Hereditary nonspherocytic hemolytic anemia and hexokinase deficiency

An 11-yr-old child with mild chronic hemolytic anemia was found to have decreased red cell hexokinase activity in spite of the reduced mean age of her red cell population. Similar decreases in red cell hexokinase activity were documented in the patient's parents and in one sib. The red cells were morphologically normal. Red cell 2,3-DPG levels were normal and ATP and glucose-6-phosphate levels were diminished. The kinetic properties, electrophoretic mobility, and thermal stability of the residual red cell hexokinase were normal or nearly so. Glucose consumption of the hexokinase-deficient cells was not appreciably decreased, probably because less of the potent inhibitor glucose-6- phosphate was present in the erythrocytes. It is likely, although not certain, that in this patient nonspherocytic hemolytic anemia resulted from hexokinase deficiency.     

Congenital nonspherocytic hemolytic anemia with an unstable hexokinase variant

We report a family with a new hexokinase variant that gives rise to nonspherocytic hemolytic anemia in one apparently homozygous family member. The variant enzyme has a normal pH optimum, normal reaction kinetics, and normal electrophoretic properties, but has reduced activity and is apparently inactivated rapidly as the affected erythrocytes age.     

Generalized hexokinase deficiency in the blood cells of a patient with nonspherocytic hemolytic anemia

In a patient with nonspherocytic hemolytic anemia, a hexokinase deficiency was detected in the red cells (residual activity about 25% of normal) and in blood platelets (20%-35% of normal activity). Although the total hexokinase activity in lymphocytes was normal, the amount of hexokinase type I was decreased to about 50% of normal. However, the deficiency was compensated for by the appearance of type III hexokinase. Compartmentation studies with controlled digitonin- induced cell lysis showed that this type III enzyme was localized in the cytosol, while almost all hexokinase activity in normal lymphocytes is particulate. No abnormal lymphocyte functions could be detected. The patient was homozygous for the defect. The parents and three of five sibs of the patient were apparently heterozygous with residual activities of 50%-67% of normal in their red cells, but did not show any clinical signs of hexokinase deficiency. The variant enzyme had a slightly decreased affinity for MgATP2- and a strongly increased inhibition constant for glucose-1,6-P2. Affinity for glucose, heat stability, and pH optimum were normal. In the electrophoretic pattern of red cell hexokinase, only one subtype of hexokinase I could be detected, while in normal red cells, at least three subtypes are present. In the heterozygous individuals, no enzymatic abnormalities could be detected, except for an aberration in the electropherogram of one sib.     

Hereditary deficiency of glucosephosphate isomerase as a cause of nonspherocytic hemolytic anemia

Summary The molecular heterogeneity of GPI deficiency is demonstrated. All variants studied are different from each other. The clinical picture is characterized by nonspherocytic hemolytic anemia.

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Zusammenfassung Die molekulare Heterogenität des Glucosephosphat-Isomerase-Mangels wird beschrieben. Alle untersuchten Varianten sind voneinander verschieden. Das klinische Bild ist durch eine nichtsphärozytäre hämolytische Anämie gekennzeichnet.

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This paper has been presented at the annual meeting of the Jugoslav Society of Hematology 1972, Novi Sad, Jugoslavia.     

Glucosephosphate isomerase deficiency type Liège: A new variant with congenital nonspherocytic hemolytic anemia

Summary GPI deficiency was detected in a three year old girl of Morrocan origin suffering, since birth, from hemolytic anemia. The defective GPI is very thermolabile and migrates on starch gel electrophoresis as a single band with a mobility of 96 % of the normal main band.The purification of the patient's GPI resulted in a 16000-fold enriched preparation, free of any other enzyme activity. The yield was 35 %. The purified enzyme was very unstable even at low temperature. The kinetic constants of the forward and backward reaction as well as the inhibitory constants of 2,3-DPG and 6-PG do not significantly differ from normal values. The bio-chemical properties of the patient's GPI indicate a new variant designated type Liège.     

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.     

HK Utrecht: missense mutation in the active site of human hexokinase associated with hexokinase deficiency and severe nonspherocytic hemolytic anemia

Hexokinase deficiency is a rare autosomal recessive disease with a clinical phenotype of severe hemolysis. We report a novel homozygous missense mutation in exon 15 (c.2039C>G, HK [hexokinase] Utrecht) of HK1, the gene that encodes red blood cell-specific hexokinase-R, in a patient previously diagnosed with hexokinase deficiency. The Thr680Ser substitution predicted by this mutation affects a highly conserved residue in the enzyme's active site that interacts with phosphate moieties of adenosine diphosphate, adenosine triphosphate (ATP), and inhibitor glucose-6-phosphate. We correlated the molecular data to the severe clinical phenotype of the patient by means of altered enzymatic properties of partially purified hexokinase from the patient, notably with respect to Mg(2+)-ATP binding. These kinetic properties contradict those obtained from a recombinant mutant brain hexokinase-I with the same Thr680Ser substitution. This contradiction thereby stresses the valuable contribution of studying patients with hexokinase deficiency to achieve a better understanding of hexokinase's key role in glycolysis.     

Hereditary nonspherocytic hemolytic disease; a study of a singular familial hemolytic syndrome

Extensive studies were performed on four cases from three unrelated kindredswith a familial hemolytic syndrome not associated with any significant red cellanomaly (hereditary nonspherocytic hemolytic disease). These cases were compared with similar ones already reported in the literature.

1. Hereditary nonspherocytic hemolytic disease appears to be transmitted asa Mendelian dominant. Frequently the gene responsible for the condition seemsto have low expressivity. In some cases, the hereditary mechanism may be dueto inheritance of a recessive gene from each parent. The basic erythrocytic defectresponsible for the condition is unknown. In view of various clinical and hematologic findings, it is likely that hereditary nonspherocytic hemolytic disease maybe a group of diseases involving more than one mechanism.

2. All criteria of hemolytic anemia (erythroid hyperplasia of the bone marrow,reticulocytosis, hyperbilirubinemia, increased fecal urobilinogen, rapid turnoverof tracer iron in the plasma) were satisfied.

3. Red cell survival time studies revealed an intraerythrocytic defect with amean life span of twelve to seventeen days. Normal red cells transfused into thepatients under study survived normally. Anemia was normochromic and normocytic or macrocytic; it varied from mild to severe.

4. Osmotic and mechanical fragility of the red cells was normal. Osmotic andmechanical fragility tests after incubation at 37 C. for 24 hours in some showed amild increase compared with normal controls. Autohemolysis of incubated oxalated blood was not marked and varied from case to case.

5. The electrophoretic mobility of hemoglobin from the patients was that ofnormal adult hemoglobin. Small increases of fetal hemoglobin were seen in several cases.

6. In contrast to the histologic findings in hereditary spherocytosis the splenicpulp was not congested, but hemosiderin deposits were heavy. Liver biopsy specimens showed deposits of hemosiderin in parenchymal and Kupffer cells.

7. Splenectomy did not arrest the hemolytic process. Mild improvement was seenin one case. In most cases the operation is of no value.

8. Diagnostic difficulties may be encountered with mild cases of hereditaryspherocytosis. Examination of rouleaux in fresh blood and an osmotic fragilitytest in 0.65 per cent sodium chloride after incubation usually establishes the differential diagnosis. The condition may present clinically as hemolytic disease ofthe newborn and must be differentiated from erythroblastosis due to Rh or otherblood group incompatibilities. Other hereditary hemolytic diseases such assickle cell anemia, Cooley’s anemia, hereditary spherocytosis, and hereditaryhemolytic elliptocytosis are easily ruled out by their typical clinical and hematologic manifestations. When a family study is negative or cannot be done, a redcell survival time determination may be necessary to rule out acquired hemolyticanemia with a negative Coombs test. Some cases that have been diagnosed asconstitutional hyperbilirubinemia (familial nonhemolytic jaundice) may actuallyrepresent mild hereditary nonspherocytic hemolytic disease.

Submitted on October 7, 1953 Accepted on November 3, 1953     

Molecular abnormality of phosphoglycerate kinase-Uppsala associated with chronic nonspherocytic hemolytic anemia.

Inherited deficiency of phosphoglycerate kinase (PGK; ATP:3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3) is associated with chronic nonspherocytic hemolytic anemia and mental disorders in man. One such variant, PGK-Uppsala, was purified to homogeneity. PGK-Uppsala had a lower-than-normal specific activity (30% of normal in the backward reaction and about 20% of normal in the forward reaction) and higher-than-normal Michaelis constants for ATP, ADP, 3-phosphoglycerate and 1,3-diphosphoglycerate. Peptide mapping analysis revealed that the structural abnormality of PGK-Uppsala is a single amino acid substitution from arginine to proline at the 206th position. Based on the known complete amino acid sequence of the normal human PGK and the three-dimensional model deduced from horse PGK, correlations between the structural and functional abnormalities of PGK-Uppsala are discussed. Structural abnormalities of PGK-II, which is an electrophoretic variant not associated with enzyme deficiency, and PGK-München, which is associated with enzyme deficiency and heat instability but not associated with hemolytic anemia, are also discussed.     

Functional analysis of pyrimidine 5'-nucleotidase mutants causing nonspherocytic hemolytic anemia

Inherited pyrimidine 5'-nucleotidase type I (P5'N-1) deficiency is the third most common erythrocyte enzymopathy that causes hemolysis. Fourteen different mutations have been identified to date. We have investigated the molecular bases of the disease by studying the biochemical properties of the recombinant wild-type human enzyme and 4 variant proteins (D87V, L131P, N179S, and G230R) bearing missense mutations found in patients affected by nonspherocytic hemolytic anemia. P5'N-1 is a relatively stable protein and has essentially identical catalytic efficiency toward cytidine monophosphate (CMP) and uridine monophosphate (UMP). All investigated mutant proteins display impaired catalytic properties and/or reduced thermostability, providing a rationale for the pathological effects of the mutations. Despite the substantial changes in the kinetic and thermostability parameters, the enzyme activity detected in the red blood cells of patients homozygous for mutations L131P and G230R exhibits moderate alterations. This suggests that P5'N-1 deficiency is compensated, possibly by other nucleotidases or alternative pathways in nucleotide metabolism. Therefore, nucleotidase activity may not be considered a prognostic indicator in patients affected by the enzymopathy.     

Analysis of pyruvate kinase-deficiency mutations that produce nonspherocytic hemolytic anemia.

The intron sequences of the human L-type pyruvate kinase gene (PKLR) were determined by using primers selected from the known cDNA sequence. Oligonucleotide primers for these determined intron sequences were used to sequence the exons. When this technique was applied to the DNA of 10 unrelated patients with pyruvate kinase deficiency, the following eight different mutations in the coding region were detected: del391-393, A401, C464, G721, A1076, T1456, T1484, A1529. The A1529 mutation was found repeatedly in unrelated individuals, even in the homozygous state. The context with respect to a polymorphism at nt 1705 was compatible with a single origin for this mutation, and it may represent a balanced polymorphism. In normal subjects, five differences from the published cDNA sequence were documented.     

Glucose-6-phosphate dehydrogenase aveiro: a de novo mutation associated with chronic nonspherocytic hemolytic anemia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked enzyme abnormality. The clinical phenotype is variable but often predictable from the molecular lesion. Class I variants (the most severe forms of the disease) cluster within exon 10, in a region that, at the protein level, is believed to be involved in dimerization. Here we describe a de novo mutation (C269Y) of a new class I variant (G6PD Aveiro) that maps to exon 8. Mutant and normal alleles were found in both hematopoietic and buccal cells, indicating the presence of mosaicism. The available model of the protein predicts that this lesion lies in proximity to the dimer interface of the molecule. A possible mechanism to explain the severity of the defect is proposed. (Blood. 2000;95:1499-1501)     

Liver cirrhosis as a consequence of iron overload caused by hereditary nonspherocytic hemolytic anemia

Nonspherocytic hereditary anemias are occasionally accompanied by significant iron overload but the significance for the development of chronic liver disease is not clear. We described two cases of patients with chronic liver disease and severe iron overload due to chronic hereditary hemolysis. Both patients have had signs of liver cirrhosis and severe hemolysis since childhood. A hereditary pyruvate kinase deficiency (PKD) was discovered as the underlying reason for the hemolysis. Sequencing of the pyruvate kinase gene showed a mutation within exon 11. Liver histology in both patients revealed cirrhosis and a severe iron overload but primary hemochromatosis was excluded by HFE-gene analysis. An iron reduction therapy with desferrioxamine led to significant decrease of serum ferritin and sustained clinical improvement. PKD-induced hemolysis may cause severe iron overload even in the absence of HFE-genotype abnormalities. This secondary iron overload can lead to chronic liver disease and cirrhosis. Therefore, the iron metabolism of PKD patients has to be closely monitored and iron overload should be consequently treated.