Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane.

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or ß-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2  = 0·15; slope = 9·09) and, less significantly, in HE (R2  = 0·021; slope = 8·92) when compared with other haemolytic disorders (R2  ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2  = 0·48; slope = -9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.

Residual pyruvate kinase activity in PKLR-deficient erythroid precursors of a patient suffering from severe haemolytic anaemia.

OBJECTIVE: Here, we present a 7-year-old patient suffering from severe haemolytic anaemia. The most common cause of chronic hereditary non-spherocytic haemolytic anaemia is red blood cell pyruvate kinase (PK-R) deficiency. Because red blood cells rely solely on glycolysis to generate ATP, PK-R deficiency can severely impact energy supply and cause reduction in red blood cell lifespan. We determined the underlying cause of the anaemia and investigated how erythroid precursors in the patient survive. METHODS: PK activity assays, Western blot and Sanger sequencing were employed to determine the underlying cause of the anaemia. Patient erythroblasts were cultured and reticulocytes were isolated to determine PK-R and PKM2 contribution to glycolytic activity during erythrocyte development. RESULTS: We found a novel homozygous mutation (c.583G>A) in the PK-R coding gene (PKLR). Although this mutation did not influence PKLR mRNA production, no PK-R protein could be detected in the red blood cells nor in its precursors. In spite of the absence of PK-R, the reticulocytes of the patient exhibited 20% PK activity compared with control. Western blotting revealed that patient erythroid precursors, like controls, express residual PKM2. CONCLUSIONS: We conclude that PKM2 rescues glycolysis in PK-R-deficient erythroid precursors.

[Analysis and prenatal diagnosis of PKLR gene mutations in a family with pyruvate kinase deficiency].

OBJECTIVE: To evaluate the feasibility of genetic and prenatal diagnosis for a family affected with pyruvate kinase deficiency (PKD). METHODS: Targeted sequence capture and high-throughput sequencing technology was used to detect the exons and exon-intron boundaries of the PKLR gene in a clinically suspected PKD patient. Meanwhile, the genotype of the pedigree was validated by Sanger sequencing. Prenatal genetic diagnosis was performed by amniotic fluid sampling after genotype of the mother of the proband was determined. RESULTS: The proband was found to harbor double heterozygous mutations, c.661G>A (Asp221Asn) and c.1528C>T (Arg510Ter), which resulted in amino acid substitution Asp221Asn and Arg510Ter. Such mutations were confirmed by Sanger sequencing. The mother and father of the proband were detected to have respectively carried c.1528C>T (Arg510Ter) and c.661G>A (Asp221Asn) mutation. The fetus was found to have carried the same mutations as the proband. Following selected abortion, analysis of fetal tissue was consistent with the result of prenatal diagnosis. CONCLUSION: The compound mutations of c.661G>A and c.1528C>T of PKLR gene probably underlie the PKD in the family. Prenatal diagnosis of the mutations analysis can facilitate detection of affected fetus in time.

Red blood cell PK deficiency: An update of PK-LR gene mutation database.

Pyruvate kinase (PK) deficiency is known as being the most common cause of chronic nonspherocytic hemolytic anemia (CNSHA). Clinical PK deficiency is transmitted as an autosomal recessive trait, that can segregate neither in homozygous or in a compound heterozygous modality, respectively. Two PK genes are present in mammals: the pyruvate kinase liver and red blood cells (PK-LR) and the pyruvate kinase muscle (PK-M), of which only the first encodes for the isoenzymes normally expressed in the red blood cells (R-type) and in the liver (L-type). Several reports have been published describing a large variety of genetic defects in PK-LR gene associated to CNSHA. Herein, we present a review of about 250 published mutations and six polymorphisms in PK-LR gene with the corresponding clinical and molecular data. We consulted the PubMed website for searching mutations and papers, along with two main databases: the Leiden Open Variation Database (LOVD, https://grenada.lumc.nl/LOVD2/mendelian_genes/home.php?select_db=PKLR) and Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/gene.php?gene=PKLR) for selecting, reviewing and listing the annotated PK-LR gene mutations present in literature. This paper is aimed to provide useful information to clinicians and laboratory professionals regarding overall reported PK-LR gene mutations, also giving the opportunity to harmonize data regarding PK-deficient individuals.

Erythrocyte pyruvate kinase deficiency: 2015 status report.

Over the last several decades, our understanding of the genetic variation, pathophysiology, and complications of the hemolytic anemia associated with red cell pyruvate kinase deficiency (PKD) has expanded. Nonetheless, there remain significant gaps in our knowledge with regard to clinical care and monitoring. Treatment remains supportive with phototherapy and/or exchange transfusion in the newborn period, regular or intermittent red cell transfusions in children and adults, and splenectomy to decrease transfusion requirements and/or anemia related symptoms. In this article, we review the clinical diversity of PKD, the current standard of treatment and for supportive care, the complications observed, and future treatment directions.

A previously unknown mutation in the pyruvate kinase gene (PKLR) identified from a neonate with severe jaundice.

We report a neonate with early and severe hemolytic jaundice and low erythrocyte pyruvate kinase enzymatic activity (<2 U/g hemoglobin, reference interval 9-22). We found her asymptomatic mother to be heterozygous for a novel PKLR mutation (c.1573delT) with an erythrocyte PK activity of 6.2 U/g hemoglobin. Her asymptomatic father was heterozygous for the common Northern European PKLR mutation (c.1529A) with an erythrocyte PK activity of 3.6 U/g. The neonate was a compound heterozygote with both mutations, but with no other mutations identified by sequencing a panel of 27 genes involved in severe neonatal jaundice.

Stem cell selection in vivo using foamy vectors cures canine pyruvate kinase deficiency.

BACKGROUND: Hematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies. Here we expanded corrected stem cells in vivo in a canine model of a severe erythroid disease, pyruvate kinase deficiency. METHODOLOGY/PRINCIPAL FINDINGS: We used a foamy virus (FV) vector expressing the P140K mutant of methylguanine methyltransferase (MGMTP140K) for in vivo expansion of corrected hematopoietic repopulating cells. FV vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy since they efficiently transduce repopulating cells and may be safer than more commonly used gammaretroviral vectors. Following transplantation with HSCs transduced ex vivo using a tri-cistronic FV vector that expressed EGFP, R-type pyruvate kinase, and MGMTP140K, we were able to increase marking from approximately 3.5% to 33% in myeloid long-term repopulating cells resulting in a functional cure. CONCLUSIONS/SIGNIFICANCE: Here we describe in one affected dog a functional cure for a severe erythroid disease using stem cell selection in vivo. In addition to providing a potential cure for patients with pyruvate kinase deficiency, in vivo selection using foamy vectors with MGMTP140K has broad potential for several hematopoietic diseases including hemoglobinopathies.

Deficiency of the alpha and beta subunits of pyruvate dehydrogenase in a patient with lactic acidosis and unexpected sudden death.

An infant with moderate muscular hypotonia and congenital lactic acidosis died suddenly at the age of 3 months. Autopsy revealed no abnormalities responsible for this unexpected death. Measurement of mitochondrial enzymes involved in energy production indicated a severely decreased total pyruvate dehydrogenase complex (PDHC) activity in muscle tissue (0.23 nmoles x min-1 x mg protein-1, control range 2.8-8.7) and moderately decreased PDHC activity in fibroblasts (0.27 nmoles x min-1 x mg protein-1, control range 0.37-2.32). The activity of the first component E1 (pyruvate dehydrogenase) in muscle tissue was 10 times lower than that of controls (0.008 nmoles x min-1 x mg protein-1, control range 0.10-0.25). The activities of dihydrolipoyl dehydrogenase (E3) and various other mitochondrial enzymes were normal. Immunochemical analysis in skeletal muscle tissue and fibroblasts demonstrated a decrease in the amount of the alpha and beta subunits of E1. The features of this patient are compared with those of other patients reported in the literature with immunochemically confirmed combined E1 alpha and beta deficiency.