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

Abstract

IntroductionPyruvate kinase (PK; EC 2.7.1.40) is a key enzyme of the glycolytic pathway responsible for irreversibly catalyzing the conversion of phosphoenolpyruvate to pyruvate. In humans, pyruvate kinase activity is provided by 4 isoenzymes encoded by 2 structural genes. 1 The PKLR gene on chromosome 1 codes for the R-type PK (RPK) (exclusively in mature red blood cells [RBCs]) and the L-type PK (in liver) isoforms under the control of tissue-specific promoters. RPK is a 200-kDa tetramer with 4 identical subunits, each consisting of 4 domains. 1 The active site of RPK occurs at a domain interface, and the allosteric site is found in domain C. 2 The PKM gene on chromosome 15 codes for 2 different M-type PK (MPK) forms, M1 (brain and skeletal muscle) and M2 (fetal and most adult tissues), produced by alternative splicing. 3,4 Undifferentiated erythroid precursor cells express the M2PK isoenzyme earlier than mature RBCs express the RPK isoenzyme. [5][6][7] Because of the tissue-specific expression patterns of the genes, only mutations in the PKLR gene lead to erythrocyte PK deficiency. 8 PK deficiency severely affects RBC metabolism, causing adenosine triphosphate (ATP) depletion, which ultimately leads to hemolysis. Clinical symptoms vary considerably from mild to severe anemia. For the patient, anemia may mean transfusion dependence and may even be life threatening. Pathologic signs of PK deficiency are usually observed when enzyme activity is less than 25% normal activity and patients are generally homozygotes or compound heterozygotes with 2 different mutant alleles. 9 Most cases of PK deficiency are caused by the production of mutant enzymes with abnormal biochemical properties. Thus far, molecular analyses have identified at least 133 different mutations in the PKLR structural gene. 10 Most are missense mutations, but there are also reports of point mutations, deletions, or insertions that led to more drastic changes, such as alterations of the splicing site, frameshift, early termination mutations, and disruption of erythroid-specific promoters. [10][11][12][13][14] Most mutations constitute single-base substitutions, predicting amino acid changes of conserved residues in structurally and functionally important domains of the RPK tetramer. 15 Mutations that affect PKLR transcription, 14,16 or processing of its premRNA, 13,[17][18][19][20][21][22] are less common. To date, only a few homozygous PK null mutations have been identified. The first reported was the relatively common "PK gypsy," consisting of a large deletion at exon 11 23 with premature termination of its translation losing 10% of the original sequence at the C-terminus (exon 11 is deleted, and 35 aberrant residues are added); afterward, 2 one-base deletions and one transition at a split site 11 were reported. More recently, the first homozygous nonsense mutation was reported, 24 which causes premature termination of translation resulting in a truncated protein lacking a 33-residue C-terminal fragment.Here, we report a novel homozygous PKLR gene...

Cholestasis and Hepatic Failure in a Neonate: A Case Report of Severe Pyruvate Kinase Deficiency

“…However, clinical symptoms are expected to be limited to RBCs, the hepatic enzyme activity usually being preserved in the hepatocytes or compensated by PK-M2 isoenzyme (dominant fetal form and most adult tissues). 1,11,12 Persistent PK-M2 isoenzyme might explain the variability in anemia severity. 13 The first mutation, 721G.T (p.Glu241*), 7,12,13,15 Animal and human models have shown a negative correlation between erythrocyte PK activity and the number of apoptotic erythroid progenitors in the spleen, providing evidence that the metabolic alteration of PKD affects erythrocyte maturation, resulting in ineffective erythropoiesis.…”

“…1,11,12 Persistent PK-M2 isoenzyme might explain the variability in anemia severity. 13 The first mutation, 721G.T (p.Glu241*), 7,12,13,15 Animal and human models have shown a negative correlation between erythrocyte PK activity and the number of apoptotic erythroid progenitors in the spleen, providing evidence that the metabolic alteration of PKD affects erythrocyte maturation, resulting in ineffective erythropoiesis. 7,13,15 If ineffective erythropoiesis has affected our patient, which is possible regarding the mutations involved, it might have prevented the compensatory effect of M2-type enzyme activity described in some cases and therefore explain the severe anemia present at birth.…”

The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis

“…Persistent expression of the PK-M2 isozyme has been reported in the red blood cells of patients (and animals; see below) with severe PK deficiency. 90,91 The survival of these patients, though not in all cases, 92 may be enabled by this compensatory increase in PK activity.…”

Mining for allosteric information: Natural mutations and positional sequence conservation in pyruvate kinase

“…This natural screen identifies only mutations that cause a functional problem and does not identify amino acid substitutions that have only minimal effects on function. Lethal mutations are not often detected (6) and the compensating expression of M 2 -PYK activity is often likely to result in a non-lethal phenotype when R-PYK is completely dysfunctional (26). In addition, most diseased individuals are heterozygous for PKD mutations making it difficult to correlate severity of disease in vivo with mutation location.…”