G6PD Deficiency in the Newborn

Practice Essentials

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 treating 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 recessive disorder and thus more often affects males. G6PD deficiency has a high prevalence in people of African, Asian, and Mediterranean descent. The condition is polymorphic, with more than 400 variants.

Patients with G6PD-deficient alleles have a selective advantage against severe malaria; hence, G6PD deficiency is highly prevalent in populations where malaria is endemic.

Signs and symptoms of G6PD deficiency

Most patients with G6PD deficiency are asymptomatic. Neonatal jaundice may be seen in newborns.

Patients may experience episodes of intravascular hemolysis and consequent anemia, triggered by infections, medicines that induce oxidative stresses, fava beans, and ketoacidosis. Hemolysis begins 24-72 hours after exposure to oxidative stress. Patients with severe hemolysis present with weakness, tachycardia, jaundice, and hematuria.

The clinical presentation of 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 depend on the level of the enzyme deficiency, which in turn depends on the G6PD variant.^{[4, 5]}

Workup in G6PD deficiency

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 test 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 tests

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]}

Spectrophotometric quantitation may fail to detect deficiency in heterozygous females, due to residual activity in G6PD-sufficient cells. Regarding the identification of G6PD-deficient, as well as G6PD-sufficient, cells by a cytochemical method or cytofluorometry, these analyses are more sensitive in testing for G6PD deficiency in females.^[9]

Management in neonates

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.

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 kernicterus (bilirubin encephalopathy).^{[10, 11, 12]}  Consultations with a hematologist are ideal for long-term follow up.

See Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency for more information on management in adults.

• References

Pathophysiology

The G6PD enzyme is part of the pentose monophosphate shunt. It catalyzes the oxidation of glucose-6-phosphate and the reduction of nicotinamide adenine dinucleotide phosphate (NADP+) to nicotinamide adenine dinucleotide phosphate (NADPH). NADPH maintains glutathione in its reduced form, with glutathione acting as a scavenger for dangerous oxidative metabolites.

The pentose monophosphate shunt is the only source for NADPH in red blood cells. Therefore, red blood cells depend on G6PD activity to generate NADPH for protection. Consequently, red blood cells are more susceptible to oxidative stresses than other cells. In persons with G6PD deficiency, oxidative stresses can denature hemoglobin and cause intravascular hemolysis. Denatured hemoglobin can be visualized as Heinz bodies in peripheral blood smears processed with supravital staining. Heinz bodies are shown in the figure below.

View Image

G6PD deficiency: Heinz bodies in a peripheral smear stained with a supravital stain. Heinz bodies are denatured hemoglobin, which occurs in G6PD defic....

Drugs, chemical agents, infections, ingestion of fava beans, and 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 toward inefficient bilirubin conjugation. Premature infants are at special risk for the bilirubin production-conjugation imbalance.

• References

Etiology

G6PD deficiency is an X-linked recessive enzymopathy caused by a missense mutation in the housekeeping G6PD gene.^[13]  The pattern of inheritance is similar to that for hemophilia and color blindness; ie, 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. There have been 186 mutations documented in the G6PD gene. Most are single-base changes that result in an amino acid substitution.^[3] These substitutions affect enzyme activity by decreasing intracellular stability of the protein or by affecting their catalytic activity.^{[14, 15, 16]}

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.^[16]

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 include G6PD A-, G6PD Mediterranean, G6PD Canton, and G6PD Union.^[16]

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, 16, 17, 18]}

G6PD Mediterranean is seen mainly in the Middle East, including Israel, and it accounts for almost all G6PD deficiency in Kurdish Jews.^{[4, 16, 19, 20, 21, 22, 23, 24, 25, 17, 18]}  It is characterized by enzyme deficiency that is more severe than that associated with 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.

Severe forms of G6PD deficiency are associated with chronic nonspherocytic hemolytic anemia. Mutations causing this anemia commonly cluster in exon 10, a region important for protein dimerization.^{[16, 26]}

The World Health Organization has classified the different G6PD variants according to the degree of enzyme deficiency and severity of hemolysis, as follows:^[27]

• Class I - Severe enzyme deficiency, chronic nonspherocytic hemolysis
• Class II - Severe enzyme deficiency (1-10% residual activity), intermittent acute hemolysis
• Class III - Moderate enzyme deficiency (10-60% residual activity), intermittent acute hemolysis
• Class IV - No enzyme deficiency (60-150% activity)
• Class V - Increased enzyme activity (>150%)

• References

Epidemiology

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 those of Sardinian ancestry), Greeks, Turkish people, Southeast Asians, people of Asian ancestry, and Sephardic Jews.^[12]

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:

• Tropical Africa
• The Middle East
• Tropical and subtropical Asia
• Some areas of the Mediterranean
• Papua New Guinea

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.

Prevalences were addressed in a study by Koromina et al, which also indicated that the frequency of genetically encoded G6PD deficiency is greatest among Africans; in the investigators’ estimate, the prevalence is 12.4% in African males and 3.3-5.9% in females. The prevalence is also considered high in Asia, being 4.4% in South Asian males and 0.5-1.2% in East Asian females. In contrast, the prevalence in Finnish females is 0.02-0.04%, and in the Amish population (males and females), 0%.^[28]

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.

• References

Prognosis

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 fatalities.^{[29, 30, 31, 26, 32]}

Kernicterus, or bilirubin encephalopathy, is a rare complication of neonatal jaundice complicated by G6PD deficiency. Kernicterus, although infrequent, has about a 10% mortality rate and 70% long-term morbidity rate, usually evident in infants with a bilirubin level higher than 20 mg/dL.^[10]

Massive hemolysis complicating G6PD deficiency has been reported in patients with hepatitis infections, specifically hepatitis A and E in the Indian subcontinent.^[33]

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).^[34]

A study by Ripoli et al indicated that G6PD deficiency is related to HbA1c deficiency in children and adolescents with type 1 diabetes mellitus. Specifically, the investigators reported that in males who are hemizygous for the deficiency gene and females who are homozygous for it, there is an average 1.3% reduction in HbA1c. For females who are heterozygous for the gene, the average reduction is much smaller, at 0.3%.^[35]

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.^[36]

• References

Patient Education

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 with the condition should be provided, especially in populations in which G6PD deficiency is highly prevalent.^[11]

If a mother is heterozygous, the chance of recurrence is 50% with every subsequent male pregnancy.^[14]

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 the provider.^[11]

Additional resources are available at G6PD Deficiency Association - Favism.

• References

History

The majority of people with glucose-6-phosphatase dehydrogenase (G6PD) deficiency may remain clinically asymptomatic. However, neonates can present with exacerbated neonatal jaundice. Episodes of acute hemolytic anemia following exposure to an oxidative agent or with chronic nonspherocytic hemolytic anemia can occur.^{[4, 16, 29, 30, 31, 26, 32]}

Patients may experience episodes of intravascular hemolysis and consequent anemia, triggered by infections, medicines that induce oxidative stresses, fava beans, and ketoacidosis. Hemolysis begins 24-72 hours after exposure to oxidative stress. Patients with severe hemolysis present with weakness, tachycardia, jaundice, and hematuria.

The clinical presentation of 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 depend on the level of the enzyme deficiency, which in turn depends on the G6PD variant.^{[4, 5]}

Neonatal jaundice/hyperbilirubinemia 

G6PD deficiency is one of the major risk factors for severe neonatal jaundice.^[10] Jaundice most often appears within the first 24 hours of life, usually earlier than physiologic jaundice but later than 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 that are associated with Gilbert syndrome) can also exacerbate neonatal jaundice.^[18]

Hazardous hyperbilirubinemia, defined as a total serum bilirubin of 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.^[37]  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.^[38]

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 lead to kernicterus and permanent neurologic damage.^{[29, 30, 31, 26, 32, 18]}

Acute hemolytic anemia 

Acute episodic hemolytic anemia occurs on exposure to oxidative stress, as in association with certain medications and chemicals, infections, and ketoacidosis, and after ingestion of fava beans. Hemolysis usually begins 24-72 hours after exposure to oxidative stress. 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, 18]}

Chronic nonspherocytic hemolytic anemia

A small percentage of G6PD-deficient patients have chronic nonspherocytic hemolytic anemia of variable severity. G6PD Brighton, G6PD Harilaou, and G6PD Serres are included in this category.^{[1, 18, 39]}

The patient with chronic nonspherocytic hemolytic anemia 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, the patients are also vulnerable to acute oxidative damage on exposure to an oxidative agent.^[18]

• References

Physical Examination

Physical examination findings may be normal in patients with G6PD deficiency. Jaundice, pallor, and splenomegaly may be present in patients with severe hemolysis. Patients may have right upper quadrant tenderness due to hyperbilirubinemia and cholelithiasis.

• References

Approach Considerations

Indications for testing for glucose-6-phosphatase dehydrogenase (G6PD) deficiency include the following:

• Unexplained severe or prolonged neonatal hyperbilirubinemia with poor response to phototherapy
• Family history suggestive of G6PD deficiency, especially among males
• Development of hemolysis after taking medications or experiencing conditions that induce oxidative stress, especially in patients of African, Mediterranean, or Asian descent
• Recurrent jaundice, splenomegaly, or cholelithiasis in patients of African, Mediterranean, or Asian descent^[4]  
• Nonspherocytic hemolytic anemia (since the underlying cause may be severe G6PD deficiency and chronic hemolysis)

In January 2020, the British Society for Haematology released guidelines for the laboratory diagnosis of G6PD deficiency that recommended re‐assay after a hemolytic episode of unknown cause to confirm that a G6PD-deficiency diagnosis was not missed.^[40]

• References

Laboratory Studies

Semi-quantitative tests

Fluorescent spot test

This 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]}  A variant of the spot test that can be interpreted by simple color change with naked eye examination is used for screening large populations in tropical areas and before starting treatment with antimalarial drugs, such as primaquine, in countries where G6PD deficiency and malaria are both endemic. The test is not reliable in heterozygous females.

Methemoglobin reduction test

This is a rapid indirect test that measures the reduced methemoglobin levels produced after NADPH oxidation. G6PD activity is assessed by first treating red blood cells with nitrite (converting oxyhemoglobin [red] to methemoglobin [brown]) and then examining the rate of NADPH-dependent methemoglobin reduction in the presence of an appropriate redox catalyst (Nile blue or methylene blue) and substrate (glucose).^[6]

Cytofluorimetric method

This 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. This assay represents a useful addition to the screening and research toolkit for G6PD deficiency, especially in malaria-endemic areas.^[7]

Quantitative tests

Spectrophotometric assay

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. In normal red blood cells, the G6PD activity ranges from 7-10 IU/g hemoglobin when measured at 30ºC.^{[6, 7, 8]}  Testing for enzyme activity should not be performed during episodes of acute hemolysis, as results may be falsely negative. Senescent red blood cells are more vulnerable to hemolysis due to their diminished G6PD levels. Compensatory increase of immature young red cells with increased G6PD levels usually occurs in a state of acute hemolysis, and results could therefore be altered.

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.^[9]

Guidelines

The British Society for Haematology guidelines on the laboratory diagnosis of G6PD deficiency include the following testing recommendations^[40] :

• Because the G6PD reaction is temperature‐dependent, an accurate cuvette temperature is essential
• Controls should be run with every sample batch; it is preferable to use a normal and deficient sample obtained through a commercial company than to employ an in‐house control
• White cells, which contain a significant amount of G6PD, ideally should be removed before assay; especially consider removal of white cells with a cellulose/”real” cotton wool column prior to assay if the count is above the lab’s reference interval upper limit
• If performed only infrequently, carry out assays in duplicate; the duplicates’ results, on normal samples, should be within 0.5 IU/g of hemoglobin of each other
• Employ in-house testing to establish a laboratory reference range
• Assay absorbance should be checked to see that it increases in a linear fashion (which may take a minute or two to achieve), and, for non-kit methods, the absorbance should be measured over 10 minutes at 20-second intervals
• Measurement of the hemoglobin concentration of the hemolysate is equal in importance to measurement of the enzyme activity, since both measurements have a comparable impact on the final result; this applies similarly to well-mixed whole blood where a kit indicates such use
• Interpret the final G6PD activity in light of the reticulocyte count measured on the same sample
• Participation in an accredited external quality assessment scheme is important for laboratories undertaking these screening tests and assays

• References

Screening for G6PD deficiency

A semi-quantitative test is usually indicated in patients with a suggestive family history or in geographic 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.

The British Society for Haematology guidelines on the laboratory diagnosis of G6PD deficiency note that screening tests should not be relied on for the diagnosis of female patients, stating that instead, G6PD activity should be measured directly by quantitative spectrophotometric assay. They also specify that in patients in general, an abnormal or borderline screening test necessitates the performance of a quantitative assay.

Genetic Testing

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.

• References

Medical Care

Most individuals with glucose-6-phosphatase dehydrogenase (G6PD) deficiency do not require any treatment. However, infants with prolonged neonatal jaundice as a result of G6PD deficiency should receive phototherapy, and 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, so that early and focused follow-up can be provided to prevent kernicterus.^{[10, 11, 12]}

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.

• References

Surgical Care

There is no evidence of selective red cell destruction in the spleen; hence, splenectomy is usually ineffective and not recommended.

• References

Consultations

Consultations with a hematologist are ideal for long-term follow-up of patients with chronic hemolysis or nonspherocytic anemia.

• References

Diet

Persons with chronic hemolysis or nonspherocytic anemia should be placed on daily folic acid supplements.

• References

Prevention

Acute hemolytic anemia in G6PD-deficient patients is largely preventable through avoidance of exposure to fava beans and drugs and chemicals that can cause oxidative stress. Identification and discontinuation of the precipitating agent is critical in the management of hemolysis in patients with G6PD deficiency.

Vaccination against hepatitis A and B is recommended in communities with a high prevalence of G6PD deficiency.^[41]

• References

Guidelines Summary

British Society for Haematology

Guidelines on the laboratory diagnosis of G6PD deficiency, published in January 2020 by the British Society for Haematology, include the following^[40] :

• Screening tests should not be relied on for the diagnosis of female patients; G6PD activity should be measured directly by quantitative spectrophotometric assay
• An abnormal or borderline screening test means that a quantitative assay must be performed
• To be certain that a G6PD-deficiency diagnosis is not missed, re‐assay after a hemolytic episode of unknown cause
• Because the G6PD reaction is temperature‐dependent, an accurate cuvette temperature is essential
• Controls should be run with every sample batch; it is preferable to use a normal and deficient sample obtained through a commercial company than to employ an in‐house control
• White cells, which contain a significant amount of G6PD, ideally should be removed before assay; especially consider removal of white cells with a cellulose/”real” cotton wool column prior to assay if the count is above the lab’s reference interval upper limit
• If performed only infrequently, carry out assays in duplicate; the duplicates’ results, on normal samples, should be within 0.5 IU/g of hemoglobin of each other
• Employ in-house testing to establish a laboratory reference range
• Assay absorbance should be checked to see that it increases in a linear fashion (which may take a minute or two to achieve), and, for non-kit methods, the absorbance should be measured over 10 minutes at 20-second intervals
• Measurement of the hemoglobin concentration of the hemolysate is equal in importance to measurement of the enzyme activity, since both measurements have a comparable impact on the final result; this applies similarly to well-mixed whole blood where a kit indicates such use
• Interpret the final G6PD activity in light of the reticulocyte count measured on the same sample
• Participation in an accredited external quality assessment scheme is important for laboratories undertaking these screening tests and assays

• References

Medication Summary

Pharmacologic therapy is not used in glucose-6-phosphate dehydrogenase (G6PD) deficiency. Prophylactic oral phenobarbital does not decrease the need for phototherapy or exchange transfusions in G6PD-deficient neonates.^[42]

The heme analogue tin-mesoporphyrin (SnMP) has been successful in inhibiting bilirubin production in newborns but remains an experimental agent.^[43]

Transcriptional upregulation of G6PD enzyme in response to histone deacetylase inhibitors in in-vitro experiments on human B cells and erythroid precursor cells has been reported by Makarona et al, which opens new areas of potential treatment in future.^[39]

• References