Introduction

 PK deficiency is the most common enzyme deficiency causing hemolysis. Although this disorder is far less common than G6PD deficiency, the vast majority of patients with G6PD deficiency never suffer a hemolytic episode, while PK deficiency has a high penetrance, although also a highly variable phenotype.

Genetics

PK enzymes consist of several isoforms. They are products of two dis tinct genes, PKLR and PKM located on different chromosomes. PKLR (encoding L, liver, and R, RBC isoenzymes) is located on chromosome 1q21. The R isoform, unique to RBCs, is 33 amino acids larger than the L isoform, which is unique to hepatocytes. Expression in RBCs versus liver is due to differential use of tissue-specific promoters, which drive expression as well as tissue-specific exon usage (use of exon 1 but not exon 2 in RBCs and exon 2 but not exon 1 in liver). Regulatory elements in PKLR-gene expression are not fully defined, but one is a key erythroid transcription factor, Kruppel Like Factor 1 (KLF1).

The PKM gene encodes the M (muscle) enzymes. There are two isoforms, M1 and M2, which are different splicing products of the PKM-gene’s single transcript. The M1 isoform is expressed in muscle, heart, brain. The M2 isoform initially predominates in fetal erythropoiesis but is progressively replaced in RBCs with the R form during fetal development. In contrast, M2 persists in leukocytes and plate lets. M2 is overexpressed in many tumors and is present in other cells such as lung, fat, retina, and pancreatic islets. PKM proteins have co-stimulatory activity with hypoxia-inducible transcription factor 1 (HIF-1). The PKM gene is located on chromosome 15q22.

Thus far, 290 PKLR mutations have been characterized by DNA sequencing (Human Gene Mutation Database [HGMD, http:// www.hgmd.org; accessed June 23, 2020]). Of these mutations, 276 are disease-causing, most of which are missense mutations.

Epidemiology

PK deficiency is distributed worldwide but is more common among people of northern European extraction. PK deficiency is an autosomal recessive disease, and affected patients are typically double heterozygotes, or, less commonly, homozygous for the same mutation. Homozygous mutations are usually seen in groups with marked consanguinity, and homozygous PK deficiency has been well studied in the Amish populations of Pennsylvania and Ohio and also in an isolated fundamental Mormon settlement at the Utah/Arizona border. Common mutations have well-defined geo graphic associations. PKLR c.1529 G>A mutation is the most common mutation in the United States, northern and central Europe with a population prevalence in the US of ~50/million, while c.1456 C>T is the most common mutation in southern Europe, and c.1468 C>T in Asia.

PK deficiency does not localize to geographic areas of malarial endemicity. However, there is in vitro evidence that PK deficiency provides protection against infection and replication of Plasmodium falciparum in human RBCs, an effect possibly mediated by reduced ATP levels in PK-deficient RBCs. PK deficiency was also shown to be protective in a mouse model of infection with Plasmodium chabaudi.

Pathobiology

PK enzymes catalyze the final step in glycolysis: the irreversible trans fer of phosphate from phosphoenolpyruvate to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP (see Fig. 1). PK is allosterically activated by the binding of fructose 1,6-diphosphate (FDP); that is, FDP binds to a site other than the active site for substrate binding, causing a conformational change in the PK enzyme. FDP is an intermediate in the glycolytic pathway, and higher concentrations of FDP increase PK activation (a process known as feedforward stimulation). PK deficiency results in decreased glycolytic ATP production and an accumulation of glycolytic intermediates, including 2,3-BPG.

Fig1. PRINCIPAL COMPONENTS OF THE ERYTHROCYTE METABOLISM WITH CLINICAL RELEVANCE. Shown are glycolysis, glutathione production (purple shaded area), pentose shunt (orange shaded area), and Rapoport-Luebering shunt (blue shaded area). 1,3-BPG, 1,3-bisphosphoglycerate; 2,3-BPG, 2,3-bisphosphoglycerate; 6PG, 6-phosphogluconate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; DHAP, dihydroxy acetone phosphate; GSH, reduced glutathione; GSSG, oxidized glutathione; NAD, oxidized form of nicotinamide adenine dinucleotide; NADH, reduced form of nicotinamide adenine dinucleotide; NADP, oxidized form of nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate.

PKLR mutations affecting the active catalytic site are associated with more severe hemolytic anemia. However, the phenotypical expression of identical mutations can be strikingly different, even within the same family. Clinical PK deficiency with hemolytic anemia is largely limited to mutations of the PKLR gene. However, a mutation in the transcription factor KLF1 that regulates PKLR transcription is a rare cause of PK deficiency (see below). Since PKM encoded proteins are present on leukocytes and platelets, using whole blood to analyze for PK deficiency may result in false-negative results.

PK-R exists as a heterotetramer. Since most PK-deficient patients are compound heterozygous for two different mutations, rather than homozygous for one, several different tetrameric forms of PK-R may be present, each with distinct structural and kinetic properties. This complicates genotype-to-phenotype correlations in these individuals, as it is difficult to infer which mutation is primarily responsible for deficient enzyme function and the clinical phenotype. There are even cases in which the activity of PK as measured in vitro is higher than normal, but a kinetically abnormal enzyme with low in vivo activity in erythrocytes is responsible for the hemolytic anemia. These mutations can only be detected if PK-enzyme activity is measured with and without FDP and at several concentrations of PK’s substrate phosphoenolpyruvate.

PK deficiency may be also caused by mutations not directly involving the PKLR gene. Mutations in the erythroid transcription factor KLF1 caused severe congenital hemolytic anemia because of a deficiency of PK. In this instance, the PKLR gene was intact and thus sequencing of the PKLR gene would miss the diagnosis of PK deficiency.

The mechanism of hemolysis in PK deficiency is not clear. The defect in ATP generation is unlikely to be the sole cause, as ATP deficiency is difficult to demonstrate in many patients and other dis orders with more severe ATP deficiency are not associated with significant hemolysis. Increased apoptosis and ineffective erythropoiesis may be features of PK deficiency, although this has only been studied in splenic erythroid progenitors. Following splenectomy, despite decreased hemolysis and improved anemia, patients paradoxically have a higher number of reticulocytes, sometimes reaching over 50%. The reasons for this phenomenon is as yet unexplained.

Tolerance of Anemia

The anemia of PK deficiency is better tolerated than a comparable level of anemia seen in patients with hexokinase deficiency, since the block in glycolysis occurs after the Rapoport-Leubering shunt (see Fig. 1). Levels of 2,3-BPG may be elevated up to two times normal in PK-deficient individuals, resulting in decreased hemoglobin oxygen affinity. This accumulation of 2,3-BPG shifts the oxyhemoglobin dissociation curve to the right, leading to better oxygen delivery to the tissue and improved tolerance of anemia than would be otherwise expected.

Clinical and Laboratory Manifestations

 Hemolysis and Anemia

Hemolysis in PK deficiency is mainly extravascular (i.e., due to phagocytosis of cells by reticuloendothelial macrophages). However, if the hemolysis is severe, there may be spillover to intravascular hemolysis. Thus, many affected patients have normal lactate dehydrogenase (LDH) levels, but most have elevated bilirubin levels and clinical jaundice, as well as reticulocytosis. The reticulocyte count invariably increases after splenectomy.

The severity of hemolysis in PK-deficient patients is highly variable, ranging from a mild, fully compensated chronic hemolytic process without anemia, to life-threatening transfusion-requiring hemolytic anemia present at birth, to rare hydrops fetalis because of homozygosity for PKLR null mutations. The disease severity is often but not always similar among siblings of a given family. In most cases the degree of hemolysis declines after infancy, by a not fully under stood pathophysiologic mechanism. Splenomegaly is often but not invariably present. Patients with severe hemolysis may be chronically jaundiced and may develop the clinical complications of chronic hemolytic states, including gallstones, transient aplastic anemia crises (caused by parvovirus infection), folate deficiency (rarely), extramedullary hematopoiesis and infrequently, skin ulcers. Pregnancy may precipitate hemolysis.

Iron Overload

As in any patient with ineffective erythropoiesis and hyperactive pro duction of early RBC precursors, iron absorption is increased and iron retention in macrophages is decreased, due to erythroferrone mediated decreases in the levels of hepcidin. Iron overload can occur, not only in transfusion-dependent patients but also in nontransfused patients, and may be severe. In some patients, clinically significant iron overload is detected in adult hood before the diagnosis of PK deficiency and chronic hemolysis.

Neonatal Icterus

Neonatal icterus may occur and is augmented by coincidental heterozygosity or homozygosity for the UGT1A1 polymorphism.

Diagnosis

 There are no characteristic RBC morphologic findings in PK deficiency; however, nonspecific changes such as echinocytes (burr cells), anisocytes, or poikilocytes may be present. PK deficiency should be suspected in patients with Coombs-negative hemolytic anemia, no RBC morphologic features of membrane disorders, and a history consistent with congenital hemolysis.

Biochemical Testing

 A rapid screening test using crude hemolysate with a single concentration of substrate has been used for the detection of pyruvate deficiency but should be avoided unless leukocytes and platelets are first filtered out. PK-enzymatic screening tests also miss some PKLR variants.

The gold standard test for diagnosing PK deficiency is spectrophotometric testing of PK activity and estimation of enzyme kinetics in RBCs free of contaminating leukocytes and platelets. In this assay, testing enzyme activity at different concentrations of the allosteric activator FDP and the substrate phosphoenolpyruvate permits detection of high Km–low-affinity mutants. However, this assay is not available at most commercial laboratories.

Genetic Testing

DNA testing for PK deficiency is increasingly available, including testing performed by commercial laboratories using heterogeneous genomic sequencing methods including next generation sequencing (NGS) of whole PKLR exomes or targeted sequencing. However, if a previously undescribed mutation is found, it may be challenging to determine if it is disease-causing or a rare polymorphism. If the familial genetic variant is not known, and no mutation found by NGS covering all exons, additional sequencing of flanking regions, the PKLR promotor, and in some instances the whole genome, may be needed. Some patients have large deletions and intronic mutations at cryptic splice sites that may not be detected by routine sequencing methods. In individuals with more than one PKLR mutation (compound heterozygotes), it is also important that parent samples be obtained, in order to determine whether the two mutations are present in cis (both from the same parent) or in trans (one from each parent). Identification of a deleterious mutation can facilitate testing of potentially affected family members, including prenatal testing. As the severity of hemolysis may be variable even among relatives with the same PKLR mutation, it is likely that in large families genetic testing will identify some affected, yet relatively asymptomatic patients, who otherwise would never be diagnosed.

However, as noted above, sequencing the PKLR gene will not diagnose the rare mutations of genes other than PKLR, such as KLF1, that have been shown to reduce PK enzymatic activity.

Therapy

Many patients do not require therapy. Some require RBC transfusions only in transient settings of increased stress, such as the perioperative period, coexistent infections, or pregnancies. However, others require chronic transfusions. Iron status should be monitored even in patients who have never been transfused. If iron overload is detected in those who are not severely anemic phlebotomy therapy, then iron chelation should be instituted.

Splenectomy has documented benefits in severe cases; the degree of hemolysis and anemia is ameliorated and the transfusion requirement is abolished or markedly decreased. The increase in hemoglobin con centration in non-transfusion-requiring patients after splenectomy ranges from 1 to 3 g/dL. It is recommended to delay splenectomy, if possible, until after the age of 3 years when the risk of infections with encapsulated organisms declines, and because in most cases the degree of hemolysis declines after infancy, by a not fully understood pathophysiologic mechanism. One PK-deficient boy with severe hemolysis was apparently cured by an allogeneic marrow transplant. Gene therapy is also actively being explored.

Mitapivat (previously called AG-348) is an orally available small molecule (a quinolone sulfonamide). It was developed using bio chemical assays for compounds that could allosterically activate the PK-R enzyme similarly to FDP, but with greater efficacy. In a study in 52 patients, mitapivat increased the hemoglobin level by more than 1.0 g/dL in 26 (50% response). Responses to mitapivat correlated with the PKLR genotype and were best in individuals with missense mutations in PKLR. Responses did not occur (or were lower) in individuals who were homozygous for PKLR^R479H or for two non-missense mutations. Of 20 individuals with a good response who continued therapy in an extension phase, 19 had a sustained increase in hemoglobin. Therapy was well-tolerated with most adverse events being grade 1 or 2 in severity. In responding patients, mitapivat was also shown to restore glycolytic pathway activity and normalize previously low ATP and previously elevated 2,3-BPG levels.

Prognosis

The clinical course is highly variable ranging from fatal hydrops fetalis, transfusion dependency from birth, and a high risk of kernicterus to normal childhood development with no or rare transfusions, or to compensated hemolysis without anemia. However, hemolysis that is fully compensated because of excessive erythropoiesis may be deceptively benign. In addition to the usual complications of chronic hemolysis such as gallstones, extramedullary hematopoiesis, and parvovirus-induced aplastic crisis, the excessive number of erythroblasts in these patients produces erythroferrone, which mediates low hepcidin and may result in typical hemochromatosis with cardiac and hepatic dysfunction.

Future Directions

New gene therapeutic techniques are being evaluated in severe PK deficiency. These include the zinc finger nucleases and CRISPR/ Cas9 technology (clustered regularly interspaced short palindromic repeats) that use noncoding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. Preclinical gene therapy has also been reported for PK deficiency based on a lentiviral vector harboring the promoter that drives the expression of the PKLR cDNA.

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