ANEMIA DUE TO ENZYME DEFICIENCY

ANEMIA DUE TO ENZYME DEFICIENCY

dr. Karjana, SpA,

Dr. dr. Nadirah Rasyid Ridha,M.Kes, Sp.A(K),

Prof. Dr. dr. H. Dasril daud, Sp.A(K)

ANEMIA DUE TO ENZIMAL DISORDERS

Anemia in children is defined as a hemoglobin level or hematocrit lower than the normal value for age in healthy children. Anemia is not a specific disease but a condition caused by various underlying diseases.
The main function of red blood cells is to carry hemoglobin throughout the circulation in a high concentration and in a functional state so that gas exchange can occur efficiently in the lungs and in the tissue capillaries. To fulfill its function, red blood cells need a supply of energy in the form of ATP and reducing resources.
Mature red blood cells (RBCs) are anucleate (thus incapable of cell division), devoid of ribosomes (thus incapable of protein synthesis) and lacking mitochondria (thus incapable of oxidative phosphorylation). Despite these limitations, RBCs survive 100 to 120 days in circulation, and effectively deliver oxygen to peripheral tissues. Glucose is the main metabolic substrate of red blood cells and is metabolized by two main pathways: the glycolytic or “energy-producing” pathway and the hexose monophosphate (HMP) bypass or “protective” pathway. Under normal conditions, about 90% of glucose flows through glycolysis, with a smaller fraction being channeled through the HMP pathway. However, the fraction of glucose entering the pentose phosphate pathway can be significantly increased under conditions of increased oxidative stress. The main products of glycolysis are ATP (the energy source for many RBC membranes and metabolic reactions), NADH (a cofactor required for the reduction of methemoglobin by cytochrome b5 reductase), and 2,3-diphosphoglycerate ([2,3-DPG], an important intermediate that modulates hemoglobin-oxygen affinity).
Adult RBCs are not capable of de novo purine or pyrimidine synthesis, although many nucleotide metabolizing enzymes are present in erythrocytes. The latter is now known to be important for in vitro RBC preservation; and it is also recognized that abnormalities in purine and pyrimidine metabolism are associated with inherited hemolytic disease.
The lack of protein synthesis in mature red cells means that none of the enzymes in the metabolic pathway can be replaced during the lifespan of the red blood cells. During 120 days of normal red cell survival, enzyme activity decreases at a variable but predictable rate. This decrease may contribute to the aging process of red blood cells. Many disorders affecting red blood cell metabolism lead to hemolytic anemia (Table 1).

 

Enzyme abnormalities manifesting hemolytic anemia

Enzyme abnormalities manifesting hemolytic anemia

The consequences of red blood cell enzymes are diverse. Several enzyme variants cause hemolytic disease, with anemia as the only enzymatic expression. In other enzyme disorders hemolysis is one of the features of a multisystem disease that affects many tissues. These RBC enzyme abnormalities are due to abnormalities in HMP shunt and glutathione metabolism, glycolytic enzyme deficiencies, and abnormalities in purine and pyrimidine metabolism.

The glycolytic pathway and its relationship to other metabolic pathways

The glycolytic pathway and its relationship to other metabolic pathways

Monophosphate Shunt Disorders And Glutathione Metabolism
The HMP shunt pathway metabolizes 5% to 10% of the glucose used by red blood cells, and this is critical for protecting red blood cells against oxidant injury (Figure 1). The HMP pathway is the only source of RBC that reduces nicotinamide adenine dinucleotide phosphate (NADPH), a cofactor important in glutathione metabolism. Red blood cells contain relatively high concentrations of reduced glutathione (GSH), a sulfhydryl-containing tripeptide (glutamylcysteineylglycine) that functions as an intracellular reducing agent that protects cells against oxidant injury. Oxidants, such as superoxide anions (O2-) and hydrogen peroxide (H2O2), are produced by exogenous factors (i.e., drugs, infections) and are also formed in red cells as a consequence of the reaction of hemoglobin with oxygen. However, when these oxidants accumulate in red blood cells, hemoglobin and other proteins are oxidized, leading to loss of function and death of RBCs. Under normal circumstances this does not happen, because GSH, together with the enzyme glutathione peroxidase (GSH-Px), rapidly inactivates these compounds. During the oxidant detoxification process, however, GSH itself is converted to oxidized glutathione (GSSG), and GSH levels fall. To maintain protection against persistent oxidizing injury, GSH levels must be maintained, and this is achieved by glutathione reductase (GSSG-R), which catalyzes the reduction of GSSG to GSH. This reaction requires NADPH produced by glucose-6-phosphate dehydrogenase (G6PD), the first enzymatic reaction of HMP shunt. Thus, it is the tight coupling of the HCH shunt and glutathione metabolism that is responsible for protecting intracellular proteins from oxidative attack. Nearly all hemolytic episodes associated with altered HMP shunt and glutathione metabolism are due to G6PD deficiency, and deficiency of this enzyme is known to affect millions of people worldwide.

Glucose 6-Phosphate Dehydrogenase (G6PD)

Definition
Glucose 6-P phosphate dehydrogenase (G6PD) deficiency is an inherited sex-linked (x-linked) enzyme disorder, in which the activity or stability of the G6PD enzyme decreases, causing the breakdown of red blood cells when an individual is exposed to exogenous substances that potential to cause oxidative damage 3,4,6.

Epidemiology
G6PD deficiency is the most common enzyme deficiency disease in humans, about 2-3% of the entire world population is estimated to be around 400 million people worldwide. The highest frequency was found in the tropics, found with varying frequencies in various Middle Eastern, Indian, Chinese, Malay, Thai, Filipino and Melanesian races5,6.
G6PD deficiency is the most common cause of jaundice and acute hemolytic anemia in Southeast Asia 5. In Indonesia the incidence is estimated at 1-14%, the prevalence of G6PD deficiency in Central Java is 15%, in isolated small islands in eastern Indonesia (Babar Island , Tanimbar, Kur and Romang in Maluku Province), stated that the incidence of G6PD deficiency was 1.6 – 6.7%.

Molecular Biochemistry and Physiological Metabolism of the G6PD . Enzyme
The G6PD enzyme is a polypeptide consisting of 515 amino acids with a molecular weight of 59.265 kilodaltons 15. The G6PD enzyme is the first enzyme of the pentose phosphate pathway, which converts glucose-6-phosphate into 6-phosphogluconate in the glycolysis process. This change produces Nicotinamide Adenine Dinucleotide Phosphate (NADPH), which reduces oxidized glutathione (GSSG) to reduced glutathione (GSH). GSH functions as a scavenger of peroxides and oxidant radicals H2O2 1-7. Under normal circumstances peroxides and free radicals are removed by catalase and glutathione peroxidase, further increasing the production of GSSG. GSH is formed from GSSG with the help of the enzyme glutathione reductase whose presence depends on NADPH. In G6PD deficiency, the formation of NADPH is reduced so that it affects the regeneration of GSH from GSSG, consequently affecting the ability to remove peroxides and free radicals 1,3,5-7.

The G6PD gene consists of 13 exons and 12 introns spread over an area of ​​more than 100 kb at the terminal end of the long arm of the X chromosome 10,12,13. G6PD deficiency results from mutations in the G6PD gene, a sex-linked disease. Males only have 1 X chromosome, so if a mutation occurs, G6PD deficiency will appear or manifest. Women have 2 X chromosomes, so if there is 1 abnormal gene due to mutation, its partner or allele can “mask” the deficiency, so G6PD deficiency can manifest or not. G6PD deficiency includes a variety of different G6PD gene mutations that do not react the same, this explains why G6PD deficient individuals react differently to the same precipitating factors 1, 3-6. The G6PD gene located on chromosome Xq28 with a length of 18 Kb, consisting of 13 exons is DNA and 12 introns are disruptive sequences, which is DNA waste that does not play a role in enzyme function. Enzyme function is determined by the sequence and size of the G6PD gene and the mRNA that characterizes the gene. PCR (polymerase chain reaction) examination can help identify the presence of mutations. Currently, there are more than 40 known mutations that are distributed throughout the entire gene coding, each of which is different and has its own characteristics 1,4.
More than 400 variants of G6PD have been reported, with various clinical appearances and/or phenotypes. The variants were differentiated based on residual enzyme activity, electrophoretic mobilization, affinity and substrate analogues, stabilization to heat and optimum pH 1.4.
WHO made a classification based on the variants found in each country, nucleotide substitution and amino acid substitution, namely 3,7:

• Class I: Non-spherocytotic hemolytic anemia (G6PD residual activity, <20). It is a rare type of G6PD enzyme deficiency. This group had severe functional abnormalities (Harilaou variant). Red blood cells are unable to defend themselves from endogenous oxidants, resulting in chronic hemolysis. Exposure to precipitating factors will cause an exacerbation of acute hemolytic anemia.
• Class II: severe deficiency (G6PD residual activity, <10). Severe G6PD enzyme deficiency group (Mediterranean variant of G6PD). Exposure to trigger factors (exogenous) will cause acute hemolysis and the process will continue as long as there is exposure to precipitating factors. This is due to the low activity of the G6PD enzyme in both old and young red blood cells.
• Class III: moderate deficiency (G6PD residual activity, 10-60). Mild G6PD enzyme deficiency group (G6PD variant A). In this group, hemolysis that occurs as a result of exposure to the precipitating factor will stop by itself even though the exposure continues. This is because the activity of the G6PD enzyme in young red blood cells is still high enough to retain oxidants, and only old red blood cells undergo hemolysis.
• Class IV: non-deficiency (G6PD residual activity, 100).
• Group without symptoms of G6PD . deficiency
• Class V: non-deficiency (G6PD residual activity, >100)

The Role of the G6PD Enzyme in Red Blood Cells
Red blood cells require a continuous supply of energy to maintain their shape, volume, flexibility (flexibility), and regulation of their sodium-potassium pump. This energy is obtained from glucose through two metabolic pathways, namely, 80% of the anaerobic glycolysis process (Emden-Meyerhof pathway) and 20% of the aerobic glycolysis process (Pentose Phosphate pathway) 2,7.

The role of the G6PD enzyme in maintaining the integrity of red blood cells and preventing hemolytic events lies in its function in the pentose phosphate pathway. In red blood cells there is a compound of reduced glutathione (GSH) which is able to maintain the integrity of the sulfhydryl group (SH) in hemoglobin and red blood cells. The function of GSH is to maintain cysteine ​​residues on hemoglobin and other proteins on the erythrocyte membrane to remain in a reduced and active form, maintain hemoglobin in ferrous form, maintain the normal structure of red blood cells, and play a role in the detoxification process, where GSH is a second substrate for enzymes. glutathione peroxidase in neutralizing hydrogen peroxide which is an oxidant that has the potential to cause oxidative damage to red blood cells 1-7

The GSH compound was originally a disulfide form of glutathione (oxidized glutathione, GSSG) which was reduced to sulfhydryl form of glutathione (reduced glutathione, GSH). The reduction of GSSG to GSH is carried out by NADPH, in the pentose phosphate pathway, where in this metabolic pathway NADPH is formed when glucose-6-phosphate is oxidized to 6-phosphogluconate with the help of the G6PD enzyme. 1-7
From the description above, it can be seen that the function of the G6PD enzyme is to provide the NADPH needed to re-form GSH. In G6PD deficiency, NADPH levels are reduced, so that exposure to oxidant stress will affect the formation of disulfide bonds, causing hemoglobin to denature and form thick particles (Heinz bodies). Heinz bodies will bind to cell membranes, causing changes in cell content, elasticity, and permeability. Red blood cells in this condition are recognized as damaged red blood cells and will be destroyed by the reticulo-endothelial system (spleen, liver and bone marrow) hemolytic processes 1,3,5.
Although the G6PD gene is present in all body tissues, the effect of deficiency in erythrocytes is very large because the G6PD enzyme is needed to produce energy to maintain erythrocyte life, carry oxygen, regulate ion and water transport into and out of cells, help remove carbon dioxide and protons formed in the cells. tissue metabolism. Because there are no mitochondria in the erythrocytes, the oxidation of G6PD only comes from NADPH, when the level of the G6PD enzyme decreases, the erythrocytes experience a lack of energy and changes in shape that facilitate lysis when there is oxidant stress 1-7.

the role of the G6PD-min enzyme

the role of the G6PD enzyme

 

Clinical and Laboratory Manifestations
Clinical Manifestations
In general, individuals with inherited G6PD enzyme deficiency do not undergo hemolysis and are often asymptomatic (and often asymptomatic), but this can occur when the patient is exposed to exogenous substances that can cause oxidative damage. Some diseases that are known to be associated with G6PD deficiency are: hyperbilirubinemia (Kern Jaundice), intravascular hemolysis, favism, hemolytic hepatitis syndrome, chronic hemolytic anemia 1,3,5. Clinical symptoms appear 1-3 days after exposure to the precipitating factor, in the form of acute hemolytic anemia with a typical picture of being fussy, irritable / looking cranky, lethargic, increased temperature > 380 C, nausea, abdominal pain, diarrhea, anemia, jaundice and abnormalities in the urine. hemoglobinuria). On physical examination, there is variable pallor and tachycardia, spleen and liver are usually enlarged. In severe cases, hypovolemic shock and heart failure occur 3,5,6.
Laboratory Overview

The laboratory findings showed that normochromic normocytic anemia varies from mild to severe, with a striking picture of anisocytosis, poikilocytosis and an increased reticulocyte count > 30%. With methyl violet staining, Heinz bodies appear. The number of leukocytes usually increases with a predominance of granulocytes, increased indirect bilirubin but liver enzymes within normal limits 3,5,6. Hemolytic anemia is generally triggered by exposure to drugs (such as sulfonamides, primaquine, chloramphenicol, chloroquine, uric acid).

nalidixate, quinacrine, nitrofurantorin, salicylates, dapsone, phenacetin, asitanicide, and antipyrine), cocoa bean diet (victa fava), chemicals (Naphthalene), pneumococcal infection, hepatitis and ketoacidosis, which principally cause a decrease in glutathione levels, where levels of already low due to G6PD deficiency itself. In malaria endemic areas of Africa and Southeast Asia hemolysis is often induced by primaquine administration. 3,5,6.
Currently, the diagnostic support that is widely used to help establish the diagnosis of G6PD deficiency is the Heinz Body test and the GSH 10 stability test. The screening test can be performed with the methylene-blue test with a color change during reduction of methemoglobin or with NADPH fluorescence. Diagnostic tests for G6PD deficiency based on enzyme activity can be detected by simple laboratory tests. Shirakawa et al screened using the formazan-ring/Hirono’s method 1 . method

Pyruvate Kinase
INTRODUCTION – Pyruvate kinase (PK) deficiency is an inherited (autosomal recessive) red blood cell (RBC) enzyme disorder that causes chronic hemolysis. This is the second most common RBC enzyme defect but is the most common cause of hemolytic anemia from RBC enzyme deficiency.
PATHOPHYSIOLOGY
Genetics – PK deficiency is an autosomal recessive disorder; Affected individuals are either homozygous for a single pathogenic mutation or compound heterozygous for two different pathogenic mutations that affect the function of PK enzymes in red blood cells (RBCs). Individuals who are heterozygous for PK deficiency have moderate enzyme levels and are not clinically affected.
PK enzymes consist of several isoforms. They are the product of two different genes, both encoding enzymes that catalyze the transphosphorylation of phosphoenolpyruvate (PEP) to pyruvate and ATP. Clinical PK deficiency with hemolytic anemia is limited to mutations of the PKLR gene:
PKLR – PKLR gene encoding isoenzymes L (liver) and R (RBC). The R isoform, unique to RBC, is 33 amino acids larger than the L isoform, which is unique to hepatocytes. Expression in red blood cells versus liver is due to differences in the use of tissue-specific promoters, which drive expression as well as use of exons. The PKLR gene is located on chromosome 1q21.
PKM – PKM gene encodes the enzyme M (muscle). This form is expressed in muscle, brain, white blood cells (WBC), and platelets. There are two isoforms, M1 and M2, resulting from differential processing of a single transcript. The M2 isoform is dominant during fetal development. After birth, the M2 isoform persists in white blood cells and platelets. In red blood cell progenitor cells, the M2 isoform is progressively replaced by the R form during fetal development. The PKM gene is located on chromosome 15q22.

More than 100 pathogenic variants and eight polymorphic sites have been reported for PKLR genes. Variants include single nucleotide substitutions as well as intronic and exonic deletions and insertions. The known variants are listed in the publicly available list of databases. The C1151T mutation, which results in the substitution of threonine for methionine at amino acid 384, is very common, even among unrelated families of different ethnicities.
The frequency of pathogenic PKLR gene mutations is much lower than mutations affecting glucose-6-phosphate dehydrogenase (G6PD), which causes G6PD deficiency. However, unlike G6PD deficiency, which can only manifest hemolysis under certain circumstances such as exposure to oxidant drugs, in PK deficiency, hemolysis is chronic. Thus, PK deficiency is the most common RBC enzyme defect causing congenital chronic non-spherocytic hemolytic anemia.

Mutations in genes other than PKLR have been shown to reduce the enzymatic activity of PK, although this is rare. For example, multiple heterozygous mutations in the gene encoding Kruppel-like factor 1 (KLF1), a hematopoietic transcription factor important for the induction of expression of adult beta globin and other erythroid genes, have been associated with severe transfusion-dependent hemolysis. anemia and PK enzyme deficiency.
Genes that increase PK activity have also been postulated, although no specific gene causing PK hyperactivity has been described.

Enzymatic function of PK – Red blood cells use several enzyme systems to maintain their viability and function. Two of the main processes under enzymatic control are energy production, in the form of ATP, and protection from oxidative injury, by compounds that act as reducing agents. PK enzymes function in the energy-producing glycolytic pathway, which metabolizes glucose to produce ATP for cells.

As shown in the figure, PK catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate by removing a phosphate group. The phosphate group from PEP is transferred to ADP to make one ATP molecule.
The PK enzyme is a homotetramer containing four molecules of the PK protein. Thus, in individuals heterozygous for two different PKLR mutations, five different compositions of tetramers will be present (e.g., tetramers containing 0, 25, 50, 75, and 100 percent of each of the two abnormal protein subunits).
PK is an allosteric enzyme (an enzyme that binds an effector to one region of

the enzyme results in an altered conformation). and/or alter enzymatic activity towards substrates that bind to other enzyme regions). The substrate for PK is PEP and the allosteric regulator is fructose 1,6-diphosphate (FDP); binding to FDP alters the conformational structure of PK and its enzymatic activity towards PEP.
As noted above, there are two different genes encoding different PK isoforms. Individuals with PK deficiency have mutations in the PKLR gene that cause reduced PK activity on red blood cells. However, PK activity in other cell types such as white blood cells, platelets, and other tissues is normal because the enzymes expressed in these cells are encoded on separate genes.

Pathogenic mutations in the PKLR gene can affect any of the following properties of PK enzymes:
Altered affinity for PEP (its substrate)
Altered affinity for FDP (its allosteric activator)
Changes in enzymatic stability

Genotype-phenotype correlations have been examined for several common mutations. The most severe clinical phenotypes are generally associated with mutations that cause premature stop codons, frame replacements, or large deletions.

Mechanism of hemolysis – The mechanism for hemolysis in PK deficiency is unclear. Although a defect in ATP generation contributes to hemolysis, it is not a sufficient explanation; this conclusion is based on the observation that ATP deficiency is difficult to demonstrate in some affected patients. In addition, other disorders with more severe degrees of ATP deficiency were not associated with significant hemolysis.

Hemolysis in PK deficiency is primarily extravascular (ie, due to phagocytosis of cells by reticuloendothelial macrophages); However, if the hemolysis is severe, there may be spillover to intravascular hemolysis.
It has been proposed that the mechanism of hemolysis in PK deficiency is similar to the unexplained destruction of young red blood cells described in individuals descending from high altitude, a process termed “neocytolysis.” It is possible that the process may involve impaired mitochondrial autophagy, resulting in increased mitochondrial mass, which in turn leads to increased production of reactive oxygen species (ROS); However, this remains speculative.

Patients with hemolytic anemia who underwent splenectomy, with the resulting decreased hemolytic process and increased anemia, had higher reticulocyte counts than they did before splenectomy. These puzzling observations suggest that knowledge about the regulation of erythropoiesis and reticulocyte kinetics is still incomplete. Significant interactions may occur between the spleen and deficient PK, young red blood cells, through an unknown mechanism influencing the premature destruction of spleen reticulocytes and young red blood cells, especially in patients with more severe PK deficiency.

The metabolic disorders in PK deficiency affect the survival of red blood cells as well as the maturation of erythroid progenitor cells in the spleen, leading to their apoptosis (referred to as ineffective erythropoiesis). Ineffective erythropoiesis in the spleen has been demonstrated in a four-year PK-deficient patient undergoing splenectomy, as well as in a PK-deficient mouse model. However, it remains to be established whether apoptosis of erythroid progenitor cells in bone marrow plays a role in PK deficiency anemia, as well as whether PK activity has a more general role in the apoptotic pathway.

The severity of hemolysis is hypothesized to be determined by the degree of enzyme disruption conferred by the specific combination of the PKLR gene mutation, as well as the possible compensatory increase in the expression of the PK M2 isoform, which is expressed from the unaffected PKM gene, although this has not been definitively demonstrated.

Increased oxygen delivery – PK deficient HRs exhibit increased oxygen delivery for the partial pressure of oxygen in the bloodstream. This is because the block in glycolysis in PK deficiency is downstream of the Rappaport-Luebering shunt, which results in the formation of the metabolic intermediate 2,3-bisphosphoglycerate (2,3-BPG; previously called 2,3-diphosphoglycerate [2,3]-DPG]). Accumulation of 2,3-BPG has been noted in PK deficiency, in contrast to low concentrations in hexokinase deficiency, which is upstream of the shunt. This increase in 2,3-BPG leads to a “right” shift of the oxygen dissociation curve for hemoglobin in patients with PK deficiency. , resulting in better oxygen delivery to the tissues. Consequently, individuals with PK deficiency tolerate anemia better than those with a defect upstream to a Rappaport-Luebering shunt that does not cause a rightward shift in the hemoglobin oxygen curve, such as hexokinase deficiency.

Mechanisms of iron overload – As in any patient with ineffective erythropoiesis and hyperactive early RBC precursor production, iron absorption is increased, and iron retention in macrophages is decreased, due to an erythroferrone-mediated decrease in hepcidin levels. These pathways are discussed in more detail separately.
Possible protection from malaria – It is unclear whether PK deficiency protects against all forms of malaria.
Evidence supporting a protective role – In vitro, RBCs from PK-deficient individuals exhibit reduced Plasmodium falciparum invasion, and RBCs from PK-deficient individuals as well as heterozygous carriers exhibit preferential macrophage clearance of ring-stage-infected RBCs. . In a mouse model, PK deficiency provides protection against malaria.

Evidence against a protective role – The geographic distribution of PK deficiency does not indicate a positive selection pressure from malaria because there are other inherited RBC gene variants such as sickle mutation, thalassemia mutation, Duffy blood group system, and some forms of hereditary elliptocytosis.

EPIDEMIOLOGY – PK deficiency is extremely rare, but its true prevalence is unknown. Based on the frequency of the PKLR 1529A gene mutation in whites and its relative abundance in patients with hemolytic anemia, a prevalence of about 51 cases per million has been estimated in the European population. However, in the experience of these authors, the true prevalence of PK deficient patients encountered by haematologists referred to RBC disorders is much lower. There are more than 200 people with PK deficiency enrolled in ongoing natural history studies.
PK deficiency has a worldwide distribution, but is more common among people of northern Europe and possibly of Chinese ancestry.

As with autosomal recessive disorders, PK deficiency may be more common in groups with a history of inbreeding or relatives. For example, a high frequency of PK deficiency has been documented in the Amish community in Pennsylvania and in the fundamentalist branch of The Church of Jesus Christ of Latter-day Saints (FLDS Church) on the Utah/Arizona border. In such isolated populations, the “founder” effect can have immediate implications. In this population, the frequency of heterozygosity may exceed 1 percent.
PK deficiency has also been described in innate mouse strains and in Basenji . dogs
SPECIAL PRESENTATION AND CLINICAL FEATURES

Overview presenting findings – PK deficiency is a lifelong condition, but the age of presentation is unpredictable and can vary widely due to significant heterogeneity in the severity of hemolysis and anemia, even among individuals of the same genotype. However, in most cases, family members with the same genetic defect tend to have similar disease severity. The disease has been reported to be particularly severe among the Amish of Pennsylvania, with occasional lethal outcomes in children unless splenectomy is performed.

The rarity of the condition and the variability of presentation make diagnostic delays common, and affected individuals may carry a diagnosis of unexplained anemia or may be misdiagnosed as having other causes of anemia. In a series of 61 patients with PK deficiency referred to a single center, the median age at diagnosis was 16 years (range, 1 day to 65 years). The frequency of findings in this group is as follows:
Anemia – 90 percent
Splenomegaly – 81 percent
Jaundice – 70 percent
Neonatal jaundice – 59 percent

The severity of the presented findings varies widely, from death in utero or shortly after birth due to hydrops fetalis, to transfusion-dependent anemia, to mild and compensatory hemolysis that does not require transfusion. The three main types of presentation are kernicterus/neonatal jaundice immediately after birth; anemia (during childhood or adulthood), which may be discovered incidentally, on the basis of a positive family history, or because of the characteristic symptoms of anemia; or complications such as iron overload caused by ineffective erythropoiesis and increased iron absorption, pigment gallstones, or transient anemia due to bone marrow insult (eg, in the setting of parvovirus infection). This presentation is discussed below.

Findings on a complete blood count (CBC) include normocytic anemia, an increased reticulocyte count, and the absence of specific red blood cell (RBC) morphological abnormalities on the peripheral blood smear. Other laboratory testing is consistent with Coombs-negative hemolytic anemia.
Unlike glucose-6-phosphate dehydrogenase (G6PD) deficiency, PK deficiency is not associated with increased susceptibility to hemolysis after exposure to oxidant agents.

Chronic hemolytic anemia from birth – The illustrative case involved a 19-year-old woman who had anemia (hemoglobin 7.5 g/dL), severe reticulocytosis (46 percent), and progressive liver failure. She had kernicterus as a neonate and was found to have hemolytic anemia without specific morphological red cell abnormalities on the peripheral blood smear. He needed blood transfusions every week. The diagnosis of PK deficiency is made on the basis of a typical presentation, the absence of a positive finding on testing for other inherited hemolytic anemias, and a positive test for PK deficiency on enzymatic testing performed by a referral laboratory.

Management included splenectomy at five months of age. No transfusion was given after splenectomy; However, he was mildly anemic (hemoglobin 7.5 to 9) g/dL) and had laboratory evidence of iron overload (ferritin >3000 ng/mL, transferrin saturation [TSAT] close to 100 percent). He was treated with iron chelation and sage phlebotomy (starting with a volume of about 50 mL and increasing in volume to 250 mL), and after two years of therapy, his liver function improved and normalized. During this time, his hemoglobin ranged from 8.2 to 9.4 g/dL and his iron markers improved (ferritin decreased to 420 ng/dL; transferrin saturation [TSAT] decreased to 62 percent). This case illustrates the combination of clinical features that can develop, their contribution to overall health, and the importance of treating iron overload.

Complications of chronic hemolysis – Some people with mild hemolysis due to PK deficiency may have no symptoms. Those with more severe hemolysis may present with (or develop) one or more of the following:
Pale
Icterus
Splenomegaly with varying degrees
Pigment (bilirubin) gallstones
Secondary folate deficiency due to increased demand
Skin boils

Hemolysis may worsen during pregnancy and after oral contraceptive use; the mechanism is unknown.
Anemia may worsen in the setting of transient bone marrow aplasia caused by infections such as parvovirus, which may not cause significant anemia in individuals without chronic hemolysis.
Individuals with gallstones requiring surgery should be evaluated for the need for splenectomy, as it may and/or benefit to perform both procedures simultaneously in certain cases.

Iron overload – Iron overload is less common than neonatal jaundice or chronic anemia as a finding that brings patients to medical care, but becomes more clinically apparent during adulthood. Causes include ineffective erythropoiesis, which leads to increased intestinal iron absorption, as well as transfusional iron overload. Similar to thalassemia, severe iron overload can occur in non-transfused patients as well.
Iron overload can appear in a number of ways depending on which organs are most affected. Examples include heart failure, liver disease, and endocrine dysfunction; this may be similar to the findings in individuals with other forms of iron overload.

Laboratory findings – PK deficiency results in chronic, Coombs-negative, non-spherocytic hemolytic anemia. The severity of hemolysis and the degree of anemia in PK deficient patients vary widely. In a series of 61 PK-deficient, non-splenectomized patients, the mean hemoglobin was 9.8 g/dL (range 2.2 to 14.4 g/dL).
Unlike G6PD deficiency, hemolysis is present all the time and is not precipitated by drug exposure.
The following findings are usually seen:
Complete blood count (CBC)

• Low hemoglobin and hematocrit
• Mean corpuscular volume is increased (MCV; increased due to reticulocytosis)
• Normal white blood cell count, differential white blood cell and platelet count
• Increased reticulocyte count (eg > 50 percent), especially if postpenectomy; This extreme reticulocytosis has not been observed in other hemolytic anemias
Peripheral blood smear
• Normal red blood cell morphology or nonspecific changes such as echinocytes (thorn cells), anisocytes, or poikilocytes

• Possibility of polychromasia associated with reticulocytosis
• Normal WBC and platelet morphology and abundance
Serum chemistry (findings consistent with non-immune hemolysis)
• Increased lactate dehydrogenase (LDH; variable)
• Increased indirect bilirubin
• Decreased haptoglobin (variable)
• Negative direct antiglobulin test (Coombs) (DAT)
• Indirect antiglobulin test negative

A rapid non-invasive method for assessing the degree of hemolysis based on measurements of exhaled carbon monoxide (eg, end-tidal carbon monoxide [ETCO]) has been described.
Echinocytes are highly variable and are not sensitive or specific for PK deficiency. Acanthocytes (spiculated RBCs) have also been reported in peripheral blood smears of Basenji dogs with PK deficiency.
Unlike congenital spherocytic hemolytic anemia, osmotic fragility of red blood cells is normal in PK deficiency. The autohemolysis test, in which hemolysis is evaluated in vitro in the presence or absence of additional glucose, is of no physiological relevance and should not be used.

DIAGNOSIS
Indications for testing – As noted above, late diagnosis of PK deficiency is common. Testing for PK deficiency is appropriate in the following settings:
Any sibling of a patient with PK deficiency who has unexplained anemia or unexplained hemolysis.
Any individual with suspected congenital hemolytic anemia who has a negative direct antiglobulin (Coombs) test and the absence of morphological abnormalities suggestive of another condition (eg, absence of profound microcytosis, sickle cell, spherocytes, basophilic determination, Heinz bodies). These findings and the conditions they suggest are briefly described below and in a separate topic review.
Prenatal testing should be offered to parents of children with hydrops fetalis or severe transfusion-dependent anemia not aborted by splenectomy.

Initial evaluation – The first step in the evaluation of a person with possible PK deficiency is to determine if hemolysis is present. This is done by measuring the reticulocyte count and serum markers of hemolysis such as lactate dehydrogenase (LDH), indirect bilirubin, or haptoglobin. Hemolytic anemia is characterized by an increase in the reticulocyte count, an increase in LDH, an increase in indirect bilirubin, and a decrease in haptoglobin. However, not all affected individuals are anemic, as some may compensate for hemolysis without anemia. In some cases, these people come to medical care when they develop an aplastic crisis (eg, in the setting of parvovirus infection) or when they present with hemolysis complications such as pigment gallstones.

The next step is to determine whether hemolysis is chronic and present at birth versus acquired, following a period of normal findings (absence of anemia and hemolysis). If hemolysis has been present throughout life, or if this information is inferred (e.g., due to family history) or cannot be verified, a peripheral blood smear is examined, with a focus on the morphology of red blood cells (RBCs) (e.g. schistocytes, elliptocytes, echinocytes, spherocytes, acanthocytes, sickle cells, RBC inclusions). This can eliminate the possibility of an inherited cause of hemolytic anemia with very characteristic findings on blood smears, such as hemoglobinopathies (eg, sickle cell disease, thalassemia) and membrane disorders (eg, hereditary spherocytosis, hereditary elliptocytosis). PK deficiency should be considered after these obvious alternative diagnoses have been eliminated.

In many cases, the presence of lifelong chronic anemia and characteristic red cell morphology strongly suggest the presence of a specific congenital abnormality of the red cell membrane, hemoglobinopathies, or enzyme deficiency, and specific diagnostic tests can be obtained. The rate of evaluation and whether tests are performed sequentially or simultaneously depends on the severity of the anemia, the patient’s symptoms, and other considerations such as the ease with which additional blood may be drawn and follow-up examinations.
If PK deficiency is suspected based on family history and/or initial evaluation, further testing may be performed using biochemical testing and/genetic testing, as discussed below.
Additional details of routine diagnostic testing that may be appropriate prior to testing for PK or PKLR activity of gene mutations are presented in a separate topic review.

PK-specific testing: Where and how to test – Testing for PK deficiency can be performed by measuring PK activity in red blood cells (biochemical testing) and/or by identifying gene-mutated PKLR pathogens (genetic testing).
We prefer biochemical testing where possible because it is the most direct evidence of functional PK deficiency. However, genetic testing may be appropriate in certain cases (eg, family members of individuals known to be affected for which variant pathogenic PKLR[s] have been identified). Another exception may be patients who have had a recent transfusion; The PK in the transfused red blood cells will have normal activity and can make the patient’s results appear normal. In the authors’ experience, two infants with PK deficiency who received monthly blood transfusions had PK enzyme activity only slightly below the normal range; However, after splenectomy and no transfusion for five months, PK activity was severely lacking.

Thus, biochemical testing should optimally be delayed for two to three months after the last transfusion; Unfortunately, this is not always possible in people who are very anemic, who need a transfusion. In patients requiring chronic transfusion, DNA testing (ie, PKLR gene sequencing) may be preferred.
Biochemical testing – The gold standard test for diagnosing PK deficiency is the assay of PK activity and estimation of enzyme kinetics of red blood cells free of white blood cell (WBC) and platelet contamination and the allosteric activator of PK activity, fructose 1,6-diphosphate (FDP).

Deletion of red blood cells and platelets is important because these cells express PK from a different PK gene (PKM) that is unaffected by the cause. PKLR gene mutation
FDP deletion is important for detecting mutants with altered allosteric interactions with FDP

White blood cells and platelets were removed by filtration by cellulose chromatography. FDP was removed by filtering the blood using a cellulose column and dialyzing the hemolysate; enzyme activity was measured in a filter, dialysate hemicate tested at different concentrations of the substrate phosphoenolpyruvate (PEP), with and without FDP. As noted above, transfused RBCs cannot be eliminated, and biochemical testing may be affected (eg, incorrectly normalized) by recent transfusions. In a series of 61 patients with PK deficiency, the median PK activity was 35 percent of normal.
However, the gold standard assay in which red blood cells, platelets, and FDP are removed is not available in commercial laboratories, and most research laboratories capable of performing these analyzes have been closed. The author is aware of Richard van Wijk’s laboratory at the University Medical Center Utrecht, the Netherlands that carried out this analysis. Mutant PK enzymes can also be analyzed via kinetic and electrophoretic studies of partially purified enzymes, although these are also not widely available.

An alternative to gold standard biochemical assays is to use rapid screening assays that are widely available in many commercial laboratories. In this assay, PK activity was measured in RBC hemolysis, without the initial steps of removing platelets, leukocytes, and FDP. This rapid test identifies most but not all patients with PK deficiency, and, if positive for PK deficiency, is sufficient to confirm the diagnosis. However, a negative test cannot be used to exclude the possibility of PK deficiency, because the contribution of WBC and PK platelets can make the PK activity of RBCs appear normal when they are not. A similar problem arises in patients who have recently received a red cell transfusion because PK activity in the transfused RBCs can cause the test to appear negative.

Apart from performing potentially less sensitive assays, routine commercial laboratories are usually unable to perform quantitative analyzes according to various concentrations of PEP substrates, which helps to screen for high Km (low affinity) mutants or to remove FDP, to detect mutations with altered FDP interactions.
Thus, if the screening test is negative and suspicion for PK deficiency is high (eg, due to Coombs-negative hemolytic anemia, negative testing for other common intrinsic RBC disorders, and/or a family history of PK deficiency), additional testing is required. The options are to use testing from specialized laboratories such as the one in Utrecht or to carry out genetic testing.
Of note, other biochemical assays such as measuring the level of the glycolytic intermediate 2,3-bisphosphoglycerate (2,3-BPG) were not used. Elevated 2,3-BPG is a common occurrence in PK deficiency but is nonspecific and highly variable.

Genetic testing – Genetic tests for PK deficiency are increasingly available, including testing performed by commercial laboratories and advanced genome sequencing methods. This assay is appropriate in circumstances where PK deficiency is suspected but initial biochemical testing is negative or borderline, and/or in families with known genetic defects. If the familial genetic variant is unknown, it may be necessary to sequence the entire gene, including all exons, flanking regions, and promoter PKLR.

Genetic testing is not usually used as an initial test because there are many genetic variants in PKLR, and their pathogenicity may not be obvious (i.e., an identifiable variant that has no clinical significance). In addition, some patients have large deletions and intronic mutations at cryptic splice sites that may not be detected by routine sequencing methods. It is important that the RBC PK ( PKLR ) gene and not the PKM gene be analyzed. In individuals with more than one PKLR mutation (possibly multiple heterozygotes), it is also important that a parent sample is obtained, to determine whether two mutations are present in cis (either from the same parent) or in trans (one from each parent).
Another benefit of genetic testing is that it can be used to identify familial variants, which in turn can facilitate testing of potentially affected family members, prenatal testing, and genetic counseling. Determination of causative mutations and their detection in parents is very important for determining the specific method of prenatal diagnosis used.
Diagnostic confirmation – The diagnosis of PK deficiency is confirmed in patients with hemolytic anemia (or compensated hemolysis) who have laboratory evidence of decreased RBC PK enzyme activity. and/or genetic evidence of pathogenic PKLR mutations.
This may include one or more of the following laboratory findings:
Low levels of RBC PK enzyme activity, either on rapid screening tests or on more sophisticated laboratory tests
Homozygosity or compound heterozygosity for missense or deletional PKLR mutations or other PKLR variants that are expected to interfere with PK enzyme activity
This test is described in more detail above.
Since the severity of hemolysis can vary even among relatives with the same PKLR mutation, it is possible that some patients who are affected but are relatively asymptomatic are never diagnosed.
DIFFERENT DIAGNOSIS – The differential diagnosis of PK deficiency includes another intrinsic red blood cell (RBC) defect that presents as a congenital non-spherocytic hemolytic anemia. as well as some other congenital anemias and acquired causes of hemolysis.
Other congenital RBC enzyme disorders – The main conditions that may be similar to PK deficiency include congenital RBC enzyme deficiency. Examples include glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is relatively common but usually manifests with isolated episodes of hemolysis rather than chronic hemolytic anemia, and deficiencies of other glycolytic pathway enzymes, which are rare (e.g. glutathione synthase deficiency, associated). with Heinz’s body; pyrimidine-5′- nucleotidase-1 deficiency, associated with administration of basophilic RBCs; glucose-6-phosphate isomerase deficiency). Evaluation and diagnosis for these other conditions are discussed separately. (
• Like PK deficiency, it is associated with chronic, non-immune hemolytic anemia (and associated symptoms and laboratory findings). Unlike PK deficiency, in G6PD deficiency, hemolysis may be intermittent and/or exacerbated by exposure to oxidant drugs or other substances.
• Unlike PK deficiency, this other condition has normal RBC PK activity.
Hemoglobinopathies or red blood cell membrane disorders – Other inherited anemias include hemoglobinopathies (disorders due to hemoglobin mutations such as sickle cell disease [SCD] or thalassemia) and RBC membrane defects (eg, disorders due to mutations in membrane components such as hereditary spherocytosis [HS] or elliptocytosis hereditary [HE]). (
• Like PK deficiency, it causes chronic hemolytic anemia, and in some cases iron overload can occur.
• Unlike PK deficiency, in this disorder, red blood cells have the classic morphological features on the peripheral blood smear suggestive of the diagnosis (eg, sickle cells in SCD, microcytosis in thalassemia, spherocytes in HS, elliptocytes in HE). In this disorder, there are diagnostic findings on hemoglobin electrophoresis or other tests such as osmotic friability, and normal PK activity.

Congenital dyserythropoietic anemia – The congenital dyserythropoietic anemia (CDAs) are a group of rare inherited RBC disorders characterized by mutations that affect RBC development in the bone marrow.
• Like PK deficiency, this disorder causes chronic anemia, jaundice, and splenomegaly, and, later in life, iron overload may develop. Similar to PK deficiency, red blood cell morphology may be normal or may show specific abnormalities.
• Unlike PK deficiency, in CDA, the reticulocyte count is low and bone marrow shows various abnormalities in developing red cell precursor cells [51].
Acquired hemolysis – The causes of acquired hemolysis or hemolytic anemia are numerous and include intrinsic (intracorpuscular) RBC defects and extrinsic (extracorpuscular) defects.
• As with PK deficiency, the age of presentation and severity of anemia varies, the reticulocyte count is elevated, and the peripheral blood smear may not reveal (eg, with normal RBC morphology or nonspecific changes); Occasional spherocytes may be present.
• Unlike PK deficiency, in acquired hemolysis and hemolytic anemia, the underlying condition or treatment can usually be identified from a medical history, medication list, or laboratory tests such as Coombs test or flow cytometry, which can reveal antibodies to red blood cells or other defects such as due to the absence of glycosylphosphatidyl linatiolositol (GPI)-containing proteins.
Some of these conditions and their sites of red cell destruction (ie, intravascular or extravascular) are listed in the table.

TREATMENT
Management review – Treatment of PK deficiency depends on the age at which the abnormality becomes apparent.
Before birth – Fetal hydrops due to severe anemia may require intrauterine transfusion.
Neonatal period – Hyperbilirubinemia during the neonatal period may require phototherapy or exchange transfusion.

Infants to adults – There is no medical treatment for anemia in PK deficiency, although small molecules are being investigated. Severe anemia in infants, children, and adults may require one or more of the following:
• Red blood cell (RBC) transfusion
• Folic acid (See ‘Folic acid’ below)
• Splenectomy (see ‘Splenectomy’ below)
• Iron chelation (see ‘Prevention/treatment of iron overload’ below)
• Investigative therapies such as hematopoietic cell transplantation (HCT), gene therapy, or small molecule AG-348

As with hereditary hemolytic anemia, it is important to thoroughly evaluate other possible causes of anemia for individuals who have changes in symptoms, hemoglobin levels, or reticulocyte counts, rather than attributing these changes to an underlying disorder. For example, decreased hemoglobin and reticulocyte counts may be a sign of parvovirus infection, new macrocytosis may be a sign of vitamin B12 or folate deficiency, and new microcytosis may be a sign of iron deficiency. At a minimum, these people demand increased monitoring, and if changes do not complete within a reasonable timeframe, additional testing may be required, as discussed in a separate topic review.
As with any genetic condition, individuals with the potential to have children may benefit from prenatal genetic counseling.

Glucocorticoids are of no value in PK deficiency. Drugs with oxidant potential appear to be safe.
Typical monitoring schedule – Patients with PK deficiency are monitored during routine medical care for symptoms of anemia and require evaluation for any changes in symptoms. The minimal evaluation includes a history of recent medications, changes in symptoms, or new medical conditions; examination for proper growth and development, signs of anemia, and findings related to other causes of anemia; and laboratory tests with complete blood count (CBC) and reticulocyte count. Additional testing may be indicated depending on the details of the presentation. Study iron is monitored periodically with ferritin levels and additional testing as appropriate.
People who have a dramatically reduced reticulocyte count may develop bone marrow insults such as parvovirus infection. In most such cases, detection of a viral infection is sufficient without the need for a bone marrow examination. If a bone marrow examination is performed, testing for parvovirus should be included.
Interventions for anemia – Supportive care for anemia can include transfusions and folic acid. Splenectomy is generally reserved for severe cases.

Transfusion and phototherapy – Phototherapy with or without exchange transfusion is indicated for severe hyperbilirubinemia during the neonatal period.
Transfusions are generally given for symptoms. The hemoglobin level should not be the sole criterion, and the need for transfusion should be individualized. As noted above, red blood cells in individuals with PK deficiency have a right-shifted hemoglobin oxygen dissociation curve, meaning that they may be less symptomatic than other individuals with similar degrees of anemia; this is the reason that hemoglobin levels alone should not be used to guide transfusion. In a series of patients with PK deficiency referred to a single center, 38 of 59 (64 percent) received blood transfusions (median rate, 15; range, 1 to >100), and 19 (32 percent) were transfusion dependent during childhood. . or until they have a splenectomy.

For those receiving chronic transfusions, chelation therapy should be initiated early, before iron overload develops.
Folic acid – Increased red blood cell turnover in PK deficiency can lead to folate deficiency in those whose fruit and vegetable intake is inadequate, but regular folate administration is not necessary in those with adequate intake of fresh fruits and vegetables or on a diet that includes whole grains added grains. For individuals who give high marks for avoiding folate deficiency, which can lead to worsening anemia, taking daily folic acid (typical dose, 1 to 2 mg daily) is safe and inexpensive, and has essentially no side effects or contraindications.

Splenectomy – Splenectomy improves hemolytic anemia, but there are several risks, including surgical complications, possible sepsis due to encapsulated organisms, and an increased risk of venous thromboembolism (VTE). Thus, splenectomy is usually reserved for individuals with severe transfusion-dependent anemia, and the decision whether to perform a splenectomy should be made on a case-by-case basis. We generally increase the likelihood of splenectomy in all chronic transfusion dependent individuals; we also evaluated the possibility of splenectomy in individuals who did not require chronic transfusion but who had a significant reduction in activities of daily living due to anemia

In a series of patients with PK deficiency, 18 of 61 (30 percent) underwent splenectomy (11 before diagnosis and seven after diagnosis). The median hemoglobin level was 7.3 g/dL in those who underwent splenectomy; this increased by approximately 1.8 g/dL (range, 0.4 to 3.4 g/dL levels) after the procedure. In comparison, those not treated with splenectomy had a median hemoglobin level of 9.8 g/dL.
For additional decision support, it is reasonable to assume that one is likely to have the same benefits as other affected family members and to follow the guidelines for splenectomy used in the more common congenital hemolytic anemias such as hereditary spherocytosis.

For individuals requiring cholecystectomy for pigmentary gallstone disease, the option of concurrent splenectomy should be discussed, and if the patient is considering splenectomy, the option of combining the procedures should be strongly recommended as these procedures can sometimes be performed simultaneously using a laparoscopic technique. Likewise, any splenectomy candidate should be evaluated for gallstones to consider cholecystectomy at the same time.

For those who choose to have a splenectomy, especially children, we try to delay the procedure for as long as possible (eg, until after age three or after age six if possible). Young children undergoing splenectomy should be treated with penicillin until they reach three years of age. Evidence to guide the optimal age of splenectomy comes primarily from observational data in other conditions.

We also ensured to provide pre-splenectomy vaccination against encapsulated organisms, similar to individuals undergoing splenectomy for other haematological conditions such as immune thrombocytopenia (ITP).
Patients should be educated about the potential risks of serious/life-threatening infection and VTE and the need to seek immediate medical attention for fever or symptoms of VTE, as discussed in detail separately.

For those undergoing splenectomy, surgeon skills are important in preventing surgical complications and enabling the laparoscopic technique, which appears to have lower rates of morbidity and mortality in other conditions such as ITP. In some individuals, it is possible to perform a partial splenectomy. A single report demonstrated the failure of partial splenectomy (80 percent) to reduce the transfusion requirements of a four-year-old patient with PK deficiency. Six months later, he successfully underwent a total splenectomy and became transfusion independent.
The beneficial effects of splenectomy on hemolysis have been well documented. Usually, hemolysis and anemia improve but are not completely relieved. In severe cases, the transfusion requirement is generally, but not always, abolished. The results of splenectomy on other family members may also be the main guide in this regard. However, there is no reliable way to predict the success of splenectomy.

Prevention/treatment of iron overload – Individuals with PK deficiency are at risk of iron overload from frequent blood transfusions as well as from increased iron absorption due to ineffective erythropoiesis. Excess iron in turn can cause serious organ poisoning in the liver, heart, and endocrine organs.

Rarely, these symptoms of organ toxicity may be responsible for bringing the patient to medical care and eventually leading to a diagnosis of PK deficiency.
In some cases, clinically significant iron overload may develop even if no transfusion is given; some of these individuals were found to have concurrent hereditary hemochromatosis.

Thus, it is important to monitor iron overload and to institute iron chelation programs in people with early signs of iron overload or in those at greatest risk of iron overload (eg, those on chronic transfusion programs).
Details of iron chelation therapy including choice of chelating agent, dosage, monitoring of iron load, and monitoring of chelating agent side effects are discussed in detail separately.

Investigative therapy – The following therapies are being investigated for individuals with PK deficiency:
Hematopoietic cell transplantation – Allogeneic hematopoietic cell transplantation (HCT) is a potential option for patients with very severe disease who continue to require chronic transfusion after splenectomy; A case report describes a five-year-old boy with severe PK deficiency who was treated with allogeneic HCT from a sibling donor human leukocyte antigen (HLA)-c.

Gene therapy – Monogenic disorders such as PK deficiency are potentially amenable to gene therapy. In hematopoietic disorders, this may require gene transduction into autologous hematopoietic stem cells and autologous HCT. Preclinical studies have been reported in which the PKLR gene was transferred to various cell lines via retroviral transduction, and an HCT mouse model for PK deficiency has been developed. There are no clinical trials using gene therapy, but attempts to correct PK deficiency in mouse models using lentiviral vectors have been reported.

AG-348 – AG-348 is an orally available small molecule (quinolone sulfonamide) developed using biochemical assays for a compound that can allosterically activate PK enzymes in red blood cells, similar to fructose 1,6-diphosphate (FDP) but with more efficacy. In an in vitro study, AG-348 was shown to increase the activity of many PKLR mutants in red blood cells from affected patients, possibly by increasing the stability of the PK enzyme multimer. In a mouse model, this agent was shown to activate normal RBC PK enzymes and restore glycolytic pathway activity and normalize ATP and 2,3-bisphosphoglycerate (2,3-BPG) levels associated with some but not all PKLR mutants. Results of clinical trials with this compound (eg, DRIVE PK ) await.

Genetic counseling and pregnancy testing – As with all inherited conditions, individuals with PK deficiency may benefit from genetic counseling regarding an affected child’s risk. This counseling may be provided by a specialist who understands the risks and can obtain a thorough family history and assess possible relatives (eg, hematologist) or genetic counsellor. Individuals with heterozygosity for PKLR mutations (eg, children of affected individuals) who have the potential to have children may benefit from discussions of testing their partners.

Individuals who have severely affected children may wish to pursue in vitro fertilization with preimplantation genetic testing or use prenatal testing to determine if subsequent pregnancies are affected. The type of genetic analysis will depend on the specific PK mutation. If desired, intrauterine transfusion of a severely affected (eg, hydropic) fetus is possible.

OVERVIEW AND RECOMMENDATIONS
Pyruvate kinase (PK) deficiency is an autosomal recessive hemolytic anemia characterized by reduced red blood cell (RBC) isoform activity of the PK enzyme, which is encoded by the PKLR gene (PK in liver and red blood cells). Affected individuals are either homozygous for a single pathogenic variant or compound heterozygous for two different pathogenic variants. PK generates ATP molecules during glycolysis, and reduced ATP may contribute to hemolysis, although the exact mechanism for hemolysis remains unclear. PK-deficient RBCs exhibit increased oxygen delivery for the partial pressure of oxygen in the bloodstream; thus, anemia is better tolerated than other inherited hemolytic anemias.

PK deficiency is rare, with an estimated prevalence of <51 cases per million population. It has a worldwide distribution, but is more common among people of northern Europe and possibly of Chinese descent.
PK deficiency is a lifelong condition, but the symptoms are variable and there is a wide age range of presentation; diagnostic delays are common. The three main presentations are with neonatal jaundice, chronic non-immune hemolytic anemia, non-spherocytes, and, rarely, iron overload symptoms. The severity of anemia varies, and findings on the peripheral blood smear are nonspecific

Testing for PK deficiency is appropriate in any sibling of a patient with PK deficiency who has unexplained anemia, or individuals with Coombs-negative hemolytic anemia and the absence of morphological abnormalities suggestive of another condition (eg, no profound microcytosis, sickle cell, spherocytes, basophilic determination, Heinz bodies). Prenatal testing should be offered to parents who have a child with hydrops fetalis or severe transfusion-dependent anemia that is not aborted by splenectomy.

PK deficiency can be diagnosed by measuring decreased PK activity in red blood cells (biochemical testing) and/or by identifying homozygous or compound pathogenic PKLR gene mutations (genetic testing). We prefer biochemical testing where possible as this is the most direct evidence of functional PK deficiency; genetic testing may be appropriate in certain cases. The gold standard biochemical assay measures PK activity and estimates the enzyme kinetics of contaminated free red blood cells (WBC), platelets, and the allosteric activator of PK activity, fructose 1,6-diphosphate (FDP); However, the availability of this test is very limited. An alternative is to use rapid screening assays that are widely available in many commercial laboratories; this test makes sense with certain caveats
The differential diagnosis of PK deficiency includes other intrinsic RBC defects that present as congenital non-spherocytic hemolytic anemia, as well as other congenital anemias (eg, congenital dyserythropoietic anemia [CDA]) and acquired causes of hemolysis.

There is no medical treatment for anemia in PK deficiency, although small molecule (AG-348) is being investigated. Severe anemia in infants, children, and adults may require red blood cell transfusions (intermittent or chronic, based on symptoms and hemoglobin level), phototherapy, and/or supplemental folic acid. Splenectomy may be indicated in certain individuals with transfusion-dependent anemia (ideally, delayed until childhood and preceded by the indicated vaccination); cholecystectomy may be required for pigment gallstones; and iron chelation may be needed to prevent or treat iron overload. Genetic counseling may be appropriate, and preimplantation genetic diagnosis or prenatal testing may be offered to individuals of childbearing age or those who have had infants who have previously suffered greatly.

Triosephosphate Isomerase
Triosephosphate isomerase deficiency is a disorder characterized by a lack of red blood cells (anaemia), movement problems, increased susceptibility to infection, and muscle weakness that can affect respiratory and heart function. Anemia in this condition begins in infancy. Because anemia results from premature destruction of red blood cells (hemolysis), it is known as hemolytic anemia. Lack of red blood cells to carry oxygen throughout the body causes extreme fatigue (fatigue), pale skin (pallor), and shortness of breath.

When red blood cells are broken down, iron and a molecule called bilirubin are released; Individuals with triosephosphate isomerase deficiency have an excess of these substances circulating in the blood. Excess bilirubin in the blood causes jaundice, which is the yellow color of the skin and the whites of the eyes.
Movement problems usually become apparent by age 2 in people with triosephosphate isomerase deficiency. Movement problems are caused by disorders of motor neurons, which are specialized nerve cells in the brain and spinal cord that control muscle movement. This disorder causes muscle weakness and wasting (atrophy) and causes movement problems typical of triosephosphate isomerase deficiency, including involuntary muscle tension (dystonia), tremors, and poor muscle tone (hypotonia).

Affected individuals may also experience seizures. Weakness of other muscles, such as the heart (a condition known as cardiomyopathy) and the muscle that separates the stomach from the chest cavity (diaphragm) can also occur in triosephosphate isomerase deficiency. Weakness of the diaphragm can cause breathing problems and eventually lead to respiratory failure. Individuals with triosephosphate isomerase deficiency are at higher risk of developing infection because their white blood cells do not function properly.

The cells of the immune system normally recognize and attack foreign invaders, such as viruses and bacteria, to prevent infection. The most common infection in people with triosephosphate isomerase deficiency is a bacterial infection of the respiratory tract. People with triosephosphate isomerase deficiency often do not survive childhood because of respiratory failure. In some rare cases, affected people without severe nerve damage or muscle weakness have lived to adulthood. Frequency Triosephosphate isomerase deficiency is probably a rare condition; about 40 cases have been reported in the scientific literature

Genetic Changes Mutations in TPI1 cause triosephosphate isomerase deficiency genes. This gene provides instructions for making an enzyme called triosephosphate isomerase 1. This enzyme is involved in the critical energy production process known as glycolysis. During glycolysis, the simple sugar glucose is broken down to produce energy for cells. TPI1 gene mutations cause the production of enzymes or enzymes that are unstable to decrease activity. As a result, glycolysis is disrupted and the cell experiences a decrease in energy supply. Red blood cells depend solely on the breakdown of glucose for energy, and without functional glycolysis, red blood cells die earlier than normal. Cells with high energy demands, such as nerve cells in the brain, white blood cells, and heart Muscle (heart) cells are also susceptible to cell death due to reduced energy caused by impaired glycolysis. Nerve cells in parts of the brain involved in coordination of movements (cerebellum) are especially affected in people with triosephosphate isomerase deficiency. Death of red and white blood cells, nerve cells in the brain, and heart muscle cells causes signs and symptoms of triosephosphate isomerase deficiency.
Pattern of Inheritance This condition is inherited in an autosomal recessive pattern, which means that both copies of the gene in each cell have mutations. The parents of an individual with an autosomal recessive condition each carry one copy of the mutated gene, but they usually do not show signs and symptoms of the condition.

Other Names for This Condition:
• Phosphotriose is isomerase deficiency
• Hereditary nonspherocytic hemolytic anemia due to triomerphosphate isomerase deficiency
• DISADVANTAGES of TPI
• TPID
• Triomer phosphate isomerase . deficiency

Rare enzyme abnormalities:
1. Hexokinase
2. Phosphofructokinase
3. Glucose Phosphate Isomerase
4. Bisphosphoglycerate Mutase

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