scholarly journals Pyruvate kinase Greensboro. A four-generation study of a high K0.5s (phosphoenolpyruvate) variant [published erratum appears in Blood 1988 Dec;72(6):2082]

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1054-1059 ◽  
Author(s):  
WN Valentine ◽  
WB Herring ◽  
DE Paglia ◽  
MC Steuterman ◽  
RA Brockway ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Clinical Manifestations ◽  
Substrate Affinity ◽  
Red Cell ◽  
Gene Products ◽  
Normal Allele ◽  
Metabolic Phenotypes ◽  
Sulfhydryl Modification ◽  
Nucleotide Specificity

The proband with lifelong hemolytic anemia has a high K0.5s phosphoenolypyruvate (PEP) erythrocyte pyruvate kinase (PK) variant substantially but incompletely normalized by the allosteric modifier fructose-1,6-diphosphate (F-1,6-P2) with conversion of sigmoidal to hyperbolic kinetics. Heterozygotes in four generations express qualitatively identical but less severely abnormal kinetics and lack overt hemolysis. Kinetic abnormalities are closely mimicked by sulfhydryl modification of normal PK. Three distinct clinical and metabolic phenotypes characterize the proband and two sisters: variant PK and hemolytic anemia, variant PK without clinical manifestations or hemolysis, and complete normality. Their mother, whose red cell PK is entirely normal except for a questionably slightly low Vmax, is postulated to express the gene products of nonidentical alleles, one encoding a product with mildly less favorable catalytic characteristics. At low PEP concentrations, the proband and heterozygotes for the PK mutant express only a very small fraction of normal PK activity despite apparent inheritance of one normal allele in the latter. Evidence suggests that disproportionately lowered PK activity may be a property of a heterotetrameric PK. Illusory abnormalities in nucleotide specificity are artifacts of diminished substrate affinity characterizing the mutant PK.

scholarly journals Pyruvate kinase Greensboro. A four-generation study of a high K0.5s (phosphoenolpyruvate) variant [published erratum appears in Blood 1988 Dec;72(6):2082]

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1054-1059
Author(s):  
WN Valentine ◽  
WB Herring ◽  
DE Paglia ◽  
MC Steuterman ◽  
RA Brockway ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Clinical Manifestations ◽  
Substrate Affinity ◽  
Red Cell ◽  
Gene Products ◽  
Normal Allele ◽  
Metabolic Phenotypes ◽  
Sulfhydryl Modification ◽  
Nucleotide Specificity

Abstract The proband with lifelong hemolytic anemia has a high K0.5s phosphoenolypyruvate (PEP) erythrocyte pyruvate kinase (PK) variant substantially but incompletely normalized by the allosteric modifier fructose-1,6-diphosphate (F-1,6-P2) with conversion of sigmoidal to hyperbolic kinetics. Heterozygotes in four generations express qualitatively identical but less severely abnormal kinetics and lack overt hemolysis. Kinetic abnormalities are closely mimicked by sulfhydryl modification of normal PK. Three distinct clinical and metabolic phenotypes characterize the proband and two sisters: variant PK and hemolytic anemia, variant PK without clinical manifestations or hemolysis, and complete normality. Their mother, whose red cell PK is entirely normal except for a questionably slightly low Vmax, is postulated to express the gene products of nonidentical alleles, one encoding a product with mildly less favorable catalytic characteristics. At low PEP concentrations, the proband and heterozygotes for the PK mutant express only a very small fraction of normal PK activity despite apparent inheritance of one normal allele in the latter. Evidence suggests that disproportionately lowered PK activity may be a property of a heterotetrameric PK. Illusory abnormalities in nucleotide specificity are artifacts of diminished substrate affinity characterizing the mutant PK.

scholarly journals Pyruvate kinase deficiency: epidemiology, molecular analyses and modern diagnostic approaches (literature review)

2020 ◽  
Vol 7 (2) ◽  
pp. 86-93
Author(s):  
A. V. Bankole ◽  
E. A. Chernyak
Keyword(s):  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Molecular Genetic ◽  
Clinical Manifestations ◽  
Diagnostic Methods ◽  
Compound Heterozygous ◽  
Red Cell ◽  
Molecular Genetic Study ◽  
Pyruvate Kinase Deficiency ◽  
Diagnostic Approaches

Red cell pyruvate kinase deficiency is the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Pyruvate kinase is the enzyme involved in the last step of glycolysis – the transfer of a phosphate group from phosphoenolpyruvate producing the enolate of pyruvate and ATP (50 % of total energy ATP of erythrocytes). ATP deficiency directly shortened red cell lifespan. Affected red blood cells are destroyed in the splenic capillaries, leading to the development of chronic hemolytic anemia. It is an autosomal recessive disease, caused by homozygous and compound heterozygous mutations in the PKLR gene. There are no exact data on the incidence of pyruvate kinase deficiency, but the estimated frequency varies from 3: 1,000,000 to 1:20,000. The clinical features of the disease and the severity are highly variable. Diagnosis of pyruvate kinase deficiency is based on the determination of pyruvate kinase activity and molecular genetic study of the PKLR gene. The variety of clinical manifestations, possible complications, as well as the inaccessibility of diagnostic methods complicate the diagnosis.

Estimating the prevalence of pyruvate kinase deficiency from the gene frequency in the general white population

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3585-3588 ◽  
Author(s):  
Ernest Beutler ◽  
Terri Gelbart
Keyword(s):  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Relative Frequency ◽  
Gene Frequency ◽  
Red Cell ◽  
White Population ◽  
Pyruvate Kinase Deficiency ◽  
Oligonucleotide Hybridization ◽  
Allele Specific ◽  
White Patients

Pyruvate kinase (PK) deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. The prevalence of this deficiency is unknown, though some estimates have been made based on the frequency of low red cell PK activity in the population. An additional 20 patients with hereditary nonspherocytic hemolytic anemia caused by PK deficiency have been genotyped. One previously unreported mutation 1153C→T (R385W) was encountered. The relative frequency of PK mutations in patients with hemolytic anemia caused by PK deficiency was calculated from the 18 white patients reported here and from 102 patients previously reported in the literature. DNA samples from 3785 subjects from different ethnic groups have been screened for the 4 more frequently encountered mutations—c.1456 C→T(1456T), c.1468 C→T(1468T), c.1484 C→T(1484T), and c.1529 G6A (1529A)—by allele-specific oligonucleotide hybridization. Among white patients the frequency of the 1456T mutation was 3.50 × 10−3; that of the 1529A mutation was 2.03 × 10−3. Among African Americans the frequency of the 1456T mutation was 3.90 × 10−3 The only mutation found in the limited number of Asians tested was 1468T at a frequency of 7.94 × 10−3. Based on the gene frequency of the 1529A mutation in the white population and on its relative abundance in patients with hemolytic anemia caused by PK deficiency, the prevalence of PK deficiency is estimated at 51 cases per million white population. This number would be increased by inbreeding and decreased by failure of patients with PK deficiency to survive.

Red Cell Enzymes

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Josef T. Prchal ◽  
Xylina T. Gregg
Keyword(s):  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Cytochrome B5 ◽  
Screening Tests ◽  
Red Cell ◽  
Developmental Abnormalities ◽  
Enzymatic Function ◽  
Cell Enzyme ◽  
Red Cell Enzymes ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract Mutations leading to red cell enzyme deficiencies can be associated with diverse phenotypes that range from hemolytic anemia, methemoglobinemia, polycythemia, and neurological and developmental abnormalities. While most of these mutations occur sporadically, some such as common glucose-6-phosphate dehydrogenase (G6PD) mutants are endemic and rarely cause disease. Common G6PD mutants likely reached their prevalence because they provide some protection against severe malarial complications. In this review G6PD, pyruvate kinase, 5′ nucleotidase, and cytochrome b5 reductase deficiencies will be discussed in greater detail. Limitations of commonly used screening tests for detection of these disorders will also be emphasized, as well as emerging knowledge about non-enzymatic function of the glycolytic enzymes.

scholarly journals Low substrate affinity of pyruvate kinase variant (PK Sapporo) caused by a single amino acid substitution (426 Arg-->Gln) associated with hereditary hemolytic anemia

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2439-2441 ◽  
Author(s):  
H Kanno ◽  
H Fujii ◽  
S Miwa
Keyword(s):  
Amino Acid ◽  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Amino Acid Substitution ◽  
Point Mutations ◽  
Alpha Helix ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Substrate Affinity ◽  
Alpha 7

A point mutation (1277 CGG to CAG) was identified in the R-type pyruvate kinase (PK) cDNA of a PK variant, PK Sapporo, associated with hereditary non-spherocytic hemolytic anemia. The mutation causes a single amino acid substitution from Arg to Gln at the 426th amino acid residue of human R-type PK; consequently, the hydrophobicity around the mutated site is drastically decreased. The amino acid change occurred in the eighth alpha helix of A domain (A alpha 8) of PK, and it has been proposed that this region as well as A alpha 7, A beta 7, and A beta 8 is a potassium (K+) binding site. Because K+ binding to the PK subunit is considered to be essential for substrate binding, the mutation might account for the decreased affinity for phosphoenolpyruvate (PEP). This is compatible with the fact that all the reported PK variants carrying point mutations in those area have a high Michaelis constant (Km) for PEP.

scholarly journals Decreased Activity and Stability of Pyruvate Kinase in Hereditary Hemolytic Anemia: A Potential Target for Therapy By AG-348 (Mitapivat), an Allosteric Activator of Red Blood Cell Pyruvate Kinase

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3506-3506
Author(s):  
Minke A.E. Rab ◽  
Brigitte A. van Oirschot ◽  
Stephanie van Straaten ◽  
Bart J. Biemond ◽  
Jennifer Bos ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Blood Cell ◽  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Red Blood Cell ◽  
Research Funding ◽  
Reticulocyte Count ◽  
Compound Heterozygous ◽  
Healthy Controls ◽  
Red Cell

Background: Reactive oxygen species (ROS) play an important role in the complex and multifactorial pathophysiology of hereditary hemolytic anemia like sickle cell disease (SCD), β-thalassemia and hereditary xerocytosis (HX). Increased intracellular levels of oxidative stress disrupt normal cell functioning and may contribute to premature red blood cell (RBC) clearance from the circulation. Pyruvate kinase (PK) is a key regulatory enzyme of glycolysis, the cell's main source of energy. Because PK is very sensitive to redox balance we hypothesized that increased levels of oxidative stress in SCD, β-thalassemia and HX impairs proper enzyme function, thereby compromizing RBC energy metabolism. This may contribute to disease pathophysiology. Aims: To investigate if secondary deficiency of PK is common in SCD, thalassemia, and HX, and to investigate if PK in these disorders is able to respond to treatment with the allosteric PK activator AG-348 (mitapivat). Methods: Enzymatic activities of red cell PK and hexokinase (HK) were measured together with PK-thermostability in order to assess relative PK activity and enzyme stability. Purified RBCs were incubated with AG-348 (3.33μM) for 24 hours after which PK activity and ATP response was measured. RBCs of SCD patients were also analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry (Rab et al, Am J Hematol, 2019). Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO2 at which RBCs start to sickle during deoxygenation under shear stress. Results: Thirty-eight patients and 21 healthy controls (HC) were included. The patient cohort consisted of patients homozygous for HbS (HbSS, n=26), patients compound heterozygous for HbS and HbC (HbSC, n=4), β-thalassemia major (regularly transfused, n=3), and hereditary xerocytosis (n=5). Patients showed reticulocytosis and, in line with this, a concomitant increase in HK activity. In contrast however, relative PK activity was decreased significantly compared to HK in HbSS, β-thalassemia and HX patients, but not in HbSC patients (Figure 1A). PK thermostability was significantly decreased compared to healthy controls in HbSS patients and patients with HX (Figure 1B). In HbSC and β-thalassemia patients, PK-thermostability was comparable to HC. PK thermostability strongly correlated with absolute reticulocyte count (ARC), indicating that patients displaying the highest degree of PK instability had the highest reticulocyte count (Figure 1C). This suggests that in general, a higher degree of PK instability is associated with more severe anemia due to a high hemolytic rate. In SCD patients, PK-thermostability inversely correlated with PoS, indicating that decreased PK stability is associated with sickling at higher pO2 (r=-0.646, p<0.001, Figure 1F). When purified RBCs were incubated with 3.33μM of the allosteric PK-activator AG-348, an increase in PK activity was seen in all patients and HCs, with a mean increase of 122% in HbSS (range 111-139%, n=6), 137% in β-thalassemia (n=1), 163% in HX (range 152-174%, n=2) and 143% in HC (range 113-173%, n=9, Figure 1E). Accordingly, ATP-levels increased in all patients and HCs, with a mean increase of 133% in HbSS (range 125-141%, n=5), 144% patient with β-thalassemia (n=1), 121% in HX (range 112-129, n=3), and 132% in HCs (range 101-149%, n=9, Figure 1E). Conclusion: PK enzyme activity and stability is compromised in patients with various forms of hereditary hemolytic anemia. This implies that PK stability and, hence, compromised red cell metabolism could contribute to the complex pathophysiology of these diseases. In SCD patients, reduced PK-thermostability is associated with higher PoS, which we previously have shown to be associated with more severe disease (Rab et al, Am J Hematol, 2019, ASH 2019 abstract ID128870). This is confirmed by the correlation of decreased PK-thermostability with increased reticulocyte count as presented in this study. Current studies are in progress to further substantiate the underlying mechanism(s) involved, and to investigate whether AG-348 may ameliorate clinical features such as hemolysis, sickling tendency and iron overload. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Kosinski:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. Kung:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding.

A Case of Congenital Red Cell Pyruvate Kinase Deficiency Associated with Hereditary Spherocytosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5272-5272
Author(s):  
Cristina Vercellati ◽  
Anna Paola Maria Luisa Marcello ◽  
Elisa Fermo ◽  
Paola Bianchi ◽  
Carla Boschetti ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Peripheral Blood ◽  
Blood Smear ◽  
Hereditary Spherocytosis ◽  
Osmotic Fragility ◽  
Peripheral Blood Smear ◽  
Red Cell ◽  
Red Cell Membrane

Abstract Abstract 5272 Pyruvate kinase (PK) deficiency, transmitted as an autosomal recessive trait, is the most common erythroenzymopathy of glycolytic pathway (prevalence of 1:20,000) associated with chronic non spherocytic hemolytic anemia from mild to severe. More than 180 mutations in the PK-LR gene have been so far reported, and genotype-phenotype correlation has been established for some of them. Hereditary Spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The main clinical features are hemolytic anemia from compensated to severe, variable jaundice, splenomegaly and cholelythiasis. The molecular defect is highly heterogeneous, caused by proteins involved in the attachment of cytoskeleton to the membrane integral domain (spectrin, ankyrin, band 3 and protein 4.2). We describe a case of PK deficiency associated with HS. The propositus was a 13 years-old Italian male with neonatal jaundice and need of blood transfusion (Hb 5.8 g/dL) during an infectious episode. At the time of the study Hb was 13.9 g/dL, MCV 81.8 fL, reticulocytes 207×109/L, unconjugated bilirubin 2.16 mg/dL, LDH 605 U/L, haptoglobin <20 mg/dL. The peripheral blood smear examination showed the presence of spherocytes (16%) and some ovalocytes (2%). The study of the most important red cell enzymes revealed reduced PK activity (59% of normal). Direct sequencing of PK-LR gene showed compound heterozygosity for the 994A mutation (Gly332Ser) and the −148T variant localized the erythroid specific promoter region. The presence of spherocytes in peripheral blood smear prompted us to investigate for the coexistence of HS. Erythrocyte osmotic fragility was decreased and SDS–PAGE analysis of red cell membrane proteins revealed a 30% spectrin reduction. Family study demonstrated a heterozygous condition for the 994A mutation in the father, who also displayed comparable enzyme deficiency, whereas promoter variant −148T was detected in the mother and in the brother. No red cell membrane abnormalities were present in the family members, although positive EMA binding test and increased osmotic fragility were found in the father and brother. The co-existence of HS and PK deficiency is very rare event, only few cases are described to date. Clinical, family and molecular studies allowed the determination of the interrelationship between the two RBC abnormalities in the patient and his relatives. The reduced PK activity in the propositus and his father is justified by heterozygous 994A mutation. The more severe clinical picture in the propositus could be caused by the coexistence of HS and by the presence of −148T mutation, that although it seems not to have effects on PK-LR mRNA expression, is often detected in PK deficient subjects with heterozygous PK mutations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The variable manifestations of disease in pyruvate kinase deficiency and their management

Haematologica ◽  
2020 ◽  
Vol 105 (9) ◽  
pp. 2229-2239 ◽  
Author(s):  
Hanny Al-Samkari ◽  
Eduard J. Van Beers ◽  
Kevin H.M. Kuo ◽  
Wilma Barcellini ◽  
Paola Bianchi ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Working Group ◽  
Disease Burden ◽  
Extramedullary Hematopoiesis ◽  
Poor Correlation ◽  
Red Cell ◽  
Hematopoietic Stem ◽  
Pyruvate Kinase Deficiency ◽  
High Index Of Suspicion

Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary non-spherocytic hemolytic anemia and results in a broad spectrum of disease. The diagnosis of PKD requires a high index of suspicion and judicious use of laboratory tests that may not always be informative, including pyruvate kinase enzyme assay and genetic analysis of the PKLR gene. A significant minority of patients with PKD have occult mutations in non-coding regions of PKLR which are missed on standard genetic tests. The biochemical consequences of PKD result in hemolytic anemia due to red cell pyruvate and ATP deficiency while simultaneously causing increased red cell 2,3-diphosphoglycerate, which facilitates oxygen unloading. This phenomenon, in addition to numerous other factors such as genetic background and differences in splenic function result in a poor correlation between symptoms and degree of anemia from patient to patient. Red cell transfusions should, therefore, be symptom-directed and not based on a hemoglobin threshold. Patients may experience specific complications, such as paravertebral extramedullary hematopoiesis and chronic debilitating icterus, which require personalized treatment. The decision to perform splenectomy or hematopoietic stem cell transplantation is nuanced and depends on disease burden and long-term outlook given that targeted therapeutics are in development. In recognition of the complicated nature of the disease and its management and the limitations of the PKD literature, an international working group of ten PKD experts convened to better define the disease burden and manifestations. This article summarizes the conclusions of this working group and is a guide for clinicians and investigators caring for patients with PKD.

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