Hematology

Glucose-6-phosphate dehydrogenase deficiency

Glucose-6-phosphate dehydrogenase deficiency

What every physician needs to know:

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary human enzyme deficiency, affecting more than 400 million people of predominantly African, Southeast Asian, Middle Eastern and Mediterranean descent and presents as neonatal jaundice and acute hemolytic anemia in its most severe forms.

G6PD is an enzyme which catalyzes the first reaction in the pentose pathway and supplies red blood cells (RBCs) with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate), which is essential for management of oxidative stress. In the presence of oxidating agents, such as certain drugs, fava beans or infection, G6PD deficient RBCs are unable to manage the overwhelming oxidative stress, which results in their quick demise. Patients present with signs and symptoms of neonatal jaundice or hemolytic anemia, which are usually self-resolving and do not require treatment, except for their most severe forms.

The gene encoding G6PD enzyme resides on the long arm of chromosome X, and the disease is inherited in an X-linked fashion, affecting all males carrying and females homozygous for G6PD mutation(s). Females heterozygous for one of G6PD mutations can also be affected, as a result of genetic mosaicism due to X-chromosome inactivation, and their clinical manifestations can range from minimal to severe. There are about 400 phenotypic variants and 140 G6PD mutations identified to date. Both phenotypic and genotypic classifications have been established. The most commonly used classification was developed by The World Health Organisation (WHO) in 1971 and is based on a combination of the level of enzyme activity and clinical manifestation, see Table I.

Table I

Classification of G6PD deficiency
  % Residual activity Clinical manifestations Notes
Class I Severely deficient (1-10%) Chronic non-spherocytic anemia Very rare. More likely to develop neonatal hyperbilirubinemia. Most sensitive to oxidating agents.
Class II Severely deficient (1-10%) More severe episodic acute hemolytic anemia G6PD Mediterranean, G6PD Canton, severe oriental variants
Class III Moderately deficient (10-60%) Less severe episodic acute hemolytic anemia G6PD A- African, G6PD Betica, G6PD Matera variants
Class IV Normal activity (60-150%) No clinical significance G6PD A+ variant, G6PD B (wild type)
Class V Increased activity (greater than 150%) No clinical significance  

Are you sure your patient has glucose-6-phosphate dehydrogenase deficiency? What should you expect to find?

Most patients with G6PD deficiency, including those carrying one of the Class II and III variants, are asymptomatic at steady state.

Symptomatic individuals present with at least one of the following:

  • An episode of neonatal jaundice

  • Hemolytic anemia in a setting of an infection, new drug or chemical exposure, ingestion of fava beans, or an episode of diabetic ketoacidosis

  • Chronic non-spherocytic hemolytic anemia (Class I variants).

Neonatal jaundice due to G6PD deficiency differs from the classic Rhesus (Rh)-related jaundice in its time of onset (peak incidence between day 2 and 3) and severity of jaundice, which outweighs the severity of anemia. Children or adults usually present with symptoms of fatigue, back pain, jaundice and shortness of breath, and are found to have evidence of hemolytic anemia (as described below). Some patients are initially diagnosed with methemoglobinemia, since oxidative injury to hemoglobin can initially result in formation of methemoglobin, which is then subsequently turned into sulfhemoglobin and denatured hemoglobin, evident on a peripheral blood smear, stained with a supra-vital dye such as brilliant cresyl blue, as red cell inclusions (called Heinz Bodies [Figure 1])

Figure 1

Heinz Bodies (oxidized, denatured hemoglobin, precipitated out of solution into dense intracellular granules, in a patient with G6PD deficiency The Heinz Bodies are revealed using a supra-vital stain, brilliant cresyl blue (courtesy of Dr Samuel Lux)

Beware of other conditions that can mimic glucose-6-phosphate dehydrogenase deficiency:

Other conditions that can mimic G6PD:

  • Hemoglobin H disease or other unstable hemoglobinopathies

- For example, hemoglobin Koln, Philly, Genova.

  • Autoimmune hemolytic anemia

  • Medication induced hemolytic anemia, in the absence of G6PD deficiency

- For example, due to dapsone.

  • Congenital deficiency of RBC glutathione, such as due to glutathione synthetase deficiency, or other RBC enzymes, such as pyruvate kinase deficiency

  • Other hereditary and acquired causes of nonimmune hemolytic anemia, including intra- and extracorpuscular causes of hemolysis

  • Hepato biliary diseases

- Such as Wilson's disease (can be associated with jaundice and hemoglobinuria; in a patient less than 40 years old, look for other associated symptoms, such as Kayser-Fleischer rings, neurologic symptoms, parkinsonism and movement disorder.

  • Paroxysmal nocturnal hemoglobinuria (PNH)

  • Hemolytic-uremic syndrome (HUS)/thrombotic thrombocytopenic purpura (TTP) - look for thrombocytopenia

- If enterohemorrhagic Escherichia colidriven HUS, look for abdominal pain and diarrhea.

Which individuals are most at risk for developing glucose-6-phosphate dehydrogenase deficiency:

Gender

Given the X-linked nature of the G6PD trait, males are more affected than females. All males carrying a G6PD mutation are hemizygous for the mutation, and can exhibit the full range of signs and symptoms, depending on the enzymatic activity of the particular variant they inherited. Similarly, females homozygous for a G6PD mutation can display the full range of signs and symptoms similar to hemizygous males. Females heterozygous for a G6PD mutation can be affected as a result of genetic mosaicism due to X-chromosome inactivation, and their clinical manifestations can range from minimal to severe.

Race

Patients of African, Southeast Asian, Middle Eastern and Mediterranean descent, areas in which malaria was once endemic, are more likely to carry one of the G6PD variants. This geographic distribution is thought to be the result of the historic anti-malarial protection afforded by RBC G6PD deficiency, whereas G6PD deficiency per se, does not block malaria parasites from infecting red cells, the subsequent intra-erythrocyte life cycle and growth of the plasmodia is impaired secondary to limiting levels of ribose derivatives, in the setting of G6PD deficiency.

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

Initial work-up should include:

  • Complete blood count (CBC) with differential and reticulocyte count to confirm and assess extent of anemia, as well as proliferative activity of the bone marrow

- Elevated reticulocyte count will reflect erythroid hyperplasia of an otherwise normal bone marrow, although it may lag in time in a setting of an acute hemolytic episode (acute favism).

  • Comprehensive metabolic panel (CMP) including direct and indirect bilirubin

- Expect to see high indirect bilirubinemia with hemoglobin degradation; may also see evidence of acute renal failure, due to acute tubular necrosis (ATN) secondary to renal ischemia or tubular obstruction by hemoglobin casts, and free plasma hemoglobin in acute favism.

  • Lactate dehydrogenase (LDH)

- Expect to be elevated in the presence of hemolysis.

  • Haptoglobin

- Low in the setting of active hemolysis.

  • Review of the peripheral blood smear

- Typically notable for bite cells and blister cells (resulting from destruction and asymmetric distribution of hemoglobin within the red cell), as well as presence of Heinz Bodies, visible with supra-vital stained blood (see Figure 1 and Figure 2) as dark precipitates of denatured hemoglobin. Evidence of reticulocytes may also be seen with supra-vital stained blood smears.

  • Direct Coombs test (DAT)

- This is performed to exclude the presence of autoimmune hemolytic anemia, which, if positive, would argue strongly against G6PD deficiency being the primary cause of hemolysis.

Whenever there is suspicion of G6PD deficiency, the work-up should include:

Measurement of the level of G6PD enzyme activity (using a quantitative spectrophotometric method): Note, a G6PD screening assay could be falsely normal/elevated if in a setting of an acute hemolytic episode, depending on the G6PD variant present in the patient. If this is a consideration, the screening assay should be repeated when the acute hemolytic event has resolved.

Figure 2

Peripheral blood smear of a child with G6PD deficiency (courtesy of Dr. Yana Pikman)

What imaging studies (if any) will be helpful in making or excluding the diagnosis of glucose-6-phosphate dehydrogenase deficiency?

No imaging studies are required to establish the diagnosis of G6PD deficiency. An abdominal ultrasound, consistent with splenomegaly and/or cholelithiasis may provide corroborative data for chronic hemolysis, though it is not diagnostic.

If you decide the patient has glucose-6-phosphate dehydrogenase deficiency, what therapies should you initiate immediately?

If G6PD deficiency is suspected in the setting of an acute hemolytic anemia, discontinue the suspected offending agent(s) immediately (see Table II).

Transfusions may be required, if severity of the hemolytic anemia exceeds the normal regenerative potential of the bone marrow. In the case of neonatal jaundice, phototherapy should be initiated immediately and bilirubin levels should be monitored closely.

Exchange transfusion may be indicated if level of hyperbilirubinemia is high (greater than 20 in the first 24 hours of life for a full term baby, lower thresholds in preterm babies) to reduce the risk of permanent neurologic damage (kernicterus). Use of prophylactic phenobarbital is advised against, based on studies showing no clinical benefit and adverse affects on cognitive development. If hemoglobinuria develops as a result of the acute hemolytic episode, adequate urine output should be maintained with the help of intravenous fluids.

Table II

Unsafe drugs in G6PD deficiency
Acetanilid
Acetylphenylhydrazine (2-Phenylacetohydrazine)
Aldesulfone sodium (sulfoxone)
Arsine
Beta-naphthol (2-Napthol)
Dapsone (diamino-diphenyl sulfone)
Dimercaprol
Furazolidone
Glucosulfone (glucosulfone sodium)
Menadiol sodium sulfate (vitamin K4 sodium bisulfite)
Menadione (menaphtone)
Menadione sodium bisulfite (vitamin K3 sodium bisulfite)
Methylthioninium chloride (methylene blue)
Napthtalene, pure (napthalin)
Niridazole
Nitrofural (nitrofurazone)
Nitrofurantoin
Pamaquine
Pentaquine
Phenylhydrazine
Primaquine
Probenecid
Stibophen
Sulfacetamide
Sulfadimidine
Sulfamethoxazole
Sulfanilamide (sulfanilamide)
Sulfapyridine
Sulfasalazine, salazosulfapyridine (Salazopyrin)
Tolonium chloride (toluidine blue)

More definitive therapies?

There is no definitive therapy for G6PD deficiency at this time. Avoidance of precipitating factors, such as medications listed in Table II, and common products, such as moth balls (napthalene), aniline dyes and henna compounds, as well as prompt treatment of infections and fever, are key.

What other therapies are helpful for reducing complications?

Supportive management and avoidance of oxidative factors are the mainstay of our current therapeutic approach to G6PD deficiency. All pregnant and nursing women who are heterozygous for G6PD, or are known to carry a G6PD deficient child, similarly to all G6PD deficient individuals, should avoid ingestion of any of the precipitating oxidative drugs or fava beans, since some of these chemicals or their metabolites can cross the placenta or be transmitted via breast milk.

Use of splenectomy and anti-oxidant agents, such as Vitamin E, have not been shown to have any benefit.

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

G6PD deficiency is rarely a life-threatening illness and does not result in decreased survival. Most acute episodes of hemolytic anemia are self-limited, and only the most severe cases require a transfusion or an exchange transfusion.

Avoidance of known hemolytic triggers and prompt treatment of infections, and control of fever, is important to prevent episodes of hemolytic anemia.

Individuals with an underlying liver problem, such as Gilbert's disease or viral hepatitis, may have an exaggerated elevation of serum bilirubin and a more complicated course.

All individuals carrying a G6PD mutations should be offered genetic counseling. The most up to date listing of safe and unsafe compounds can be found on the G6PD deficiency association website, listed in the references section.

"What if" scenarios.

If you strongly suspect G6PD deficiency in a patient with an acute hemolytic attack and the spectrophotometric assay is consistent with normal G6PD enzyme activity, this may be a false negative result (young RBCs from patients with certain G6PD variants have higher levels of G6PD enzyme than older cells, which are selectively destroyed during hemolysis) and the G6PD enzyme activity should be repeated again in 2 to 3 months. Alternatively, testing family members may provide corroborative evidence.

If patients with a known history of G6PD deficiency or a suspected diagnosis of G6PD deficiency develop methemoglobinemia, use of methylene blue is strongly contraindicated, since the oxidative properties of methylene blue will worsen hemolysis. If necessary, transfusion may be indicated in this case.

If faced with a strong clinical indication for continuing the administration of an oxidating agent, it can be done safely in certain variants of G6PD deficiency (for example, the milder, Class III forms, such as the G6PD A- variant) under close monitoring and with reduced dosing. Please refer to the G6PD deficiency website (listed under references) for the most up to date listing of safe and unsafe medications for different classes of G6PD deficiency.

If a simple or exchange transfusion is indicated for treatment of neonatal jaundice in an area where Class II G6PD variants are common, donor blood should be screened to exclude G6PD deficiency (in the donor blood), given high risk of complications.

Pathophysiology

G6PD is an enzyme that catalyzes the first step in the pentose phosphate shunt and provides RBCs with NADPH, need to curtail oxidative stress. The active form of G6PD is a dimer that contains tightly bound NADP. The gene encoding G6PD enzyme resides on the long arm of chromosome X, and the disease is inherited in an X-linked fashion, affecting all males carrying, and females homozygous for G6PD mutation(s). Females heterozygous for one of G6PD mutations can also be affected as a result of genetic mosaicism due to X-chromosome inactivation, and their clinical manifestations can range from minimal to more severe.

There are about 400 phenotypic variants and 140 G6PD mutations identified to date. Majority of the G6PD variants are due to missense point mutations.

Class I variants carry mutations in the G6P or NADP binding site of the enzyme. Class II variants, such as G6PD Mediterranean variant, have a markedly reduced catalytic activity and result in a formation of an unstable enzyme (half-life of a few hours versus 62 days in a wild type G6PD B variant). Class III variants, such as the G6PD A- variant, result in formation of an unstable enzyme (half-life of 13 days versus 62 days in a wild type G6PD B variant). Class IV and V variants result in formation of enzymes with a normal catalytic function, as well as a normal half-life, which is why they carry no phenotypic consequences for the affected individual.

The exact cellular mechanisms of hemolysis in G6PD deficiency are still not fully elucidated. It has been observed that hemolysis in patients carrying one of the Class III G6PD variants is restricted to older RBCs only. In contrast, both young and old RBCs of patients carrying one of the more severe Class II variants exhibit hemolysis in a setting of an appropriate oxidative exposure. The extravascular and intravascular hemolysis that ensues, is a result of oxidation of sulfhydryl groups on hemoglobin, formation of methemoglobin and denatured sulfhemoglobin, which alters the structure of RBCs and makes them more likely to be destroyed by the reticuloendothelial system in the spleen, liver and bone marrow.

What other clinical manifestations may help me to diagnose glucose-6-phosphate dehydrogenase deficiency?

Inquire about history of choledocholithiasis (pigment gallstones can be a result of a long-standing hemolytic anemia) as well as leg ulcers.

None of the following physical exam signs are diagnostic, but may support the diagnosis of anemia:

  • Splenomegaly

  • Early systolic (flow) murmur

  • Pale conjunctiva

  • Hyperkinetic apical pulse

What other additional laboratory studies may be ordered?

Routine screening for G6PD deficiency is not performed in the United States outside of the District of Columbia although many pediatricians will check for G6PD deficiency in their African American patients, or patients of Mediterranean descent. There are, however, a few case reports of severe complications resulting from simple and exchange transfusions using blood from G6PD-deficient donors, which makes screening donors a serious consideration in those areas of the world where G6PD deficiency is more prevalent.

What’s the evidence?

Beutler, E. "Glucose-6-phosphate dehydrogenase deficiency: a historical perspective". Blood. vol. 111. 2008. pp. 16-24.

[American Society of Hematology 50th anniversary historical review, encompassing the discovery of G6PD deficiency, its clinical manifestations, detection, population genetics, and molecular biology.]

Cappelini, MD,, Fiorelli, G. "Glucose-6-phosphate dehydrogenase deficiency". Lancet. vol. 371. 2008. pp. 64-74.

[Most recent comprehensive review of G6PD deficiency, including discussion of malaria protection hypothesis.]

Yoshida, A, Beutler, E, Motulsky, AG. "Human glucose-6-phosphate dehydrogenase variants". Bull World Health Organ. vol. 45. 1971. pp. 243-53.

[First classification of G6PD variants and their clinical features.]

. "WHO working group. Glucose-6-phosphate dehydrogenase deficiency.". Bull World Health Organ. vol. 67. 1989. pp. 601-11.

World Health Organization update on new developments in methodology of detection of G6PD in the community, as well as prevention of hemolytic episodes.]

Janssen, WJ. "Clinical problem-solving. Why "why" matters". NEJM. vol. 351. 2004. pp. 2429-34 .

[New England Journal of Medicine clinical problem solving article discussing a case report of a 38-year old woman with methemoglobinemia and G6PD deficiency, presenting with jaundice and shortness of breath.]

. "G6PD Deficiency Association". http://www.g6pd.org..

[Physician and patient resources, including most up to date listing of safe and unsafe drugs in G6PD deficiency.]
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