Red cell enzymopathies

What every physician needs to know:

Red cell enzymopathies can result in diverse phenotypes. Some are associated with chronic or intermittent hemolysis, others with cyanosis due to methemoglobinemia, and others with no discernable erythrocyte phenotype, but with multiple organ abnormalities such as seen in some glycogen storage diseases. Yet others have no disease phenotype at all.

The two most common red cell enzymopathies resulting in hemolysis, are glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase (PK) deficiency. Although The World Health Organisation (WHO) divides G6PD deficiency into 5 different categories, only types 1 and 2 are clinically significant.

Type 2 variants are by far the most common and are endemic in different parts of the world. The best studied are the African and Mediterranean variants. These are moderate to severe defects with less than 10% enzymatic activity. The affected individuals have no evidence of hemolysis, unless they are exposed to certain oxidant drugs or chemicals , ingest fava beans (typical for Mediterranean but not African variant), are infected with certain organisms, or have ketoacidosis.

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Type 1 G6PD deficiency is associated with chronic hemolytic anemia, but is extremely rare. PK deficiency is far less common than the variants causing G6PD deficiency but is the most common cause of chronic, clinically significant, hemolysis. Other red cell enzyme deficiencies causing hemolysis are very rare and include glucose phosphate isomerase deficiency, the second most common cause of chronic hemolytic anemia arising from red cell enzymopathies and 5′ nucleotidase deficiency, a condition associated with distinct red cell morphological abnormalities (basophilic stippling, triosephosphate isomerase, and hexokinase).

Type I cytochrome b5 reductase (b5R) deficiency (NADH methemoglobin reductase), in which the b5R deficiency is confined to red cells, causes chronic congenital asymptomatic cyanosis due to methemoglobinemia. At times, individuals heterozygous for the mutant gene may develop symptomatic acute methemoglobinemia on ingestion of certain drugs or exposure to some chemicals. The rarer form, type II b5R deficiency, has deficient enzyme activity in all cells and results in multiple organ defects, cyanosis, and typically death in infancy.

Are you sure your patient has a red cell enzymopathy? What should you expect to find?

The vast majority of G6PD deficient individuals are asymptomatic. Acute extravascular and rarely intravascular hemolysis develops in patients with type 2 deficiency after exposure to offending agents and is usually self limited and short lasting. If the offending agent is reintroduced within a few weeks, the hemolysis does not recur as the oldest erythrocytes with the lowest G6PD activity have been destroyed. However, if the offending agent is introduced after a longer lag time, approximately 2 months, hemolysis will recur.

Individuals with the extremely rare G6PD deficient mutants comprising type 1 deficiency have chronic hemolytic anemia, but the degree of hemolysis can further increase after exposure to offending agents.

PK deficiency causes chronic hemolytic anemia of variable severity, from mild and clinically insignificant, to severe transfusion requiring anemia, and, in rare occasions, is incompatible with life. It often presents in newborns with severe hyperbilirubinemia at times leading to kernicterus.

Individuals who are homozygous or compound heterozygous for deficient variants of b5R have most often asymptomatic congenital cyanosis due to methemoglobinemia (type I b5R deficiency). The hemoglobin level in some individuals is elevated, often leading to unnecessary work up for polycythemia.

Type II b5R deficiency is less common. It presents in neonates with cyanosis and failure to thrive with global neurological defects and affected individuals generally die in the first year of life. Heterozygous patients for deficient variants of b5R are asymptomatic but on exposure to certain drugs or chemicals can develop acute toxic methemoglobinemia, see chapter “Methemoglobinemia”.

Beware of other conditions that can mimic red cell enzymopathies:

Acute hemolytic anemia can also been seen in newly-diagnosed autoimmune hemolytic anemia and fragmentation hemolytic syndromes, in acute exacerbation of autoimmune hemolytic anemia, in patients with unstable hemoglobins (after ingestion of certain drugs and chemicals), and in hereditary spherocytosis (during infections).

Chronic hemolytic anemia may be caused by rarer red cell enzyme deficiencies such as glucose phosphate isomerase, 5′ nucleotidase, hexokinase, and others, and also occurs in patients with unstable hemoglobins or hereditary spherocytosis, as well as in various types of autoimmune hemolytic anemias.

The differential diagnosis of chronic cyanosis includes not only methemoglobinemia, but also diseases associated with desaturated hemoglobin and sulfhemoglobinemia.

Which individuals are most at risk for developing red cell enzymopathies:

G6PD deficiency is endemic in many parts of the world but sporadic and very rare in other parts of the world.
Both males and females may be affected. Males typically have more severe hemolysis than heterozygous females, unless the affected female is homozygous for the defect. Exposure to certain drugs, fava beans, and other oxidative substances may induce acute hemolytic episodes.

PK and b5R deficiency are both autosomal recessive disorders, more common in certain populations and ethnic groups, but sporadic worldwide. They are seen with higher frequency among children of consanguineous unions, where the affected individual is more likely homozygous for the mutation. In sporadic cases, the affected individual is more likely compound heterozygous for the mutated gene.

What laboratory studies should you order to help make the diagnosis and how should you interpret the results?

Laboratory tests for suspected hemolysis include complete blood count (CBC), reticulocyte count, total and indirect bilirubin. Lactate dehydrogenase (LDH), haptoglobin, urine hemoglobin, and urine hemosiderin are abnormal in intravascular hemolysis. It should be noted that in severe extravascular hemolysis, more sensitive measures of intravascular hemolysis such as LDH may also be abnormal.

Screening tests for G6PD deficiency are widely available, but do not discriminate among hundreds of possible G6PD variants and at times may miss the diagnosis in heterozygous females and in males shortly after a hemolytic episode. Specific DNA based screening tests are being developed and some are now available.

There are no specific or even consistent morphological abnormalities in G6PD deficiency, despite some reports to the contrary. During acute hemolytic episodes, Heinz bodies can be seen, but these are also seen in hemolysis due to some other enzyme deficiencies or unstable hemoglobins. Heinz bodies are more likely to be seen when a Heinz body preparation is performed.

Screening tests for PK deficiency will identify most of the deficient variants. However, since PK is an allosteric enzyme that has a binding site not only for its substrate, phosphoenolpyruvate, but also for its allosteric activator, fructose diphosphate, more sophisticated enzyme analyses are needed when a diagnosis of PK deficiency is suspected but not confirmed by the screening tests.

Cyanotic patients without a clear cut cause should be tested for an elevated methemoglobin level. In those patients with an autosomal dominant family history, globin mutants (hemoglobin M) should be sought for first, see chapter “Methemoglobinemia”. If a globin mutation is excluded, decreased red cell activity of b5R confirms the diagnosis of b5R deficiency. In cyanotic infants with methemoglobinemia, whose red cell b5R activity is decreased and who also have neurological or developmental abnormalities, b5R activity should also be evaluated in non-erythroid tissues, such as leukocytes, platelets, fibroblasts. If these tissues are also deficient in b5R, type II b5R deficiency is confirmed.

Isolated erythroid enzyme deficiency is indicative of type I b5R deficiency.

What imaging studies (if any) will be helpful in making or excluding the diagnosis of red cell enzymopathies?

No imaging studies are specific for red cell enzymopathies. However, evaluation for gallstones is recommended in patients with chronic hemolysis.

If you decide the patient has red cell enzymopathies, what therapies should you initiate immediately?

Supportive measures, such as red cell transfusion, should be initiated in patients with symptomatic anemia and G6PD, or PK deficiency.

In chronically cyanotic patients with methemoglobinemia due to b5R deficiency, no immediate therapy is required as these patients are not symptomatic from the methemoglobinemia. Acute toxic methemoglobinemia seen in some patients who are heterozygous for b5R mutants is usually symptomatic and at times, life threatening. The administration of methylene blue usually rapidly corrects the defect, but caution should be used in patients with concomitant G6PD deficiency as this agent can induce acute hemolytic crisis. In those patients for whom methylene blue cannot be administered, red cell exchange transfusions and/or hyperbaric chamber should be considered.

More definitive therapies?

In patients with PK deficiency who require red cell transfusions, splenectomy ameliorates but does not abolish hemolysis. Bone marrow transplantation may be considered in patients with life threatening or severely disabling PK deficiency .

In patients with type I b5R deficiency who are bothered by the cosmetic appearance of the cyanosis, chronic administration of methylene blue has been shown to be effective. Benefit from riboflavin and vitamin C have also been reported, but these agents are less well studied. Methylene blue is not effective in patients with congenital methemoglobinemia due to globin mutations (hemoglobin M).

In type II b5R deficiency, while methylene blue can correct cyanosis, it has no benefit on the neurological and other systemic complications.

What other therapies are helpful for reducing complications?

G6PD deficient patients should be counseled to avoid agents known to precipitate hemolysis.

In those rarely encountered patients with PK deficiency who have marginal folate levels, folate supplementation may be useful. Some patients with severe hemolysis from PK deficiency, with or without chronic transfusion therapy, may develop severe iron overload, which may require intervention such as chelating therapy. In some of these patients, administration of erythropoietin may increase hemoglobin levels that will permit treatment of iron overload by judicious phlebotomies.

What should you tell the patient and the family about prognosis?

Patients with chronic hemolytic anemia should be counseled about the possibility of acute aplastic crisis due to parvovirus infection, see chapter “Aplastic anemia”. The aplastic crisis may not be self limiting in some patients, in which case, intravenous immunoglobulin may be effective. The increased availability of parvovirus vaccination means this preventative therapy may be contemplated in some situations.

The prognosis in type I b5R deficiency is excellent. However, those patients with elevated hemoglobin levels should not be phlebotomized, since the elevated red cell mass is a physiologic response to tissue hypoxia. Unfortunately, the prognosis in type II b5R deficiency is abysmal. A theoretical benefit to liver transplantation has been raised but not properly evaluated. Bone marrow transplantation would not be expected to be of benefit.

What if scenarios.



G6PD deficiency

G6PD generates reduced nicotinamide adenine dinucleotide phosphate (NADP), which is essential for the regeneration of reduced glutathione (GSH) from oxidized glutathione (GSSG). This high GSH to GSSG ratio prevents the sulhydryl groups of many proteins, including hemoglobin, from forming disulfide bridges. When hemoglobin is subjected to oxidation, the oxidant-induced conformational changes of hemoglobin result in its decreased solubility. Since hemoglobin is present in red cells in almost saturated solution, this decreased solubility of hemoglobin leads to its precipitation in the form of Heinz bodies. This results in acute extravascular hemolysis.

In some instances, when hemolysis is particularly acute and often compounded by concomitant independent hemolytic stress, such as malarial infection, intravascular hemolysis with hemoglobinuria may be observed (blackwater fever). However, as the deficiency of G6PD in all endemic variants is relatively mild, there is usually sufficient generation of NADPH (reduced form of NADP) maintaining an almost normal GSH to GSSG ratio. Only with increased oxidative stress is the diminished G6PD activity insufficient to protect protein -SH groups from the decreased GSH to GSSG ratio and hemolysis ensues.

PK deficiency

PK enzymology is complex as there are two PK genes in different chromosomal locations. One, PK-M, is expressed in muscle and other non-erythroid tissues, but also in leukocytes. PK-M has two isoforms, M1 and M2. One of the PK-M products, M2, has been shown to be a potent regulator of hypoxia inducible transcription factor (HIF) 1 activity. Neonatal red cells have detectable PK-M enzyme that is later replaced by adult PK-R, which is a product of a different gene, PK-LR. The enzyme products of PK-LR gene are also not identical. The PK-L messenger RNA (mRNA) expressed in liver uses a different transcription initiation site than the PK-R mRNA expressed in erythroid cells.

The mechanism of hemolysis in PK deficiency is not known. However, reticulocytes and young red cells have been shown to be preferentially destroyed in PK deficiency, a phenomenon known as neocytolysis. Thus, after splenectomy, while hemolysis decreases and the hemoglobin level increases, there is a seemingly paradoxical increase in reticulocyte count. Whether neocytolysis is caused by disregulation of genes controlled by HIFs is under investigation.

b5R deficiency

During release of oxygen from oxyhemoglobin, superoxide, and other oxygen radicals are formed, resulting in autooxidation of ferrohemoglobin to ferrihemoglobin, that is, methemoglobin. Since methemoglobin cannot reversibly bind oxygen, the normal physiological response is a continuous reduction of methemoglobin to oxyhemoglobin by the b5R enzyme. When b5R activity is not sufficient, either because of its deficiency or because its activity is overwhelmed by certain drugs and chemicals, methemoglobin can still be efficiently reduced by the activation of another red cell enzyme, NADPH-methemoglobin reductase, which is normally not enzymatically active as it lacks an electron acceptor. Methylene blue and other compounds provide an extrinsinic electron acceptor, resulting in rapid reduction of methemoglobin.

What other clinical manifestations may help me to diagnose red cell enzymopathies?

G6PD deficiency is more often seen in African-American and Mediterranean males, and to a lesser degree in females. A personal or family history of hemolysis after exposure to offending agents may be elicited.

Consanguinity may be present in families of patients with PK or b5R deficiency.

Jaundice or scleral icterus may be present in acute hemolytic episodes. There are no specific physical findings in G6PD or PK deficiency.

Congenital cyanosis is noted in individuals with type I and type II b5R deficiency, and acute onset cyanosis may at times occur in acute toxic methemoglobinemia episodes in patients heterozygous for b5R mutants.

What other additional laboratory studies may be ordered?

For families with an affected child with type II b5R or severe PK deficiency, prenatal diagnosis may be considered for the next at risk pregnancy. However, accurate genotyping of the fetus generally requires knowledge of the exact mutation(s) of the gene in the previously affected child and preferably also confirmed in the parents.

What’s the evidence?

Agarwal, N, Prchal, JT, Kaushansky, K, Lichtman, MA, Kipps, TJ, Beutler, E, Seligsohn, U, Prchal, JT. “Methemoglobinemia and other causes of cyanosis”. Williams Hematology. 2010. pp. 743-755. [A thorough review of enzyme defects.]

Gregg, XT, Prchal, JT, Hoffman, R, Benz, E. “Red cell enzymopathies”. Hematology Basic Principles and Practice. 2009. pp. 611-623. [A comprehensive summary of the many enzyme defects with practical clinical advice.]

Luo, W, Hu, H, Chang, R. “Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypxia-inducible factor 1”. Cell. vol. 145. 2011. pp. 732-744. [Important study relating glucose metabolism to growth control.]

Maran, J, Guan, Y, Ou, CN, Prchal, JT. “Heterogeneity of the molecular biology of methemoglobinemia; a study of eight consecutive patients”. Haematologica. vol. 90. 2005. pp. 687-689. [Shows the range of clinical manifestations in inherited defects producing methemoglobinemia.]