Gene:

G6PD

glucose-6-phosphate dehydrogenase

Background

Molecular Structure and function

The G6PD enzyme is conserved throughout evolution, with human G6PD sharing around 93% amino acid identity with rat and 37% with E. coli [Articles:2606104, 8119488]. G6PD is encoded by a gene on the X chromosome (Xq28) [Articles:6930669, 2194676, 2364435], contrary to an early report describing the G6PD enzyme as a fusion protein encoded by genes on chromosomes 6 and X [Article:2758468]. The G6PD gene is around 18kb in length and consists of 13 exons and 12 introns, and was originally cloned in 1986 [Articles:3515319, 3012556, 2428611]. The G6PD gene is found on the minus chromosomal strand - please note that for standardization, the PharmGKB presents all allele base pairs on the positive chromosomal strand, therefore the alleles within our variant annotations will differ (in a complementary manner) from those in this VIP summary that are given on the minus strand as reported in the literature.

The promoter region of the G6PD gene shares some sequence homology with other housekeeping genes, and contains elements for tissue-specific expression which regulate transcription in response to oxidative stress, hormones, nutrients and growth factors [Articles:2428611, 8119488]. Alternative transcriptional start sites and mRNA splice variants have been described [Articles:2910917, 2836867, 3515319, 2428611, 8466644]. The G6PD mature peptide of 514 amino acids in length (59KDa) is active as a dimer or tetramer, and 1 molecule of NADP+ is bound per protein subunit [Articles:10745013, 8466644, 5781270, 7857286, 921782]. The binding of NADP+ is thought to be integral to the enzyme's stability and thus its function, as point mutations close to the NADP+ and dimer interface result in severe G6PD deficiency, revealed by the crystal structure of the Canton variant [Article:10745013] and site directed mutagenesis studies [Article:9492308].

G6PD is a cytoplasmic protein and has two main roles within the cell: the production of NADPH and Ribose-5-phosphate (reviewed in [Articles:17611006, 20122995]. Both are synthesized by steps within the Pentose Phosphate Pathway (PPP), also known as the Hexose Monophosphate Shunt (HMPS), e.g. [Article:539595] (reviewed in [Articles:18177777, 17611006]. NADPH is essential to maintain the redox state of the cell and relieves oxidative stress through the reduction of glutathione, which in turn reduces hydrogen peroxide and oxidative free radicals (reviewed in [Articles:2633878, 8119488, 18177777, 20122995, 17611006]. Ribose-5-phosphate is required for glycolysis and for DNA and RNA biosynthesis (reviewed in [Articles:18177777, 17611006, 2633878, 8119488, 20122995]). Alternative pathways can be utilized for the biosynthesis of nucleic acids, but G6PD is essential for a cell's ability to cope with oxidative stress [Article:7489710]. Tumor suppressor protein p53 has been shown to regulate the PPP by binding to G6PD, preventing dimer formation and thus NADP+ binding, inhibiting NADPH production [Article:21336310]. Several p53 mutants associated with tumors were shown to lack this inhibitory property, and therefore disregulation of G6PD in cancer cells may result in increased cell growth through unregulated glucose biosynthesis and the production of NADPH [Articles:21336310, 20122995].

G6PD is expressed in all cells, but its role is particularly important in red blood cells (rbcs), which do not have mitochondria and are therefore dependent upon G6PD as the only source of NADPH to relieve oxidative stress and protect the hemoglobin beta chain from oxidation (See the PharmGKB Oxidative Stress Regulatory Pathway (reviewed in [Articles:17611006, 18177777, 2633878]). In addition, enzyme levels fall during the rbc lifespan [Article:2633878]. When the required levels of NADPH cannot be maintained, the amount of reduced glutathione falls, resulting in oxidative damage which can ultimately lead to lysis of rbcs (reviewed in [Articles:17611006, 19233695, 18177777]). Under normal conditions, G6PD activity in rbcs is only around 2% of its capacity, inhibited through a negative feedback loop with NADPH (reviewed in [Articles:2633878, 9581796]). However under oxidative pressure, oxidation of NADPH releases the inhibitory effect and G6PD enzyme activity increases, enabling enhanced reducing activity to deal with the additional stress (reviewed in [Articles:2633878, 9581796]). In G6PD deficient rbcs where enzyme activity can be below 10% of the normal value, homeostasis can be maintained and most G6PD deficient individuals remain asymptomatic (reviewed in [Article:2633878]). However, the deficiency becomes apparent under oxidative stress conditions when an increased demand in NADP/NADPH turnover cannot be met (reviewed in [Article:2633878]).

G6PD as an important pharmacogene

We have known for more than 2000 years that the ingestion of fava beans can have dire consequences in some individuals, and could indeed be why Pythagoras imposed abstinence from beans amongst his followers (Brumbaugh and Schwartz, 1980) [Article:11678777]. However, it wasn't until the 20th century that a deficiency in the G6PD enzyme was discovered to be the underlying cause of 'Favism', and the connection that agents other than fava beans can cause similar adverse events in G6PD deficient individuals (discussed in [Articles:18177777, 13618370]). In the 1950s, it was observed that a subset of African-American soldiers were more likely to develop an adverse reaction to the anti-malarial drug primaquine, compared to their Caucasian counterparts [Articles:14945981, 14945980]. This susceptibility to primaquine-induced intravascular hemolysis led to the discovery of a deficiency in G6PD enzyme activity in rbcs [Article:13360274].

More than 400 variations of the G6PD enzyme have now been described, based on clinical manifestations and biochemical properties, and G6PD deficiency is the most prevalent enzyme deficiency in the world (reviewed in [Articles:18177777, 17611006, 7949118, 12064901, 8364584]), affecting an estimated 4.9% of the world's population (more than 300 million people) [Article:19233695]. Polymorphic variants in G6PD are those of significant frequencies (1-70%) in specific human populations - these fall into World Health Organization (WHO) class II and III (see Table I) [Articles:2633878, 5316621, 17611006, 7949118]. Different polymorphic variants have arisen in different geographical areas, for example the Canton variant is predominantly found in Chinese and South East Asian populations [Articles:1953767, 12064901, 17018380, 11499668], reviewed in [Article:17611006]. It is hypothesized that higher population densities of humans due to the development of agriculture facilitated malaria endemisms, and introduced a selective pressure for the spread of G6PD variants (reviewed in [Article:17611006]). Nevertheless, G6PD deficient variants can also occur sporadically due to de novo mutations (reviewed in [Article:17611006]). Most of the genetic variants tend to be single point mutations, and the lack of large or out-of-frame deletions may indicate that total absence of enzyme activity is fatal [Articles:17611006, 2633878, 8364584, 7949118, 3393536]. In-frame deletions are usually associated with the most severe clinical manifestations (class I) (reviewed in [Articles:17611006, 8364584, 7949118]), such as the novel G6PD Tondela variant, a 6 amino acid deletion identified in a heterozygous woman with chronic hemolytic anemia (see Table 1) [Article:21397531].

The mechanism underlying the G6PD deficient phenotype may vary depending on the location of the mutation in the enzyme's 3D structure, and include alterations to protein assembly, dimer formation and stability, interaction with substrates, and protein turnover (reviewed in Bautista, J.M. and Luzzatto, L., Glucose 6-phosphate dehydrogenase. In: Swallow D.M. & Edwards. Y.H., Editors. Protein Dysfunction in Human Genetic Disease. Oxford: BIOS Scientific Publishers; 1997, p. 33-56). Studies suggest that most G6PD variants result in G6PD enzyme instability (reviewed in [Articles:17611006, 5316621]), and variants which cause the most severe deficiency (resulting in CNSHA) are found predominantly at or near the dimer interface of the G6PD protein in exon 10 [Article:8639919], though not exclusively. Different gene variants can confer similar effects on enzyme function and clinical manifestations, and conversely, the same genetic variation can result in different molecular and clinical phenotypes [Articles:8364584, 12064901, 7906668]. Therefore G6PD variants based on enzyme biochemistry may have been characterized differently yet have the same underlying genetic mutation [Articles:2572288, 2912069, 2602358]. The term 'haplotype' is used in this review to define a set of linked alleles in G6PD that are inherited together. The B haplotype is considered the 'wild type' precursor sequence, as alignment of Human and Chimpanzee DNA sequences have shown [Article:2572288]. Variants which differ from the B wild type are often named with the region where the population in which the variant was found [Articles:12064901, 6075369]. Even though attempts have been made to standardize nomenclature since 1967 [Article:6075369], there are still instances where haplotype names have been used to refer to more than one set of variations (see text on haplotype A-). It should be noted that many studies do not screen the whole G6PD gene, and therefore some genetic variants may be unreported and may be a factor underlying differences in biochemical properties.

Exogenous agents can trigger hemolytic anemia in G6PD deficient individuals by inducing oxidative stress in rbcs (reviewed in [Articles:17018377, 18177777, 17611006]). These include certain food items, therapeutic drugs, infections, and exposure to chemicals (for example hair dye containing naphthol) [Articles:17018377, 18177777, 17611006, 20085579]. G6PD variants have been classified into 5 WHO categories according to the severity of clinical manifestation resulting from the genotype (see Table 1), with class II and III the most common type of polymorphic G6PD deficient variant [Articles:8364584, 2633878]. Due to lower rbc G6PD activity, patients carrying class I sporadic variants (associated with CNSHA) are highly susceptible to hemolytic anemia caused by the same drugs that can induce adverse reactions in carriers of polymorphic G6PD variants (reviewed in PMID: 17611006].

Table 1: Classification of G6PD variants

Table based on [Articles:18177777, 2633878, 8364584, 7949118, 5316621].

Testing for G6PD deficiency

G6PD variants that result in enzyme deficiency confer a G6PD deficient phenotype in hemizygous males (with one copy of the G6PD gene) and homozygous females (for example; [Articles:20520804, 10747271]). To diagnose a phenotype of G6PD deficiency in heterozygous females is more difficult, as the extent of enzyme deficiency activity can vary greatly within heterozygous individuals, due to X-linked mosaicism [Articles:7949118, 13868717, 10747271]. This pattern of gene inactivation is random therefore female heterozygotes will have G6PD deficient rbcs combined with those expressing normal G6PD activity, and the population sizes of these cells can vary from 50:50, to minimal levels or a majority of G6PD deficient cells [Articles:2633878, 13868717, 7949118]. Genotyping is therefore essential to establish heterozygosity in females; however this can make prediction of drug response difficult without phenotypic information of G6PD enzyme activity levels. For example, 75% of females genetically heterozygous for the Mediterranean variant had normal G6PD activity, whereas 25% were enzyme deficient, as assessed by a colorimetric test [Article:20520804]. Testing for both genotype and enzyme function is the ideal method, however due to various factors, including the impracticalities and costs of genotyping in the field, many studies and clinics have solely tested G6PD enzyme activity, making the association of causative variants difficult. In a meta-analysis of 280 studies, less than 8% used DNA analysis to assess G6PD deficiency [Article:19233695].

G6PD and therapeutic drug response

The WHO recommends testing of drugs to predict for risk of hemolysis in G6PD deficient individuals if the drugs are to be prescribed in areas of high prevalence of G6PD deficiency [Article:2633878]. As a consequence of adverse reactions in individuals with G6PD deficiency, the FDA has introduced warnings or precautions on the drug labeling of primaquine, chloroquine, dapsone, rasburicase, avandaryl tablets (glimepiride + rosiglitazone maleate) and glucovance tablets (metformin + glibenclamide) (FDA website). These highlight the possible risk of hemolytic anemia in G6PD deficient individuals upon drug intake. It should be noted that numerous factors can contribute to drug-induced hemolytic anemia in G6PD deficient individuals, including high dosage, other drugs taken in combination, concurrent infections and other genetic variants, as discussed in [Articles:1984194, 20701405, 18177777]. Therefore it may be that many drugs which have been reported to cause hemolysis in individual case studies can be taken safely by G6PD deficient individuals, for example aspirin, vitamin C and chloroquine (discussed further below). These drugs however should be administered with caution especially in combination with other drugs or at high doses, with possible monitoring of rbc or hemoglobin levels [Articles:1984194, 20701405]. Readdressing the safety of drugs in these individuals could make effective therapeutics available (as discussed in [Article:20701405]). More information and comprehensive advice on unsafe drugs in G6PD deficient individuals can be found at http://www.favism.org. Outlined below are several drug subsets and the possible consequences of drug intake for G6PD deficient individuals:

G6PD deficiency and anti-malarial drugs

It is hypothesized that variants associated with G6PD deficiency have remained prevalent in the human population due to positive selective pressures, in particular resistance to uncomplicated and severe malaria (reviewed in [Articles:17611006, 18177777]). At the same time G6PD deficiency confers susceptibility to hemolytic anemia triggered by some anti-malarial drug treatments [Article:19233695]. The prevalence of G6PD deficiency in malaria endemic regions where anti-malarial drugs are required is an important public health issue [Articles:21311583, 19233695]. It should be noted that hemolysis is also a phenotype induced by malaria infection; therefore distinguishing whether this is truly a drug-induced effect may be difficult (as discussed in [Articles:15183620, 20194698]). Another important consideration in areas where malaria is endemic is the financial and practical costs of G6PD screening [Articles:20520804, 18177777]. However, the wide-scale drug-based eradication of malaria is likely to require G6PD deficiency testing [Article:21311583], and thus the application of pharmacogenomics in the treatment of G6PD deficient individuals.

Race-specific differences in sensitivity to hemolytic anemia after treatment with aminoquinolines have been reported since the 1920s (reviewed in [Article:13618370]). In 1956 a deficiency in the G6PD enzyme was determined as the underlying cause of 'primaquine-sensitivity' [Articles:14945981, 14945980, 13360274]. Metabolism of primaquine mediated by CYP proteins is thought to be one of the mechanisms behind the drug sensitivity, as the resulting metabolites induce formation of methemoglobin and reactive oxygen intermediates [Article:19616568]. Alternatives to primaquine have been sought for use in areas where G6PD deficiency is prevalent. However, unfortunately numerous anti-malarial drugs can also induce hemolysis in G6PD deficient individuals, including dapsone which has FDA drug labeling precautions [Articles:20194698, 19690618, 19112496] (reviewed in: [Article:20701405]. The WHO released guidelines after a Technical Consultation in 2004 for the use of Lapdap™ (a combination of chloroproguanil and dapsone) due to safety concerns in G6PD deficient individuals, recommending use only if malaria infection is confirmed, testing for G6PD deficiency and avoiding drug treatment in these individuals, as discussed in [Article:20599264].

FDA labeling of chloroquine advises that caution should be taken when administering this drug to G6PD deficient individuals, though it is not contraindicated. Treatment of normal rbcs with chloroquine in vitro does not boost the PPP, and does not reduce the survival of G6PD deficient rbcs transferred into wild type recipients [Article:1247492] (Chan, T.K., Todd, D., Tso, S.C., Red cell survival studies in glucose 6 phosphate dehydrogenase deficiency. Bulletin of the Hong Kong Medical Association, 1974. 26(1): p. 41-48). However, a high dose of chloroquine (600mg) for the prophylaxis of malaria was reported to induce severe hemolytic anemia in 50 soldiers, all G6PD deficient [Article:79931]. G6PD deficiency may also increase the risk of side affects such as pruritus induced by chloroquine [Article:15027776]. Chloroquine treatment combined with primaquine for P. vivax infection has been associated with significantly decreased hemocrit levels [Article:16969059], though the development of hemolysis in G6PD deficient individuals has been affiliated with primaquine administration rather than chloroquine [Article:20963329]. Severe hemolysis has been reported in several children treated with combinations of chloroquine, chloramphenicol, aspirin and primaquine [Article:1708959]. On the other hand, chloroquine treatment combined with methylene blue or elubaquine does not induce severe adverse effects in individuals with G6PD deficiency [Articles:15655011, 16179085, 16969059]. To conclude, the safety of chloroquine in G6PD deficient individuals is indefinite, and seems to depend on numerous factors including dosage, concurrent drugs and infections, as discussed in [Articles:1984194, 20701405].

When comparing G6PD deficient and G6PD 'normal' children with uncomplicated malaria infection in a trial of artesunate+amodiaquine or artemether-lumefantrine (also known as Artemisinin-based Combination Therapy (ACT)), no significant differences in adverse events were observed, indicating these drug combinations are a promising alternative (see PharmGKB Artemisinin and Derivatives, Pharmacokinetics Pathway) and Amodiaquine Pathway, Pharmacokinetics [Article:18575626].

G6PD deficiency and cancer therapeutics

Several agents involved in the treatment of cancer patients have the potential to result in severe adverse side effects in G6PD deficient individuals, due to induction of oxidative stress in rbcs. Carmustine (BCNU) treatment results in a deficiency in glutathione reductase in erythrocytes, platelets and leukocytes [Articles:539595, 870569]. This lowers levels of reduced glutathione and leads to insufficient removal of hydrogen peroxide and susceptibility to oxidative hemolysis, particularly in rbcs that are G6PD deficient [Articles:539595, 870569]. Doxorubicin (Adriamycin) stimulates the PPP but to a lower extent in G6PD deficient rbcs, and results in oxidative stress through the accumulation of hydrogen peroxide due to an inability to increase G6PD activity (see PharmGKB Doxorubicin Pathway (Cancer Cell), Pharmacodynamics [Articles:539595, 21048526]. This response can also be mirrored in "normal" rbcs when cells are pretreated with carmustine before doxorubicin treatment, resulting in diminished glutathione stability and enhanced susceptibility to oxidative stress [Article:539595]. A number of cases of methemoglobinemia and acute hemolysis after treatment with rasburicase in G6PD deficient individuals have been described [Articles:20196170, 16204390, 19654083]. The FDA labeling has contraindicated rasburicase for G6PD deficient individuals.

The anti-leukemia drug daunorubicin is metabolized into the less-potent form daunorubinol, a process dependent on NADPH via G6PD [Article:8648264]. This biotransformation was greatly reduced in rbcs from A- or Mediterranean G6PD deficient individuals, and thus may have implications in the clinic, raising the issue of whether or not the drug is more effective in these individuals due to prolonged exposure to the more active form and possible toxicity concerns [Article:8648264]. NADPH inhibitors, including primaquine aldehyde, were also shown to reduce daunorubicin metabolism and daunorubinol appearance [Article:8648264].

Aspirin

A child carrying the Mediterranean variant who was diagnosed with systemic arthritis and prescribed a daily dose of 100mg/kg aspirin subsequently developed severe hemolytic anemia, with no sign of viral or bacterial infection [Article:2502894]. Hemolysis was reported in a male administered 1.5g aspirin for a fever (likely a viral infection), who later was found to be G6PD deficient and had a family history of Jaundice [Article:13836342]. However, in 22 healthy G6PD deficient individuals, normal therapeutic doses of aspirin for 4 days (50mg/kg daily) had no effect on rbc count or hemoglobin levels [Article:993904]. Therefore the effect of aspirin on G6PD deficient individuals may be dependent on the variant type and pre-existing clinical conditions (such as an infection or inflammatory disease) [Article:714540]. Dosage is also likely to contribute, as exemplified in the above studies and demonstrated by in vitro studies in which higher levels of aspirin were required to see a drop in GSH levels in the blood from sensitive patients [Article:13836342].

G6PD deficiency and diabetes mellitus treatment

Glibenclamide (glyburide) has been shown to induce acute hemolysis in diabetic patients carrying the A- haplotype or the Mediterranean variant [Articles:8562390, 15126005], though it should be noted that these seem to be sporadic case studies, as discussed in [Article:20701405]. FDA labeling of glucovance tablets, which contain glyburide and metformin HCl, cautions use in G6PD deficient individuals as sulfonylurea agents can result in hemolytic anemia, and advise using a non-sulfonylurea alternative (FDA MedWatch).

G6PD Variants and Haplotypes

A selection of the most studied G6PD variants and known haplotypes are discussed in the separate VIP variant sections (see Variant Summaries), and their association with drug response is summarized in Table 2.

Table 2: Polymorphic G6PD Variants and Haplotypes associated with drug response

Table key:
*: For each reference, details of whether the study was a single case study, or total study numbers and percentage of individuals carrying the indicated G6PD allele, are given in the reference column.
#: Please note in this study heterozygous 202A individuals were considered G6PD A, and hemizygous/ homozygous 202A individuals were classified as G6PD A-.

The G6PD gene is found on the minus chromosomal strand. Please note that for standardization, the PharmGKB presents all allele base pairs on the positive chromosomal strand, therefore the alleles within our variant annotations and haplotypes will differ (in a complementary manner) from those in this VIP summary that are given on the minus strand as reported in the literature.

Appendix

A database with G6PD mutational and structural data is available at http://www.bioinf.org.uk/g6pd/ established by Dr Andrew C.R. Martin's Group at UCL. A list of pharmacogenomic biomarkers in Drug labels, including G6PD-deficiency, can be found at: http://www.fda.gov/Drugs/ScienceResearch/ResearchAreas/Pharmacogenetics/ucm083378.htm and information on drug safety at MedWatch; http://www.fda.gov/Safety/MedWatch/default.htm. Information related to genetic tests are available for G6PD deficiency; http://www.ncbi.nlm.nih.gov/sites/GeneTests/lab/clinical_disease_id/2339.