Diagnosis of pyruvate kinase deficiency in a transfusion‐dependent patient with severe hemolytic anemia

Abstract

In a 2-yr old girl a hemolytic anemia was present since birth requiring multiple blood transfusions. Pyruvate kinase deficiency was suspected on the basis of a marginal enzyme activity, but could not be established due to the presence of massive numbers of donor cells in her peripheral blood. However, by density fractionation we succeeded in the isolation of a small fraction of the patient's own cells, in which a severe pyruvate kinase deficiency could be detected. In contrast hexokinase and glucose-6-phosphate dehydrogenase activities were extremely high, which is indicative that a very immature cell population is present in this fraction. In immunofluorescence studies a clear crossreaction was apparent with anti M2-type pyruvate kinase antibodies, whereas only a faint reaction with anti L-type could be detected. Despite the presence of a slight amount of L-type immunoreactive material, the residual activity in the patient's cell fraction could only be attributed to M2-type pyruvate kinase as was shown by cellulose acetate electrophoresis.

Life-threatening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression

“…However, the persistent expression of M2-type PK in mature RBCs has been reported in a few patients with severe PK deficiency. 8,17,[34][35][36][37][38][39] In these patients, M2PK activity is assumed to be a compensatory mechanism that ameliorates the PK deficiency.…”

“…8,17,35,38,39 This hypothesis of compensatory PK activity provided by an isozyme was first formulated to explain the results of zymogram analysis by thin-layer electrophoresis of red cell hemolysates from a patient who had PK deficiency with extreme reticulocytosis. In this study, the PK activity band with RPK mobility was not observed; rather, an activity band with the mobility of M2PK was detected.…”

“…However, the persistent expression of M2-type PK in mature RBCs has been reported in a few patients with severe PK deficiency. 8,17,[34][35][36][37][38][39] In these patients, M2PK activity is assumed to be a compensatory mechanism that ameliorates the PK deficiency.Rabbit anti-human RPK polyclonal antibodies also reacted with the MPK protein, probably because of the high proportion (69%) of shared amino acid identity. Using these antibodies in Western blots, the M2PK subunit band (58.7 kDa) was detected in PBMCs obtained from a healthy control and from our patient, but not in their corresponding hemolysates ( Figure 2B).…”

Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency

“…PKR-RE1 is involved in DNA-protein interaction. Electrophoretic mobility shift assay was performed with K562 nuclear extract and oligonucleotide probes designed according to the native core binding motif (PKWT; lanes 1-7) and mutated PKR-RE1 (PKmut; lanes [8][9][10][11][12][13][14]. Absence (Ϫ) and increasing amounts of unlabeled wild-type and mutant competitor are indicated.…”

“…13 PK activity and activity measurements of the red blood cell age-related enzymes, glucose-6-phosphate dehydrogenase (G6PD) and hexokinase (HK), were determined according to standardized procedures. 14 …”