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A G-6PD deficiency was detected in a Negro patient from Senegal suffering from congenital nonspherocytic hemolytic anemia. The main characteristics of this variant were: profound defect of G-6PD activity in the red cells, decreased immunologic specific activity, fast electrophoretic mobility, decreased Km-G-6P and normal Km-NADP+, normal inhibition by ATP and NADPH, slightly increased utilization of the substrate analogues, slightly biphasic pH curve, high heat lability, subnormal activation energy. The characteristics of this variant being unique, it was called 'G-6PD Hôtel Dieu.'

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.

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.

A new variant of G6PD associated with chronic nonspherocytic hemolytic anemia (CNSHA) in a Japanese male is described. This variant is unique in that it has a normal electrophoretic mobility, normal Km for glucose-6-phosphate and NADP, and a normal utilization of the substrate 2-deoxyglucose-6-phosphate and deamino-NADP. It also shows a decreased thermal stability and a biphasic pH curve. These characteristics distinguish this variant from previously reported variants associated with CNSHA and we have called it G6PD Kanazawa.

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.

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 biochemical properties of the patient's GPI indicate a new variant designated type Liége.

A new variant of G-6-PD was found in a Thai boy who had typical clinical and hematologic findings of congenital nonspherocytic hemolytic anemia. This mutant has normal electrophoretic mobility, normal affinity for G-6P and NADP, and is very labile to heat. It has an activity of about 5 per cent of normal and shows increased utilization of 2-deoxyglucose-6-phosphate and NAD and decreased utilization of deamino NADP. It is different from previously described mutants, and is named G-6-PD Bangkok.

Two brothers with congenital nonspherocytic hemolytic anemia and neurologic disease were found to have erythrocyte and leukocyte phosphoglycerate kinase deficiency. Elevated levels of red cell dihydroxyacetone phosphate and 2,3-diphosphoglycerate were documented as well. Erythrocyte phosphoglycerate kinase deficiency is X-chromosome linked, and the present report brings the number of male hemizygous individuals to five. All are mentally retarded and some have additional neurologic defects

HEREDITARY spherocytosis with its associated congenital hemolytic anemia is generally easily recognized and satisfactorily treated with splenectomy. A rarer group of congenital hemolytic anemias, however, is sometimes confused with hereditary spherocytosis because of the occasional presence of small numbers of spherocytes in association with varying degrees of abnormality in the incubated osmotic fragility test. In this later group of "congenital nonspherocytic hemolytic anemias" splenectomy has often been performed but usually does not produce a clinical cure. Because splenectomy is generally of inconsistent value, this type of anemia must be accurately differentiated for the sake of proper treatment and accurate prognosis. . . .

1) A case of congenital nonspherocytic hemolytic anemia was reported. A metabolic abnormality in the erythrocytes from the patient was demonstrated, consisting of a low content of reduced glutathione, an abnormality in the reduced glutathione stability test, and a low activity of glucose-6-phosphate dehydrogenase. These abnormalities were also found in the erythrocytes from the mother to a milder degree, while there was no abnormalities in the erythrocytes from the father. This is the first case in Japan, of this type of hemolytic anemia in which an enzymatic anomaly of the erythrocytes was demonstrated. 2) The treatment with large dose of vitamin B12 brought about a relief from jaundice, when an increase of reduced glutathione content in the erythrocytes and an improvement of reduced glutathione instability were observed, although the activity of glucose-6-phosphate dehydrogenase in the erythrocytes remained in a constantly low level. 3) The in-vitro effect of vitamin B12 on gulcose-6-phosphate dehydrogenase was investigated. The relationship between vitamin B12 and glutathione metabolism was discussed. 4) Genetic mode of the disease was discussed.

Two families of congenital nonspherocytic hemolytic anemia were reported. A metabolic abnormality in the erythrocytes from the patients was demonstrated, consisting of a low content of GSH, an abnormality in the GSH stability test and a low activity of G6PD. These abnormalities were also found in the erythrocytes from the mothers in a milder degree, while there was no abnormalities in the erythrocytes from the fathers. An aggravation of hemolysis and metabolic disturbances mentioned above was observed following administration of Domian (6-sulfanilamido-2, 4-dimethylpyrimidine) to the patient. It was also argued by way of experiment that the difference between the so-called congenital nonspherocytic hemolytic anemia and drug-induced hemolytic anemia is not essential, but only quantitative, so that both disorders belong highly probably to the same entity of enzymopathy fromm the viewpoint of pathogenesis of hemolysis.

Glucose Phosphate Isomerase Deficiency with Congenital Nonspherocytic Hemolytie Anemia: A New Variant (Type Nordhorn). II. Purification and Biochemical Properties of the Defective Enzyme

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 G6PD with total enzyme deficiency associated with nonspherocytic hemolytic anemia in a 60 year old Frenchman is characterized. Partially purified enzyme revealed slow electrophoretic mobility, decreased G6P affinity, thermal instability, abnormal pH curve with a single peak at pH 5.0, abnormal utilization of 2-deoxy-G6P and deamino NADP. This variant differs from all previously reported variants associated with chronic nonspherocytic hemolytic anemia. Accordingly this variant is designated Gd(-) Rennes.

Glucosephosphate isomerase deficiency as the cause of macrocytic congenital nonspherocytic hemolytic anemia is described in three unrelated families. The biochemical properties of the variant glucosephosphate isomerases indicate that the patients have new variants, designated as GPI Kiel, GPI Hamburg, and GPI Homburg. The severity of the clinical symptoms depended on the amount of residual GPI activity and the biochemical properties of the variant enzyme. Thus the patient with GPI Kiel (34% residual activity) whose variant GPI was slightly unstable showed a mild chronic hemolytic anemia. The patient with GPI Homburg (7% residual activity) whose variant enzyme was stable and had a reduced specific activity, suffered from severe congenital hemolytic anemia and neuromuscular symptoms. Due to the special properties of GPI Homburg, we assume that both the hematological and neuromuscular symptoms of the patient with GPI Homburg are caused by his GPI deficiency. The twins with GPI Hamburg (27% residual activity) had a distinctly unstable variant enzyme and had suffered from hemolytic crises since birth. Only GPI Homburg showed an altered electrophoretic mobility and an increased affinity for fructose-6-phosphate. The other two variants had normal values.

4. Granoff DM, and Roskes S: Urinary tract infection due to Haemophilus influenzae type b, J PEDIATR 84:414, 1974. 5. Doolittle KH, et al: Epididymitis in the prepubertal boy, J Urol 96:364, 1966. 6. Haahr J, et al: Epididymitis in children, Acta Paediatr Scand 60:16, 248, 1971. 7. McDonald JH, et al: Acute pneumococcal epididymitis, Ill Med J 95:304, 1949. 8. Davis WH, and Scardino PL: Meningitis presenting as epididymitis, Southern Med J 65:9360, 1972. 9. DeLeon W: Report of a case of meningitis, arthritis, and epididymitis due to mixed Pfeiffer bacillus and pneumococcus infection, J Phillipine Islands Med Assn 1:195, 1921. 10. Samellas W: Acute epididymo-orchitis associated with bacteremic shock, J Urol 89:370, 1963.

The clinical, hematologic and biochemical characteristics of the known cases of glucosephosphate deficiency are reviewed. This hereditary disorder of erythrocyte metabolism is the third most common enzyme defect resulting in congenital nonspherocytic hemolytic anemia. Its unique features relative to other erythroenzymopathies are emphasized.

Two new variants of glucose 6-phosphate dehydrogenase (G6PD) deficiency associated with chronic nonspherocytic hemolytic anemia were discovered in Japan. Gd(-) Tokushima was found in a 17-years-old male whose erythrocytes contained 4.4% of normal enzyme activity. Partially purified enzyme revealed a main band of normal electrophoretic mobility with additional two minor bands of different mobility; normal Km G6P, and Km NADP five-to sixfold higher than normal; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; marked thermal instability; a normal pH curve; and normal Ki NADPH. The hemolytic anemia was moderate to severe. Gd(-) Tokyo was characterized from a 15-year-old male who had chronic nonspherocytic hemolytic anemia of mild degree. The erythrocytes contained 3% of normal enzyme activity, and partially purified enzyme revealed slow electrophoretic mobility (90% of normal for both a tris-hydrochloride buffer system and a tris-EDTA-borate buffer system, and 70% of normal for a phosphate buffer system); normal Km G6P and Km NADP; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; greatly increased thermal instability; a normal pH curve; and normal Ki NADPH. These two variants are clearly different from hitherto described G6PD variants, including the Japanese variants Gd(-) Heian and Gd(-) Kyoto. The mothers of both Gd(-) Tokushima and Gd(-) Tokoyo were found to be heterozygote by an ascorbate-cyanide test.

A new glucose-6-phosphate dehydrogenase (G6PD) variant with total deficiency associated with congenital nonspherocytic hemolytic anemia was found in a Costa Rican family. The study of the partially purified enzyme revealed thermal instability, increased G6P affinity, abnormal pH optimum, increased utilization of analogues, and a chromatographic behavior that differs from all the variants previously described. Thus, this new variant was designated G6PD Puerto Limón.

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.

In a 1-year-old German boy a GPI deficiency was found to be the cause of a chronic nonspherocytic hemolytic anemia with recurrent hemolytic crises. Because of consanguinity of the parents, the patient is true homozygote. The investigation of the biochemical properties of the deficient enzyme revealed an altered electrophoretic behavior, pronounced thermolability, an increased affinity for G6P, an increased affinity for the competitive inhibitor 6-PG, and slightly changed pH optima for both substrates. Electrophoresis after freezing and thawing the hemolysate indicates that the genetic modification of the subunit involves the mechanism of transforming the main band into the secondary bands. The properties of the new deficient GPI indicate a new variant designated GPI Augsburg.

Three new glucose-6-phosphate dehydrogenase (G6PD) variants, which showed electrophoretically normal mobility and were associated with chronic nonspherocytic hemolytic anemia, were found in Japan. G6PD Ogikubo, found in a 17-year-old male whose red cells contained 3% of normal enzyme activity, had normal Km G6P, normal Km NADP, normal utilization of deamino-NADP, decreased heat stability, and a normal pH curve. G6PD Yokohama, characterized from a 15-year-old male, had 1.9% of normal enzyme activity, normal Km G6P, normal Km NADP, low Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NADP, decreased heat stability, and normal pH curve. G6PD Akita, characterized from a 56-year-old male, had an undetectably low activity when hemolysate was examined, normal Km G6P, normal Km NADP, normal Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NADP, decreased heat stability, and normal pH curve. The degree of hemolytic anemia was moderate to mild in all three patients.

In a presumptive heterozygote for congenital nonspherocytic hemolytic anemia caused by glucose-6-phosphate dehydrogenase deficiency, a fraction of short-living red blood cells was demonstrated by administration of nitrofurantoin in the 22nd day of the 59Fe study of erythrocyte life span. A small percentage of deficient red blood cells in peripheral blood in this case (4%) may originate in the shorter life span of deficient cells and in the proportion of the deficient to normal red blood cells produced.

The unusual occurrence of both congenital nonspherocytic hemolytic anemia and hemochromatosis in a brother and sister is reported. The strikingly similar clinicopathologic abnormalities, including extraordinarily severe cardiac lesions in these relatively young siblings, is discussed.

An 11-year-o'.d Negro boy with a history of neonatal jaundice requiring exchange transfusion had a severe hemolytic episode at 8 years of age. A diagnosis of nonspherocytic congenital hemolytic anemia due to G-6-PD deficiency was made at that time. The red cells of the patient contained a level of G-6-PD activity approximating 14 per cent of normal. The residual enzyme was partially purified and characterized. It proved to be electrophoretically rapid. The Michaelis constant (K m ) for G-6-P was diminished and that for NADP increased. The enzyme had greatly increased utilization of deamino-NADP and slightly increased utilization of 2-deoxyglucose-6-phosphate. The pH optimum curve was slightly bimodal. Like most other G-6-PD variants associated with congenital nonspherocytic hemolytic anemia, the enzyme was thermally labile. This enzyme represents a new deficent variant, G-6-PD Charleston. The patient continues to have chronic severe hemolysis. Two brothers presumably died of the same enzyme deficiency. The erythrocytes of two living brothers contain G-6-PD A-, The mother is heterozygous for the two variants.

A specific diagnosis must be established in congenital nonspherocytic hemolytic anemia, for guidance in therapy and family counseling. Diagnostic methods have recently progressed rapidly and include determination of deficiencies in the activity of certain enzymes. Further definitions of enzymatic deficiencies are anticipated.

"in vitro" test for identifying primaquine sensitivity, Bul. W.H.O. 22: 633, 1960. 3. Kirkman, N. H., and Riley, H. D.: Congenital nonspherocytic hemolytic anemia. Studies on a family with a qualitative defect in glucose-6phosphate dehydrogenase, Am. J. Dis. Child. 102: 313, 1961. 4. Valaes, T., Karaklis, A., Stravrakakis, D., Bavela-Stravrakakis, K., Perakis, A., and Doxiadis, S. A.: Incidence and mechanism of neonatal jaundice related to glucose-6-phosphate dehydrogenase deficiency, Pediatr. Res. 3: 448, 1969.

It has been well demonstrated in man and in other species that the fundamental expression of a hereditary chemical disorder may be the deletion of a single metabolic or enzymic step. The extent to which such loss in enzymic activity is due to dimimished amounts or to a qualitative inefficiency of the enzyme has been a matter of considable interest. The latter possibility has appeared particularly tenable since the outstanding demonstrations by Pauling, Itano, et al1and by Ingram and Hunt2,3of the qualitative changes in sickle hemoglobin. Erythrocytic glucose-6-phosphate dehydrogenase (G-6-PD) has been studied in 2 varieties4,5of a hereditary defect of this enzyme, primaquine-sensitive hemolytic anemia,6-9and recently10a certain type of congenital nonspherocytic hemolytic anemia. Although the active G-6-PD in primaquinesensitive erythrocytes from Negro males is qualitatively similar, if not identical, to that from normal persons, the G-6-PD of a Caucasian male

Hemolytic anemia or potentially hemolytic disorders due to metabolic errors of the erythrocyte are being recognized with increasing frequency. These disorders are characterized either by congenital nonspherocytic hemolytic anemia or by drug-inducible hemolysis. For identification of such disorders, most clinical laboratories will find it expedient to depend primarily on simple, specific screening tests which usually permit a precise etiologic diagnosis to be established as reliably as, and more economically than, by more tedious quantitative assays. The specific screening tests can be satisfactorily performed in laboratories with limited facilities. A test for peroxidative denaturation of hemoglobin is very sensitive, but nonspecific, and may be useful in screening for a variety of metabolic defects and for some of the unstable hemoglobinopathies. In unexplained cases of congenital nonspherocytic hemolytic anemia, a thermolability test should be performed for unstable hemoglobin.

Previous communications1,2concerning A2hemoglobin in parents of subjects with thalassemia major reported normal levels in a small minority. Fetal hemoglobin percentage was determined in some of these individuals and was noted to be unexpectedly high in all but one. The single exception was the mother of 2 children originally diagnosed as having thalassemia major. Later investigation of this family resulted in reclassification of diagnosis to congenital nonspherocytic hemolytic anemia. It was suggested2"that patients diagnosed as having Mediterranean anemia do not represent a homogeneous group as regards pathogenesis and heredity." The present report details further observations concerning these families and presents evidence for heterogeneity of thalassemia major. Material and Methods The pedigrees of 3 subjects with thalassemia major who have been followed in the Pediatric Hematology Clinic of the Babies Hospital have been investigated for evidence of heredity transmission of the gene for thalassemia minor.