G-6-PD Tripler: A Unique Variant Associated with Chronic Hemolytic Disease

A 52-year-old Caucasian was hospitalized for recurring jaundice. Laboratorytests established a compensated hemolytic anemia due to partial erythrocyteG-6-PD deficiency. Biochemical characterization of the affected enzyme uncovered a unique variant: G-6-PD Tripler. Its significant properties includemarked thermal instability, an electrophoretic mobility slower than the normalB variant in all systems studied, and slightly increased affinity for the substrate G-6-P.

Submitted on February 9, 1970 Revised on February 9, 1970 Accepted on February 17, 1970     

G6PD Varadero A New Variant of Glucose-6-Phosphate Dehydrogenase Associated with Congenital Nonspherocytic Hemolytic Anemia

Abstract. A glucose-6-phosphate dehydrogenase (G6PD) variant was studied in a mulatto patient with chronic nonspherocytic hemolytic anemia. This variant has reduced activity, increased thermolability, a reduced Michaelis constant for glucose-6-phosphate, slightly increased electrophoretic mobility, a biphasic pH activity profile, high 2-deoxy-glucose-6-phosphate utilization, normal diamino nicotinamide adenine dinucleotide phosphate utilization and a peak of elution profile after G6PD B. The electrophoretic, kinetic, and chromatographic properties of this erythrocyte G6PD variant allow the conclusion that G6PD Varadero is probably a new variant.     

A New Variant of Hereditary Hemolytic Anemia With Stomatocytosis and Erythrocyte Cation Abnormality

A new variant of congenital hemolyticanemia associated with stomatocytosis,reticulocytosis, decreased osmotic fragility, type I autohemolysis and shortened erythrocyte survival without specific splenic sequestration was discoveredin three siblings of Swiss-German ancestry. Increased intracellular sodium(two to three times normal) and slightlydecreased intracellular potassium weredetected. Total sodium efflux was eight-fold greater than normal but total potassium influx was normal and ouabain-sensitive potassium influx was decreased.The ouabain-sensitive sodium efflux:potassium influx ratio was 26:1 ratherthan the 3:2 ratio noted in normal cells.The consanguineous parents, four othersiblings, and 44 other family membershad mild stomatocytosis, reticulocytosis,and, when studied, decreased osmoticfragility, increased autohemolysis, intermediate abnormalities of cation content,cation flux, and moderate shortening oferythrocyte survival. Autosomal dominant inheritance was suggested. Noabnormalities of RBC enzymes, hemoglobin or lipids were observed. No abnormalities of membrane protein weredetected on acrylamide gel. Substratedepletion of these hypermetabolic cellsresulted in intracellular dehydrationwith potassium loss in excess of sodiumgain and decreased deformability. Although the exact nature of the defectresponsible for hemolysis is unknown,this syndrome differs from other hereditary hemolytic anemias associated withstomatocytosis.

Submitted on December 21, 1970 Revised on March 16, 1971 Accepted on March 29, 1971     

G-6-PD Walter Reed: possible insight into "structural" NADP in G-6-PD

A new G-6-PD variant, G-6-PD Walter Reed, causing hereditary nonspherocytic hemolytic anemia is characterized. This variant is unusual in that its stability requires the presence of high concentrations of NADP, while its Km for NADP is normal. This finding is consistent with the suggestion that G-6-PD has two separate binding sites, a high affinity "structural" site and a lower affinity catalytic site. The mutation in G-6-PD Walter Reed, like that of the previously described variant, G-6-PD Torrance, may be due to a mutation of the "structural" site for NADP.     

Glucosephosphate Isomerase (GPI) Deficiency Mutations Associated with Hereditary Nonspherocytic Hemolytic Anemia (HNSHA)

ABSTRACT: Five unrelated patients with hereditary glucosephosphate isomerase (GPI) deficiency resulting in nonspherocytic hemolytic anemia were studied. Three new mutations were found in the coding region of the GPI gene: two patients were heterozygous for 223 A→G (R75G) and 898 G→C(R300P), respectively and one was homozygous for 1415G→A(R472H). Surprisingly, 2 previously reported mutations, 286 C→T and 1039 C→T, were found in 2 and 3 patients respectively. Until now only 4 of 18 GPI mutations had been found more than once in unrelated patients and these 4 in only 2 patients each. Eleven of the 20 known point mutations have occurred at CpG “hot spots” and the 286 C→T and 1039 C→T are among these. The 489 G/A polymorphism in the GPI coding region was used to demonstrate unequivocally that the 1039 C→T mutation occurred in both haplotypes and therefore probably originated more than once. Because no common GPI mutation has been found we suggest that heterozygosity for GPI confers little if any selective advantage.     

Glucose-6-phosphate dehydrogenase (G-6-PD) mutations in Mexico: four new G-6-PD variants

Screening for mutations at the G-6-PD gene by PCR-SSCP combined with restriction enzyme analysis and DNA sequencing was performed in nine G-6-PD deficient individuals with negative results for the presence of the most frequent G-6-PD mutations previously observed in Mexican population. The variants G-6-PD Valladolid(406T), G-6-PD Durham(713G), and G-6-PD Viangchan(871A) and four new G-6-PD mutant alleles were identified. The new mutations are located at cDNA nt 376 A --> T (126 Asn --> Tyr), nt 770 G --> T (257 Arg --> Leu), nt 1094 G --> A (365 Arg --> His), and nt 1285 A --> G (429 Lys --> Glu) and they were named G-6-PD San Luis Potosi, G-6-PD Zacatecas, G-6-PD Veracruz, and G-6-PD Yucatán, respectively. To date, a total of 18 different G-6-PD variants have been observed in Mexico and several of them are common in Africa, South Europe, and Southeast Asia.     

Congenital Nonspherocytic Hemolytic Anemia, Associated with Glutathione Deficiency of the Erythrocytes: Hematologic, Biochemical and Genetic Studies

1. A new biochemical defect of erythrocytes is described: glutathionedeficiency (reduced glutathione less than 10 per cent of the amount of reducedglutathione in normal erythrocytes).

2. The defect is associated with a clinical picture of congenital nonspherocytic hemolytic anemia which is fairly well compensated.

3. The results of a family study are consistent with an autosomal recessivepattern of inheritance.

4. Labeling with Na₂Cr⁵¹O₄ has a damaging effect on glutathione-deficienterythrocytes. The erythrocyte life span, as estimated by a serological method(Ashby), was markedly shortened (30 days instead of 100-120 days).

5. Red cell destruction could be increased by the administration of primaquine.

6. Secondary to the glutathione deficiency, low glyoxalase activity wasobserved. The glutathione-reducing capacity, glycolytic activity, and the ATPlevel of the abnormal red cells were found to be within the normal range.

7. On incubation of the glutathione-deficient erythrocytes in vitro withglycine-C¹⁴ and glutamine-C¹⁴, no formation of labeled glutathione could bedemonstrated.

Submitted on October 7, 1964 Accepted on June 29, 1965     

Three new variants of glucose-6-phosphate dehydrogenase associated with chronic nonspherocytic hemolytic anemia: G-6-PD Lincoln Park, G-6-PD Arlington Heights, and G-6-PD West Town

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency was identified in three children who were evaluated because of chronic nonspherocytic hemolytic anemia. One child is of German extraction, another Puerto Rican, and the third Mexican. In each of the patients the hemolytic process was well compensated, but each had one or more episodes of anemia following exposure to an oxidant drug or with infections. The electrophoretic, functional, and kinetic properties of the mutant enzymes, derived both from the patients' erythrocytes and from cultured fibroblasts, allowed each to be distinguished from G-6-PD variants previously described.     

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     

Hereditary Hemolytic Anemia Associated With Abnormal Membrane Lipids: Mechanism of Accumulation of Phosphatidyl Choline

In order to study the mechanism of theaccumulation of phosphatidyl choline(PC) in erythrocytes with abnormal erythrocyte phospholipids from patientswith a hereditary hemolytic anemia, thephospholipids of the erythrocytes werelabeled radioactively. Labeling of phosphatides was achieved by both passiveequilibration with preformed phosphatides, and active "acylase"-mediated incorporation of fatty acid (FA) in thepresence of glucose, ATP and coenzymeA. The labeled cells were then reincubated in fresh compatible sera and thecatabolism of the labeled erythrocytephospholipids was followed. In addition,total acylase capacity of erythrocytestroma was determined under optimalconditions in a system with excesslysophosphatide, FA, ATP, CoA, andMg⁺⁺. No differences in passive uptakeor release of phosphatides were found between the patients’ erythrocytes andcomparable reticulocyte-rich controls. Onthe other hand, overall active incorporation of FA into PC was abnormally increased in the patients’ erythrocytes,whereas incorporation of FA intophosphatidyl-ethanolamine (PE) was decreased. However, acylase capacity forboth lysophosphatidylcholine (LPC) andlysophosphatidylethanolamine (LPE) wasnormal in the patients’ cells. This apparent paradox could be explained by thesubsequent turnover of actively incorporated PC-FA which was found to bereduced. A brief labeling experiment designed to approximate pulse-chase conditions and to label primarily PC showed aconsiderable inhibition of the subsequenttransfer of PC-FA to PE upon reincubation in fresh serum. This transfer haspreviously been shown to be responsiblefor a significant portion of PC-FA catabolism. Reincubation in hyperlipemicsera obtained from patients with liverdisease or artificially enriched with PCdid not influence the abnormal outflow ofphosphatide-FA in actively labeled cells.The findings were consistent with theconcept that PC accumulated in thesecells because of a defect in the catabolismof actively incorporated PC-FA. This defect appeared to be in the transfer ofPC-FA to PE prior to final release fromthe cell. Passive exchange pathways andthe active anabolic acylase pathway werenot abnormal in these patients’ erythrocytes.

Submitted on March 4, 1971 Revised on May 4, 1971 Accepted on May 10, 1971     

Case Report: Autoimmune Hemolytic Anemia in Chronic Granulocytic Leukemia

A patient with chronic granulocytic leukemia developed autoimmune hemolytic anemia. He was found to have a cold agglutinin and a nonspecific warmpanagglutinin. Therapy with prednisone appeared to control the hemolyticcrisis and did not affect the granulocytic process. The patient died of bronchopneumonia, and at autopsy no other neoplasm was found.

Submitted on March 2, 1967 Accepted on April 17, 1967     

Hb Stara Zagora: A New Hyper-Unstable Hemoglobin Causing Severe Hemolytic Anemia

We describe a new hyper-unstable β chain variant (codons 137–139, ? 6 bp) in a 2-year-old Bulgarian boy. The abnormal hemoglobin (Hb) is associated with severe hemolytic anemia as a consequence of its hyper instability. The child was admitted to the Pediatric Clinic (Faculty of Medicine, Stara Zagora, Bulgaria) at the age of 2 months. Because of anemia (Hb 6.9 g/dL) and high serum iron level (58 μM/L) the child was transfused. However, a month later his Hb level had dropped to 7.5 g/dL, and since then he has been on a regular monthly blood transfusion regimen. Hemoglobin analysis of a blood sample collected 2 months after the last transfusion at the age of 2 years, revealed no abnormalities except for the presence of inclusion bodies after incubation of peripheral blood with brilliant cresyl blue. Sequencing of the β-globin gene revealed heterozygosity for a 6 bp deletion (–TGGCTA) at codons 137 [the second and third base pair (bp)], 138 and 139 (the first bp), forming a new codon at position 137 (GAT). This event eliminates three amino acids (Val-Ala-Asn) and introduces a new residue (Asp). It creates a new restriction site for HphI. The parents and his dizygotic twin brother had no history of hemolysis. The paternity of the child was confirmed by DNA analysis.