Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells, affecting 400 million people worldwide.[1] Paul Carlson and colleagues first reported G6PD deficiency in 1956 while working on a patient previously identified as "primaquine sensitive."[2]
G6PD is an enzyme involved in the pentose monophosphate pathway. G6PD deficiency leads to free radical–mediated oxidative damage to red blood cells, which in turn causes hemolysis.[3] It is an X-linked disorder with high prevalence particularly in people of African, Asian, and Mediterranean descent. G6PD deficiency is polymorphic, with more than 400 variants.
Patients with G6PD-deficient alleles have selective advantage against severe malaria; hence, it is highly prevalent in populations where malaria is endemic.
The clinical presentation of glucose-6-phosphate dehydrogenase (G6PD) deficiency includes a spectrum of hemolytic anemia ranging from mild to severe hemolysis in response to oxidative stress. The likelihood of developing hemolysis and its severity depends on the level of the enzyme deficiency, which in turn depends on the G6PD variant.[4, 5]
Semi-quantitative tests
The fluorescent spot test is a direct test that measures the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) from nicotinamide adenine dinucleotide phosphate (NADP+); the test is positive if the blood spot fails to show fluorescence under ultraviolet light. It is rapid, simple, sensitive, and inexpensive.[6, 7, 8]
The methemoglobin reduction test is a rapid indirect test that measures the reduced methemoglobin levels produced after NADPH oxidation.[6]
The cytofluorimetric method is a cytochemical typing assay that provides a fluorometric readout of the classic methemoglobin reduction test at the level of an individual red blood cell.[7]
Quantitative test
Quantitative tests for G6PD activity are considered the criterion standard. The rate of NADPH generation is spectrophotometrically measured at a wavelength of 340 nm. The G6PD activity is finally expressed as G6PD IU/red blood cell and G6PD IU/hemoglobin ratios.[6, 7, 8]
Most individuals with G6PD deficiency do not require any treatment. Acute hemolytic anemia in G6PD-deficient patients is largely preventable by avoiding exposure to fava beans, drugs, and chemicals that can cause oxidant stress. Identification and discontinuation of the precipitating agent is critical in the management of hemolysis in patients with G6PD deficiency.
Anemia secondary to mild to moderate hemolysis in G6PD deficient patients is usually self-limited and often resolves in 8-14 days. Transfusion is rarely needed in cases of severe anemia.
Infants with prolonged neonatal jaundice as a result of G6PD deficiency should receive phototherapy. Exchange transfusion may be necessary in cases of severe neonatal jaundice or hemolytic anemia caused by favism.
Systematic assessment for the risk of severe hyperbilirubinemia should be performed before discharge in neonates in whom G6PD deficiency is suspected to provide early and focused follow-up to prevent bilirubin encephalopathy.[9, 10, 11]
Persons with chronic hemolysis or nonspherocytic anemia should be placed on daily folic acid supplements. Consultations with a hematologist are ideal for long-term follow up.
The G6PD enzyme catalyzes the oxidation of glucose-6-phosphate and the reduction of nicotinamide adenine dinucleotide phosphate (NADP+) to nicotinamide adenine dinucleotide phosphate (NADPH) in the pentose monophosphate shunt. NADPH is important in maintaining glutathione in its reduced form, which protects the red blood cell against oxidative stress.
Red blood cells carry oxygen and hence are more susceptible to oxidative stress than other cells. The pentose monophosphate shunt is the only means of NADPH generation in red blood cells and therefore crucial in protecting red cells against oxidative damage.
In a G6PD deficient patient, oxidative stresses can denature hemoglobin and cause intravascular hemolysis.
Drugs, chemical agents, infections, ingestion of fava beans, or ketoacidosis can trigger oxidative stress leading to hemolysis.
Jaundice in G6PD-deficient neonates is considered to be due to an imbalance between the production and conjugation of bilirubin, with a tendency towards inefficient bilirubin conjugation. Premature infants are at special risk of the bilirubin production-conjugation imbalance.
G6PD deficiency is prevalent worldwide. In the United States, African Americans are primarily affected, with a prevalence of about 10%; however it is also seen among Italians (especially Sardinian ancestry), Greeks, Turks, South East Asians, people of Asian ancestry, and Sephardic Jews.[11]
Internationally, the geographic prevalence of the disorder correlates with the distribution of malaria. The highest prevalence rates (with gene frequencies from 5-25%) are found in the following regions:
The heterogeneity of polymorphic G6PD variants is proof of their independent origin, and it supports the notion that they have been selected by a common environmental agent, in keeping with the concept of convergent evolution.
G6PD deficiency affects all races, although the severity of G6PD deficiency varies significantly among racial groups. The highest prevalence is among the people of African, Asian, or Mediterranean descent. Variants producing severe deficiency primarily occur in the Mediterranean population. African populations have milder hemolysis due to higher enzyme levels.
G6PD deficiency is an X-linked inherited disease that primarily affects men. Women may be affected if they are homozygous, which occurs in populations in which the frequency of G6PD deficiency is quite high. Heterozygous women (carriers) can experience clinical disease as a result of X chromosome inactivation, gene mosaicism, or hemizygosity.
Many people with G6PD deficiency are asymptomatic. However, case reports of acute massive hemolysis with jaundice have been reported especially in the neonatal period, leading to kernicterus and fatality.[12, 13, 14, 15, 16]
Kernicterus or bilirubin encephalopathy is a rare complication of neonatal jaundice complicated by G6PD deficiency. Kernicterus, although infrequent, has about 10% mortality and 70% long-term morbidity usually evident in infants with a bilirubin level higher than 20 mg/dL.[9]
Massive hemolysis complicating G6PD deficiency has also been reported in patients with hepatitis infections, specifically hepatitis A and E in the Indian subcontinent.[17]
A literature review by Lai et al suggested that G6PD deficiency is a risk factor for diabetes, with the risk being greater in men than in women (odds ratio of 2.22 vs 1.87, respectively).[18]
A study by Rostami-Far et al indicated that G6PD deficiency increases the likelihood of neonatal sepsis. The study involved 76 neonates with sepsis and 1214 without sepsis, with the prevalence of G6PD deficiency being significantly greater in the sepsis group than in the controls.[19]
The X linked pattern of inheritance of G6PD deficiency and its clinical severity should be discussed with parents and counseling with regard to their risk for having other children should be provided, especially in populations in which G6PD deficiency is highly prevalent.[10]
If a mother is a heterozygote, the chances of recurrence is 50% with every subsequent male pregnancy.[20]
Parental-child G6PD deficiency self-care discussions are associated with better child health, and parental involvement in these discussions is facilitated by the thoroughness and clarity of patient education received from provider.[10]
Additional resources are available at G6PD Deficiency Association - Favism.
The majority of people with G6PD deficiency may remain clinically asymptomatic. However, they can present with exacerbated neonatal jaundice or with episodes of acute hemolytic anemia following exposure to an oxidative agent or with chronic non-spherocytic hemolytic anemia.[4, 21, 12, 13, 14, 15, 16]
Neonatal jaundice/hyperbilirubinemia
G6PD deficiency is one of the major risk factors for severe neonatal jaundice.[9] Jaundice usually appears within first 24 hours of life, usually earlier than physiologic jaundice but later compared to jaundice seen in blood group alloimmunization.
Jaundice can be very severe in some G6PD-deficient babies, especially in association with prematurity, infection, and/or environmental factors (such as naphthalene-camphor balls used in babies' bedding and clothing). Coexistence of a mutation in the uridyl transferase gene (UGT1A1; the same mutations are associated with the Gilbert syndrome) can also exacerbate neonatal jaundice.[22]
Hazardous hyperbilirubinemia defined as a total serum bilirubin greater than 30 mg/dL is a rare event, occurring in 5 per 100 000 live births after universal bilirubin screening. G6PD deficiency is the leading cause of hazardous hyperbilirubinemia when an etiology is identified.[23] A retrospective study evaluating neonates readmitted to the hospital for hyperbilirubinemia indicated G6PD deficiency to be the most frequent and severe risk factor for hyperbilirubinemia in regions where prevalence of the deficiency is high.[24]
Some G6PD-deficient neonates, if undiagnosed soon after birth, could present later in the first week of life with generalized jaundice, poor feeding, lethargy, breathing difficulty, or seizures. If inadequately managed, neonatal jaundice associated with G6PD deficiency can produce kernicterus or bilirubin encephalopathy and permanent neurologic damage.[12, 13, 14, 15, 16, 22]
Acute hemolytic anemia
Acute episodic hemolytic anemia occurs on exposure to oxidant stress like certain medications, chemicals, infections, ketoacidosis, or after ingestion of fava beans. Hemolysis usually begins 24-72 hours after exposure to oxidant stress and in cases of severe hemolysis, patients present with malaise, irritability, weakness, jaundice, tachycardia due to moderate to severe anemia, and often dark urine (cola- or tea-colored) due to hemoglobinuria usually within 6-24 hours. The onset can be extremely abrupt, especially with favism in children.
Acute hemolysis is usually self-limited and resolves within 8-14 days due to the compensatory production of young red blood cells, which have high levels of G6PD enzyme. Young red blood cells are not vulnerable to oxidative damage and, hence, limit the duration of hemolysis. Acute renal failure is a rare complication of acute hemolytic anemia in adults.[4, 22]
Chronic nonspherocytic hemolytic anemia (CNSHA)
A small percentage of G6PD-deficient patients have chronic nonspherocytic hemolytic anemia (CNSHA) of variable severity. G6PD Brighton, G6PD Harilaou, and G6PD Serres are included in this category.[1, 22, 25]
The patient is usually a male with a history of neonatal jaundice who may present with anemia, unexplained jaundice, or gallstones later in life. Although they have chronic hemolysis, they are also vulnerable to acute oxidative damage on exposure to an oxidative agent.[22]
Jaundice, pallor, and splenomegaly may be present in patients with severe hemolysis. Patients may have right upper quadrant tenderness due to hyperbilirubinemia and cholelithiasis.
G6PD deficiency is an X-linked recessive enzymopathy caused by a missense mutation in the housekeeping G6PD gene.[26] The pattern of inheritance is similar to that of hemophilia and color blindness: males usually manifest the abnormality and females are carriers. Females can be symptomatic if they are homozygous or if their normal X chromosome is inactivated.
The G6PD gene is located in the distal long arm of the X chromosome at the Xq28 locus. More than 160 mutations in the G6PD gene (OMIM #305900) have been reported.[26] Most are single-base changes that result in an amino acid substitution. These substitutions affect enzyme activity by decreasing intracellular stability of the protein or by affecting their catalytic activity.[20, 27, 21]
A large deletion in the G6PD gene is incompatible with life. Although small deletion mutation is rare, it has been reported and presents with severe G6PD deficiency.[21]
Specific G6PD alleles are associated with G6PD variants with different enzyme levels and, thus, different degrees of clinical disease severity. The variation in G6PD levels accounts for differences in sensitivity to oxidants.
The most common G6PD variants includes G6PD A-, G6PD Mediterranean, G6PD Canton, and G6PD Union.[21]
G6PD A- occurs in high frequency in Africa, Southern Europe, and North and South America. It is associated with lower enzyme levels and acute intermittent hemolysis.[4, 21, 28, 22]
G6PD Mediterranean is seen mainly in the Middle East, including Israel, and it accounts for almost all G6PD deficiency in Kurdish Jews, India, and Indonesia.[4, 21, 29, 30, 31, 32, 33, 34, 35, 28, 22] It is characterized by enzyme deficiency that is more severe than G6PD A- alleles. Hemolysis after ingestion of fava beans (Favism) is most frequently associated with the Mediterranean variant of G6PD deficiency.
G6PD Canton is seen mainly in China and G6PD Union is seen worldwide.
G6PD B is the wild type of allele (normal variant). The G6PD A+ variant is associated with high enzyme levels and, hence, no hemolysis.
In addition, severe forms of G6PD deficiency are associated with chronic nonspherocytic hemolytic anemia. Mutations causing severe chronic non-spherocytic hemolytic anemia commonly cluster in Exon 10, a region important for protein dimerization.[21, 15]
The World Health Organization has classified the different G6PD variants according to the degree of enzyme deficiency and severity of hemolysis, into classes I-V:[36]
Indications for testing for glucose-6-phosphatase dehydrogenase (G6PD) deficiency include the following:
Tests to diagnose hemolysis include the following:
Tests to rule out other causes of hemolysis and hemoglobinuria include the following:
Complete blood cell count will show mild to severe anemia depending on the G6PD variant and the type of oxidant stress. Increase in reticulocyte count represents bone marrow response to hemolysis by producing young red cells. Increase in indirect serum bilirubin and LDH indicate hemolysis. Low or absent haptoglobin levels, hemoglobinemia, hemoglobinuria, and presence of urinary hemosiderin indicate severe intravascular hemolysis, which is the main contributor to pathophysiology and diagnosis of G6PD deficiency. A part of hemolysis can be extracellular where damaged red cells are recognized as abnormal and undergo extravascular hemolysis by reticulo-enothelial system.[20]
On the peripheral smear, routine staining may reveal polychromasia, representing increased red blood cell production. Another typical feature is the presence of “hemighosts,” red cells that appear to have unevenly distributed hemoglobin, and “bite cells” or “blister cells,” red cells that appear to have a portion of them bitten away. Blister cells are characteristic of acute hemolysis induced by oxidative stress.[22]
Denatured hemoglobin can be visualized as Heinz bodies in peripheral blood smears processed with supravital staining. Heinz bodies are shown in the figure below.
Heat stability and/or heat denaturation and high-performance liquid chromatography can be used to identify unstable hemoglobin and thereby rule out G6PD deficiency.
Semi-quantitative tests:
Quantitative test:
A study by Peters et al indicated that in the detection of heterozygously G6PD-deficient females, spectrophotometry, cytofluorometry, and chromate inhibition have a sensitivity of 0.52, 0.85, and 0.96, respectively, and a specificity of 1.00, 0.88, and 0.98, respectively. The investigators stated that although routine means of assessing total G6PD activity can miss heterozygously G6PD-deficient females in whom a larger percentage of red blood cells is G6PD-sufficient, chromate inhibition and cytofluorometry can detect most of these cases.[37]
A semi-quantitative test is usually indicated in patients with a suggestive family history or in geographical areas with a high prevalence of the disorder. Positive screening results should be confirmed by quantitative tests. Diagnosis of G6PD may be difficult in females, who may be hemizygous or have skewed X chromosome inactivation or G6PD gene mosaicism.
G6PD activity is higher in premature infants than in term infants. This should be considered when testing for G6PD deficiency in infants.
Abdominal ultrasound may be useful in assessing for splenomegaly and gallstones. These complications are typically limited to patients with severe chronic hemolysis.
Genetic testing consists of DNA-based genotyping and sequencing, which helps in the identification of hundreds of mutations associated with G6PD deficiency worldwide, including many region-specific common variants. The molecular analysis may be useful for population screening, family studies, females, and prenatal diagnosis.
Most individuals with G6PD deficiency do not require any treatment.
Acute hemolytic anemia in G6PD-deficient patients is largely preventable by avoiding exposure to fava beans, drugs, and chemicals that can cause oxidant stress. Identification and discontinuation of the precipitating agent is critical in management of hemolysis in patients with G6PD deficiency.
Anemia secondary to mild to moderate hemolysis in G6PD deficient patients is usually self-limited and often resolves in 8-14 days. Transfusion is rarely needed in cases of severe anemia.
Infants with prolonged neonatal jaundice as a result of G6PD deficiency should receive phototherapy. Exchange transfusion may be necessary in cases of severe neonatal jaundice or hemolytic anemia caused by favism.
Systematic assessment for the risk of severe hyperbilirubinemia should be performed before discharge in neonates in whom G6PD deficiency is suspected to provide early and focused follow-up to prevent bilirubin encephalopathy.[9, 10, 11]
Persons with chronic hemolysis or nonspherocytic anemia should be placed on daily folic acid supplements. Consultations with a hematologist are ideal for long-term follow up.
Vaccination against hepatitis A and B is recommended in communities with high prevalence of G6PD deficiency.[38]
Transcriptional upregulation of G6PD enzyme in response to HDACi (histone deacetylase inhibitors) in in-vitro experiments on human B cells and erythroid precursor cells has been reported by Makarona K et al, which opens new areas of potential treatment in future.[25]
There is no evidence of selective red cell destruction in the spleen; hence splenectomy is usually ineffective and not recommended.
Consultations with a hematologist are ideal for long-term follow up of those with chronic hemolysis or nonspherocytic anemia.