by approximately normal maximum reaction velocities but had markedly decreased affinity for the substrate, phosphoenolpyruvate (PEP), with 5-fold to 10-fold increases in half-saturation constants and decreased interaction between substrate binding sites. Two distinctly separable kinetic patterns for PEP were observed with multiple specimens. Concentrations of fructose 1,6-diphosphate (FDP) required for half-maximal activation were two orders of magnitude greater than controls, and optimal pH was lowered to 6.5. Stability at 4°C was markedly decreased, but the lost enzyme could be reactivated by FDP for periods even longer than normal controls.

Electrophoretic characterization of human phosphoribosylpyrophosphate synthetase

A method has been developed for the electrophoretic separation of 5-phosphoribosyl-1-pyrophosphate synthetase on cellulose acetate. The human enzyme in extracts of various tissues appears as a narrow symmetrical peak, indicating a single genotype. Similar results were obtained with rabbit and rat tissues. A mutant erythrocyte 5-phosphoribosyl-1-pyrophosphate synthetase from two siblings having gout associated with excessive purine production was found to have normal electrophoretic mobility.

Electrophoretic and Kinetic Studies of Mutant Erythrocyte Pyruvate Kinases

Abstract Thin-layer polyacrylamide electrophoresis and kinetic studies were used to differentiate three different mutant erythrocyte pyruvate kinase (PK) in congenital nonspherocytic hemolytic anemia. In the first family, proband and father had PK which migrated faster than the normal and high Michaelis—Menten constants (Km), whereas mother had normal migration and kinetics, but low PK activity. In the second family, proband and mother had PK which migrated slower than the normal. Proband had markedly high Km and mother intermediately high Km. In the third family, proband and mother had faster migrating PK than the normal. Proband had high Km and mother had intermediately high Km. The parents in the second and third families were consanguineous. Inherited molecular abnormalities of erythrocyte PK were proven to exist by abnormal electrophoretic mobilities, as well as abnormal results of kinetic studies.

Human Carbonic Anhydrases: IV. PROPERTIES OF A MUTANT B TYPE ISOZYME

A mutant erythrocyte B type carbonic anhydrase has been found in a Caucasian. The isozyme was isolated in crystalline form and various of its properties were explored. It was similar in electrophoretic properties to the P isozyme previously found in pooled erythrocytes of normal individuals. It differs from the normal B and P isozymes in that 1 aspartic acid residue is substituted by valine. This variation was localized to a chymotryptic undecapeptide whose sequence was determined.

Kinetic studies of mutant human erythrocyte adenine phosphoribosyltransferases.

The kinetic constants and reaction mechanism of human erythrocyte adenine phosphoribosyltransferase from individuals whose enzyme activities have high, intermediate, or low degrees of stability to heat inactivation, and in a human mutant with reduced activity of this enzyme, have been measured. The Michaelis constants for one or both substrates are different from normal in seven mutants.