Hisaichi Fujii

Seven Pyruvate Kinase Variants Characterized by the ICSH Recommended Methods

Summary Seven new red‐cell pyruvate kinase (PK) variants were characterized by the methods recently recommended by the International Committee for Standardization in Haematology. The cases were all true homozygote as evidenced by consanguineous marriages of the parents; all are Japanese. These variants were designated as PK Tokyo, PK Nagasaki, PK Sapporo, PK Maebashi, PK Itabashi, PK Fukushima and PK Aizu, respectively. Low substrate affinity (high K0.5S for phosphoenolpyruvate) and thermal instability appear to play major roles in causing defective enzyme function, resulting in chronic haemolytic anaemia. Product inhibition of PK by ATP may also play an additional role in causing haemolysis in more than half the cases.

Change of pyruvate kinase isozymes from M2- to L-type during development of the red cell.

Summary. The relationship between erythroid cell maturation and the change of pyruvate kinase (PK) isozymes was studied using fluorescent antibody techniques. The fluorescence of M2‐type PK was strongest in the proerythroblast stage, and then steeply declined with red cell maturation so that only weak fluorescence was seen in orthochromatic erythroblasts. On the other hand, the fluorescence of L‐type PK was seen throughout the maturation of the erythroblasts, with stronger fluorescence in the basophilic or polychromatic erythroblasts than in proerythroblasts.

Chromatographic analysis of human erythrocyte pyrimidine 5′‐nucleotidase from five patients with pyrimidine 5′‐nucleotidase deficiency

Human erythrocyte pyrimidine 5′‐nucleotidase (P5N) was separated into two subclasses, P5N‐I and P5N‐II, by DEAE Bio‐Gel A column chromatography. Their enzymological properties were studied using five normal subjects and five patients with different P5N deficiencies. Study of the normal subjects showed that P5N‐I and P5N‐II have distinctive properties, and P5N‐II is similar to the 5′‐nucleotidase in rat liver cytosol. The P5N‐II from the five subjects with this deficiency had normal activity and other normal enzymological properties. However, the P5N‐I from these patients had abnormal properties, including reduced activity. These variant enzymes had a high Michaelis constant for substrate cytidine 5′‐monophosphate and were heat stable. The optimum pH was shifted towards the acidic side in two patients, towards the basic side in one, and was unchanged in the other two. These results strongly suggest that the main cause of P5N deficiency is an abnormality of P5N‐I, probably arising from a structural gene mutation.

Molecular basis of Japanese variants of pyrimidine 5′‐nucleotidase deficiency

The type‐I isoform of pyrimidine 5′‐nucleotidase (P5N‐I) has an important role in the catabolism of pyrimidine mononucleotides during erythroid maturation. Two alternatively spliced forms of P5N‐I mRNA have been identified, and we found another alternatively spliced form in reticulocytes, which included an additional 87‐bp sequence. The sequence is located 6·2‐kb downstream of the exon 2 and 2·7‐kb upstream of the exon 3 sequence; consequently, the P5N‐I gene encodes 11 exons, which span approximately 48 kb. We identified five novel mutations in nine families with P5N‐I deficiency: two missense mutations (425C, 721C), one splice mutation (339C), one 1‐bp insertion (251‐insA‐252) and one 9‐bp deletion (del 192‐200). All patients were homozygous for each mutation. The mutant P5N‐I with 721C (G241R) had lower affinity for cytidine monophosphate, suggesting that Gly241 is important for substrate binding. Haplotype analysis showed that 721C, which had been identified in five unrelated families, was a founder mutation. The mutant P5N was then expressed in Cos‐7. The degradation of P5N with 425C (L142P) was significantly faster than a wild‐type control, and proteasome inhibitors restored the stability of L142P. These data suggest that L142P increases susceptibility to the degradation by the ubiquitin‐proteasome pathway.

A novel missense mutation (1060G → C) in the phosphoglycerate kinase gene in a Japanese boy with chronic haemolytic anaemia, developmental delay and rhabdomyolysis

Summary. We report the case of a 3‐year‐old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had anaemia and jaundice at birth, necessitating exchange transfusions for 2 d. After one red blood cell transfusion at age 2 months, his Hb level was 8–9 g/dl, his reticulocyte counts were 300–500 × 109/l, and his total bilirubin level was 25·65–42·75 µmol/l. The patient suffered two episodes of respiratory infection‐associated haemolytic crisis and rhabdomyolysis during early infancy. At age 3·0 years, his developmental milestones (developmental quotients measured using the Tsumori–Inage methods) score was 49% (normal 74–131%), and his height was below average by −2·0 standard deviations. The diagnosis of PGK1 deficiency was made based on his remarkably low (< 10% of normal) erythrocyte PGK enzyme activity level and the identification of a novel missense (1060G→C) PGK1 gene mutation. This mutation results in the Ala‐353Pro amino acid substitution, which has been designated PGK Kyoto. The patient developed the full clinical symptoms of PGK1 deficiency including haemolytic anaemia, myopathy, central nervous system disorder and growth retardation, which is unusual.

Molecular abnormality of a Japanese glucose-6-phosphate dehydrogenase variant (G6PD Tokyo) associated with hereditary non-spherocytic hemolytic anemia

SummaryThe 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.

Splenic infarction after Epstein-Barr virus infection in a patient with hereditary spherocytosis.

We describe the first patient with hereditary spherocytosis (HS) known to have developed splenic infarction following infectious mononucleosis (IM). An 18-year-old Japanese man was referred to our hospital in November 2004 because of continuous fever and icterus. He had undergone cholecystectomy at the age of 14 years. On patient admission in November 2004, a physical examination showed marked hepatosplenomegaly, icterus, and jaundice. He had a white blood cell count of 14.9 x 109/L with 9.5% atypical lymphocytes, a red blood cell count of 2.93 x 1012/L, and a hemoglobin concentration of 7.8 g/dL. Microspherocytes were observed in the patient’s peripheral blood smear, and immunoglobulin M antibody to Epstein-Barr virus (EBV) viral capsid antigen was detected. The patient’s diagnosis was HS with IM. On day 4 of admission, the patient complained of severe abdominal pain. Abdominal computed tomography scanning revealed findings of splenic infarction.Two months after the occurrence of splenic infarction, a splenectomy was performed. A pathohistologic examination of the resected spleen revealed no evidence of thrombosis or arterial occlusion. We assume that the cause of splenic infarction was insufficient blood flow to oxygenate the entire spleen during the acute enlargement of the spleen.

Red blood cell enzymes and their clinical application.

Red blood cell enzyme activities are measured mainly to diagnose hereditary nonspherocytic hemolytic anemia associated with enzyme anomalies. At least 15 enzyme anomalies associated with hereditary hemolytic anemia have been reported. Some nonhematologic disease can also be diagnosed by the measurement of red blood cell enzyme activities in the case in which enzymes of red blood cells and the other organs are under the same genetic control. Progress in molecular biology has provided a new perspective. Techniques such as the polymerase chain reaction and single-strand conformation polymorphism analysis have greatly facilitated the molecular analysis of erythroenzymopathies. These studies have clarified the correlation between the functional and structural abnormalities of the variant enzymes. In general, the mutations that induce an alteration of substrate binding site and/or enzyme instability might result in markedly altered enzyme properties and severe clinical symptoms.

Glucose 6-Phosphate dehydrogenase variants: A unique variant (G6PD Kobe) showed an extremely increased affinity for galactose 6-phosphate and a new variant (G6PD Sapporo) resembling G6PD Pea Ridge

SummaryTwo new glucose 6-phosphate dehydrogenase (G6PD) variants associated with chronic nonspherocytic hemolytic anemia were discovered. G6PD Kobe was found in a 16-year-old male associated with hemolytic crisis after upper respiratory infection. The enzyme activity of the variant was about 22% of that of the normal enzyme. The main enzymatic characteristics were slower than normal anodal electrophoretic mobility, high Km G6P, increased thermal-instability, an acidic pH optimum, and an extremely increased affinity for the substrate analogue, galactose 6-phosphate (Gal-6P).G6PD Sapporo was found in a 3-year-old male associated with drug-induced hemolysis. The enzyme activity was extremely low, being 3.6% of normal. In addition, this variant showed high Ki NADPH and thermal-instability.G6PD Kobe utilized the artificial substrate Gal-6P effectively as compared with the common natural substrate, glucose 6-phosphate. In G6PD Sapporo, NADPH could not exert the effect of product inhibition. The structural changes of these variants are expected to occur at the portions inducing conformational changes of the substrate binding site of the enzyme.

Gene Expression and Biological Significance of Hexokinase in Erythroid Cells

Red blood cells (RBCs) express two hexokinase (HK) isoforms, HK-I and HK-R. Both isozymes are generated from the HK-I gene by use of an alternate promoter. Gene structure and exon-intron organization of the HK-I gene have been elucidated from a sequence of three contiguous genomic clones localized at human chromosome 10. The sequence spans about 131 kb, and consists of 25 exons, which include 6 testis- and 1 erythroid-specific exons. HK-R has been shown as an erythroid-specific isozyme whose expression is turned on in the early erythroid-progenitors and is significantly induced during their differentiation. HK-R unfolds major HK activity in immature RBCs and is rapidly degraded during the maturation process. HK-I has a porin-binding domain in its N-terminus. Recent studies have shown that HK isozymes with a porin-binding domain play a role in mitochondrial integrity, suggesting that HK-I-deficient erythroid cells might be eliminated by apoptosis. It is most likely that RBCs are most labile as a result of HK-I/R deficiency since the HK-I gene but not the other isozyme genes are expressed in fetal and adult RBCs.