Introduction

 G6PD deficiency was the first described and is the most common and best-studied RBC enzyme deficiency. G6PD deficiency is more common where Plasmodium falciparum malaria is or has been endemic. It was discovered in the 1950s as a result of investigations into a self limited hemolysis that occurred after administration of the antimalarial drug primaquine, most commonly in individuals of African or Mediterranean ethnic origin. These early studies also determined that G6PD deficiency is X-linked. Subsequent studies in carrier females led to the discovery of X-inactivation, a phenomenon that has been exploited to study the hierarchy of hematopoiesis and the clonality of malignant neoplasms.

Epidemiology

G6PD deficiency is the most prevalent human enzyme deficiency in the world, affecting about 500 million people, although the vast majority of affected individuals never become symptomatic. Although most prevalent in individuals of African, Mediterranean, and southeast Asian ethnic origins, it has been found in almost every population. The highest prevalence is in the tropical belt of sub Saharan Africa (>32%) and the Arabian Peninsula. In other populations, its prevalence ranges from less than 1 in 1000 among northern European populations to 50% of Kurdish Jews. The distribution across Asia is heterogeneous. Nearly 20% of males in Thailand are affected by one of the five prevalent variants. It is common in south ern China (~5% in Hong Kong) and rare in other parts of China, while in India the prevalence varies from 0% to 27% in different regions, caste, ethnic, and linguistic groups. In the United States, G6PD deficiency affects about 10% of African American males, a lower proportion of individuals of Italian, Greek, Spanish, Corsican, and Sardinian ancestries and a variable proportion of Middle-Eastern and recent Asian immigrants. G6PD deficiency is virtually nonexistent among indigenous peoples of the Americas and Asian high landers. The variable geographic distribution of G6PD deficiency implies that it confers a selective advantage and, as it coincides with the geographic distribution of endemic malaria, suggests protection from lethal malaria, although the exact mechanism has not been fully elucidated.

The wild-type enzyme is designated G6PD B. The most common G6PD low activity allelic variants in the United States are G6PD A− and G6PD Mediterranean; however, Asian variants are being encountered in the US population with increasing frequency. G6PD A− accounts for approximately 90% of G6PD deficient variants in Africa but is also prevalent in North and South America, the West Indies, Italy, the Canary Islands, Spain, Portugal, and the Middle East. The G6PD A− mutation (G202A; c.202 C>T) arose on a G6PD A+ chromosome (A376G, c.C376C>T); these two missense changes are in cis orientation. G6PD A+ has no obvious hematologic phenotype and has a gene frequency similar to that of G6PD A− among African Americans. G6PD Mediterranean is found in highest frequencies in the southern part of Italy, Greece, Spain, Sardinia and Corsica, as well as the Middle East, Iran, and the Arabian Peninsula, India, and Indonesia. G6PD Mediterranean is not homo geneous, but is composed of several distinct mutations, which G6PD Mediterranean (c.C563T) predominates. Several G6PD variants are pandemic in Asia. There are more than 100 different mutations in various Asian populations.

The high frequency of the most common G6PD variants and the diversity of the variants suggest selection of the variants, presumably because of protection from malaria, given the geographical distribution of these variants in areas of the world where malaria is or has been endemic. However, data on which genotype confers protection from malaria and the mechanism of the protection has been conflicting. The conflicting epidemiological data may be in part due to the large number of variants and phenotypical heterogeneity. The most common African variant G6PD A− (c.202 C>T) and possibly other variants associated with more severe G6PD deficiency is associated with a decreased risk of cerebral malaria in male hemizygotes and female heterozygotes, but an increased risk of severe malarial anemia in male hemizygotes and female homozygotes. The mechanism of malarial protection remains to be established. However, deficient cells infested with malaria parasites appear to be phagocytized more efficiently than normal cells. The host RBCs’ impaired ability to restore intracellular NADPH to maintain a high GSH/GSSG ratio may also mean that malarial parasites in G6PD-deficient RBCs are more vulnerable to ROS.

Pathobiology

G6PD is a housekeeping enzyme that catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate in the first reaction of a pentose shunt, which reduces NADP+ to NADPH (see Figs. 1 and 2). In RBCs, the pentose shunt is the only source of NADPH, which is crucial to maintaining high cellular levels of GSH to protect the cell from oxidative, stress-induced damage. Within the RBCs, oxidant injury leads to the oxidation of sulfhydryl (SH) groups on the hemoglobin molecule. This results in the formation of disulfide bridges (-S-S-), which in turn leads to decreased hemoglobin solubility and ultimately the irreversible precipitation of oxidized hemoglobin. Under normal conditions these oxidized -S-S- groups of hemoglobin and other RBC proteins are reduced by GSH to SH-groups by glutathione peroxidase, which in turn is oxidized to GSSG that is restored back to GSH by glutathione reductase in a reaction requiring NADPH. NADPH levels are maintained by G6PD, meaning in G6PD-deficient RBCs, GSH is not restored to adequate levels under oxidative stress, leading to a buildup of free radicals and insoluble hemoglobin within the cell. Precipitated hemoglobin is disruptive to the structure and function of the RBC membrane and leads to increased membrane permeability, osmotic fragility, and cell rigidity. This compromised integrity of the RBC membrane in G6PD deficient RBCs results in extravascular hemolysis from the rapid removal of these cells within the splenic pulp and also in varying degrees of intravascular hemolysis.

Fig1. PRINCIPAL COMPONENTS OF THE ERYTHROCYTE METABOLISM WITH CLINICAL RELEVANCE. Shown are glycolysis, glutathione production (purple shaded area), pentose shunt (orange shaded area), and Rapoport-Luebering shunt (blue shaded area). 1,3-BPG, 1,3-bisphosphoglycerate; 2,3-BPG, 2,3-bisphosphoglycerate; 6PG, 6-phosphogluconate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; DHAP, dihydroxy acetone phosphate; GSH, reduced glutathione; GSSG, oxidized glutathione; NAD, oxidized form of nicotinamide adenine dinucleotide; NADH, reduced form of nicotinamide adenine dinucleotide; NADP, oxidized form of nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate.

Fig2. GLUTATHIONE PATHWAY. ADP, Adenosine diphosphate; ATP, adenosine triphosphate; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; NADP, oxidized form of nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate.

The G6PD gene localizes to Xq28, spans 18 kb, and contains 13 exons. The active G6PD enzyme exists as a tetramer or dimer. Stability of the multimeric structures is crucial for optimal G6PD activity. G6PD activity decreases significantly as RBCs age, with a half-life of about 60 days. Reticulocytes have five times higher enzyme activity than the oldest RBC subpopulation. An even greater decrease in G6PD activity with aging is present in some mutant variants and is particularly pronounced in the African G6PD A− mutant.

G6PD variants can be divided into three categories based on the type of hemolysis they cause: acute intermittent (most common), chronic (rare), or none. The World Health Organization also classifies the different G6PD variants according to the degree of enzyme deficiency and severity of hemolysis. Class I deficiencies are the most severe and cause chronic hemolysis. Less severe G6PD Mediterranean is a class II deficiency. The even less deficient G6PD A− is a class III deficiency. Classes IV and V do not cause hemolysis and are of no clinical significance.

Over 400 variants of the G6PD enzyme have been identified by biochemical methods. However, this is likely an overestimate, as some previously described different mutations were found to be caused by the same mutation through molecular genetic studies. Currently, 237 G6PD mutations have been characterized by DNA sequencing in the Human Gene Mutation Database (HGMD, http://www.hgmd.org; accessed June 23, 2020); 212 of these cause disease. The majority are missense mutations but small deletions (13), splicing mutations (4), and small indels and gross deletions (4) also exist. Only one regulatory domain substitution has so far been identified. Of these mutations, at least 100 have the prevalence of a polymorphism (a rate of at least 1% in tested population). Mutations associated with chronic hemolysis tend to cluster in the vicinity of the NADP-binding domain of the G6PD gene and cause more severe deficiency, whereas those associated with acute intermittent hemolysis or no hemolysis are scattered throughout the gene. Unlike disease-causing mutations of other genes, total gene deletions and insertions causing frameshift and stop codon mutations are not observed. These events would be expected to be fatal, since G6PD is a housekeeping gene essential for basic cellular functions.

Clinical Manifestations

 Acute Hemolysis Individuals with the most common forms of G6PD deficiency have no anemia or other clinical manifestations, unless they are exposed to triggers of acute hemolysis such as oxidant drugs, infection, or ingestion of fava beans. Exposure of red cells to certain drugs results in the formation of low levels of hydrogen peroxide as the drug interacts with hemoglobin; other drugs may form other ROS that oxidize GSH without the formation of peroxide as an intermediate. Hemolysis begins in hours to 1 to 3 days after exposure to the offending drug. However, many drugs implicated in acute hemolysis in G6PD deficiency may not be true culprits because infection and other stressors can also provoke hemolysis in these people; this was elegantly reviewed by Beutler in Blood in 1994. In 2010 Youngster and colleagues critically evaluated extensive literature from the Cochrane database, MEDLINE, and PubMed, as well as leading pediatric, adult, pharmacology, and hematology textbooks. They concluded that solid evidence for drug induced hemolysis in G6PD-deficient individuals exists only for these seven drugs: dapsone, methylthioninium chloride (methylene blue), nitrofurantoin, phenazopyridine, primaquine, rasburicase, and tolonium chloride (toluidine blue).

The mechanism of hemolysis induced by infection is not well understood, but the generation of hydrogen peroxide by phagocytizing leukocytes or the diffusion of oxidants from neutrophils undergoing oxidative bursts, leading to the formation of disulfide bridges of hemoglobin may be causative factors.

Depending on the G6PD variant, the duration of hemolysis can be brief or protracted. The RBCs of G6PD A- contain only 5% to 15% of the normal amount of enzyme activity and the age-dependent decline of the activity renders old RBCs severely deficient and susceptible to hemolysis. As this subpopulation is eliminated, younger RBCs and reticulocytes produced in response to hemolysis have higher G6PD activity and are typically not hemolyzed. Thus, the hemolytic process is self-limited, even when the offending agent is continued. In contrast, in G6PD Mediterranean the enzyme activity of the young RBCs is lower than in G6PD A− and hemolysis continues longer, even a few days after discontinuation of the culprit drug, albeit it is also self-limiting.

Favism

Fava beans are a staple food in many parts of the world where G6PD deficiency is found at a high gene frequency. The hemolysis precipitated by fava bean ingestion known as favism, occurs only in people who are G6PD-deficient. It is most frequently associated with the more severe G6PD Mediterranean and G6PD Cairo variants, but is also seen with lower frequency with G6PD A−. Not all individuals with G6PD Mediterranean are susceptible to favism and a tendency toward familial occurrence suggests that additional genetic factors may be important. Favism is more common in children undiagnosed with G6PD deficiency than in adults, perhaps because persons who have experienced favism are likely to avoid this exposure. Hemolysis usually occurs one to several days after fava bean consumption, but onset within the first hours after exposure has been reported.

Chronic Hemolysis

The rare G6PD variants causing chronic hemolytic anemia occur sporadically. The severity of the hemolysis ranges from mild to transfusion-dependent. Exposure to the oxidants that cause hemolysis in the acute hemolytic G6PD variants may further exacerbate hemolysis.

Neonatal Jaundice

Neonatal jaundice, which may result in kernicterus, is the most common and often the most serious consequence of G6PD deficiency. The icterus is not only caused by hemolysis but also to inadequate processing of bilirubin by the immature liver of the G6PD-deficient infant. Further, the genetically unrelated coinheritance of polymorphic UDP-glucuronosyltransferase 1 (UGT1A1) promoter alleles (Gilbert syndrome) exacerbates the icterus. Neonatal screening for G6PD deficiency and early phototherapy treatment in endemic areas has been associated with a decreased incidence of kernicterus.

Non-Erythroid Effect of Glucose-6-Phosphate Dehydrogenase Deficiency

Although patients with the common endemic G6PD variants are not at an increased risk for infections, neutrophil dysfunction has been described in some patients with rare severely deficient G6PD variants. Occasionally, cataracts have been observed in patients with some rare variants of G6PD that produce chronic hemolytic anemia. Small studies from the Middle East that remain to be confirmed suggest that decreased G6PD activity may predispose to the development of diabetes. Splenomegaly is generally not seen in G6PD-deficient individuals.

Laboratory Manifestations

 Under normal conditions, most G6PD-deficient individuals are not anemic and have no laboratory evidence of hemolysis. In the setting of oxidative stress, laboratory findings indicative of acute hemolysis including anemia and reticulocytosis are seen. As the hemolysis is mainly extravascular with a variable intravascular component, variable degrees of hyperbilirubinemia, increased LDH, and decreased haptoglobin occur. Heinz bodies may be visible in the erythrocytes during an acute hemolytic episode, but not under normal circumstances. “Bite cells” have been described and purported to be indicative of G6PD deficiency, but they are not specific for G6PD deficiency and these cells are usually not present in acute hemolytic states of patients with common G6PD variants or in G6PD-deficient patients with chronic hemolysis.

Individuals with the chronic hemolytic G6PD variants have varying degrees of anemia and reticulocytosis.

Diagnosis

A biochemical diagnosis of G6PD deficiency can be made using quantitative spectrophotometric analysis to measure the generation of NADPH from NADP in RBC hemolysates. A more convenient rapid fluorescent screening test can be used to test at-risk populations. False negative results are not unusual, however, especially if enzymatic analysis is performed during or shortly after resolution of acute hemolytic episodes, or in heterozygous females. After acute hemolysis, reticulocytes and young RBCs, which have much higher enzymatic activity, predominate. These false-negative test results are more likely to occur when a screening test rather than a quantitative spectrophotometric analysis of the enzyme activity is used. However, this obstacle can be overcome when hexokinase (HK) activity, another glycolytic enzyme with higher enzymatic activity in reticulocytes and young RBCs than in aged RBCs, is concomitantly measured; the HK/G6PD enzyme activity ratio can then be used to diagnose or rule out G6PD deficiency.

Females heterozygous for G6PD are particularly difficult to diagnose because of their mosaicism for this X-chromosome enzyme and may have total RBC enzymatic activity ranging anywhere from hemi zygote to normal. However, these females have a variable mixture of deficient and nondeficient RBCs and their deficient RBCs are subject to same hemolytic destruction as those of males. Since the nucleotide substitutions of most G6PD-deficient isoenzymes have been identified, molecular diagnostic methods are more reliable for the accurate diagnosis of females who are heterozygous for G6PD deficiency and also in males with ongoing or recent acute hemolysis, or after transfusion.

Severe sporadic variants causing chronic hemolytic anemia may be considered for prenatal diagnosis in some circumstances.

Prognosis

Most patients with G6PD deficiency have normal life spans with no clinical sequelae. Neonatal icterus with resultant kernicterus that may lead to neurological deficits and mental retardation has the gravest consequences.

Therapy

Treatment of an acute hemolytic crisis includes withdrawal of any offending agent and supportive care, which in severe cases includes RBC transfusions. Folic acid supplementation is advocated for patients with chronic hemolysis, but even without folic acid supplementation, significant folate deficiency rarely occurs. Neonatal icterus associated with G6PD deficiency is treated in the same manner as neonatal icterus arising from other causes; G6PD deficiency should be considered in any neonate with hyperbilirubinemia, especially those of high-risk ethnic descent. The chronic hemolytic anemia in some exceedingly rare G6PD variants may be severe enough to require chronic transfusions and iron chelation.

Future Directions

In certain areas of the world where G6PD deficiency reaches epi demic proportions and ingestion of fava beans is staple, screening for endemic G6PD-deficient variants and avoiding fava beans in those that are G6PD deficient reduces hospitalizations and RBC transfusions. Screening of neonates in endemic areas can also reduce mortality (from kernicterus).

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