TK Chan

Introduction

Glucose-6-phosphate Dehydrogenase (G6PD) deficiency was discovered by Alving and coworkers¹ when they investigated the unusual haemolytic reaction that occurred in ethnic Black individuals following the administration of primaquine, an 8-aminoquinoline, for the radical treatment of malaria. Such "primaquine sensitivity" was latter observed in other ethnic groups as well. By the 1960's, four syndromes, which included massive intravascular haemolysis as an idiosyncratic reaction to multiple drugs and chemicals; haemolysis after ingestion of fava bean (Favism); severe haemolysis as an unusual complication of illnesses ; and severe neonatal jaundice causing kernicterus, were all explained by their occurrence predominantly in those who have inherited the G6PD deficient genotype.

Biochemical Characteristics

G6PD catalyzes the entry step of G6P into the Pentose Phosphate Shunt (PPS), first described by Warburg and Christian in 1931. In the red cells, this alternate anaerobic pathway for glucose metabolism is the only source for reduced NADP (NADPH), which is required for methaemoglobin reductase activity and the maintenance of the level of reduced glutathione (GSH). NADPH and GSH in turn maintain an effective redox potential protecting cell membrane sulphhydryl groups, enzymes and haemoglobin against oxidative stress and injury. In view of the metabolic relationship of these intermediate enzymes, rare occurrence of G6PD, glutathione reductase and glutathione peroxidase deficiencies produce the same susceptibility as G6PD deficiency.

G6PD in the active state consists of identical subunits of dimer and tetramer, the proportion of the two forms are pH dependent. The primary structure of the single subunit has been determined from cDNA sequence and consists of 515 aminoacids with a molecular weight of 59,265 daltons.² Difference in G6PD from liver and leukocyte represents N-terminal post-translational changes.³ Furthermore, enzymes isolated from erythrocytes and leucocytes in normal and deficient variants showed the same enzyme characteristics.⁴ Hence the enzyme in all three tissues and probably those in other tissues of the body are under the same genetic control. Nucleotide sequences of the G6PD gene from a variety of organisms revealed regions of conserved aminoacids, probably corresponding to important functional domains.⁵ This comparative biology information is important in understanding how various mutations result in different phenotype.

Standardized technique for purification of red cell G6PD enzymes and determination of enzyme characteristics have been published.⁶ The electrophoretic mobility (EM) of the purified enzyme, Michaelis constant (Kin) for substrate G6P, G6P analogue utilization (e.g. 2dG6P), pH optima, and thermostability of the enzymes served to characterize more than 400 G6PD variants. In the Chinese, about five variants account for the majority of the enzymes characterised.⁷ Unfortunately with enzyme characterisation, there is a range for the parameters measured and different variants characterised using such criteria, especially those reported from different laboratories, have been proven to be due to the same nucleotide mutation. On the other hand, a single well-characterised variant, e.g. G6PD A- in the Blacks, can result from at least four different mutations.⁸

Molecular Basis of G6PD Variants

The G6PD gene, located on chromosome Xq28 region, is 18 Kb long consisting of 13 exons transcribed to a 2.269 Kb messenger RNA with 1.545 Kb of coding regions.^2,9,10 The commonest variant in South China, G6PD Canton, has been sequenced and was found to be due to a mutation at nucleotide (nt) position 1376 of cDNA, G to T, resulting in a missense mutation in amino acid position 459, Arg to Leu.¹¹ With improved DNA technology, the whole cDNA sequence can be amplified and screened for mutation directly. Chang et al¹² have used PCR technique and restriction analysis to characterize the mutations in 94 Chinese G6PD deficient subjects in Taiwan and have shown that five mutations account for 90% of the cases (Table I).

While most of the G6PD deficient variants resulted in compensated haemolysis and no anaemia in the steady state, some cause chronic nonspherocytic hemolytic anaemia (CNSHA). Minor deletion or missense mutation near putative substrate binding sites, around exon 10, are responsible for these rare variants.^13,14 Unlike the thalassaemias, no gross deletion, nonsense mutations nor splicing defects have been described in over 90 variants characterised to-date. This is probably because such mutant will not be compatible with life ,since G6PD is a housekeeping gene expressed in all tissues.

World Incidence and Distribution of G6PD Deficiency

G6PD deficiency in male subjects can be detected easily by a number of screening tests. The simplest one is the fluorescent spot test developed by Beutler and Mitchell¹⁵ which relied on the fluorescence of NADPH, generated by an adequate amount of G6PD enzyme. This test can also be done on blood sample dried on filter paper similar to the Guthrie cards. In Hong Kong, the routine screening of newborns have included test for G6PD deficiency. Table IIa showed the incidence of G6PD deficiency in some major ethnic groups of the world and Table IIb in peoples of South East Asia.

Populations where G6PD deficiency is common, i.e., an incidence of over 1%, are distributed in the Mediterranean regions, across the Middle East, India, Indochina, South China as well as middle Africa. This distribution is similar to that of the thalassaemias and is thought to be due to the selective advantage of these phenotypes against endemic malaria infection in the past. In fact, Luzzatto et al¹⁶ have shown that in the heterozygote G6PD deficient subjects, malaria parasite are preferentially found in G6PD normal red cells. This suggests that in the heterozygote female, the density of malaria infection, which correlates with the incidence of mortality, is lower and, therefore, has a survival advantage. That G6PD deficient males are not immune to malaria was explained by the adaptation of plasmodium falciparum to such deficient cells by the production of G6PD enzyme endoded by the parasite itself.¹⁷

Genetics and X - Inactivation

G6PD gene is located in the terminal region of the long arm of the X chromosome (Xq28), less than 2 centi-Morgan centrometric to the Factor VIII gene. The gene frequency in a population can be determined by studying the rate of hemizygote males where, if p = incidence of normal trait and q = incidence of abnormal trait, then p+q = 1. The corresponding incidence in females can be predicted by the Hardy-Weinberg formula p2+2pq+q2 = 1 (2pq = heterozygous and q2 = homozygous state in the female population). For example, in Hong Kong where the deficient gene frequency (q) is 5.5% in the males, the incidence of heterozygous female (2pq) is 2 0.945 x 0.055 = 10% and homozygote female (q2) is 0.055 x 0.055 = 0.3%.

Beutler et al¹⁸ showed that in G6PD deficient females, the red cells are mosaic with two populations: one G6PD normal level and the other G6PD deficient level. This was postulated to be due to the random inactivation of one of the two X chromosomes in the female. When this occurs in the multicellular stage of human embryo development, the haematopoietic self-renewal stem cells are variably X-inactivated, hence accounting for the mosacism of the progeny cells in adult life. In fact the same phenonema was proposed by Mary Leon who studied X-linked coat colour in rodents. In practice, this information explains the finding of severe enzyme deficiency in some heterozygote females due to extreme degree of X inactivation of the normal chromosome. Furthermore, even in females with 50% activity, half of the mosaic red cells with G6PD deficiency are prone to haemolysis from drugs similar to their male counterpart. Hence, females with G6PD deficiency are clinically important although the degree of haemolysis is generally milder.

The other practical aspect of X-inactivation is as a marker for monoclonality, a characteristic of malignant growth. Linder and Gartler¹⁹ first used G6PD A+/B+ in Black females to demonstrate the multicentric origin of uterine fibromyomata. Subsequently, the clonal origin of cells in leukaemia and lymphoma and malignant tumours were shown to arise from only one of the X-inactivated cell.^20,21 More recently, DNA markers on the X chromosomes have been used, where the inactive X-genes are hypermethylated and can be detected by restriction enzymes e.g., Msp I and Hpa II.²² However, as discussed above, extreme lyonization can occur in certain females and the use of these parameters to indicate monoclonality have to be confirmed by demonstrating two X-inactivated populations in normal tissue of the same individual.²³

Clinical Spectra

The majority of the G6PD deficient variants, including those found in Hong Kong Chinese, only manifest when these individuals took drugs or chemicals that trigger the massive haemolysis. Classically, within two days of ingestion of the offending agent, the patient will develop fever, dark brown to black, "Coca-Cola", urine, jaundice and anaemia. Acute tubular necrosis may complicate such severe haemolytic episode, especially in those with underlying diseases of the liver such as hepatitis. Maintenance of adequate renal blood flow, e.g. by forced alkaline diuresis, can prevent this complication. In those with compromised renal blood flow as evidenced by low urine output, exchange transfusion to remove the irreversibly damaged red cells that block the microcirculation, can also avert the renal complication. In some patients, disseminated intravascular coagulation (DIC) may complicate such massive intravascular haemolysis and need appropriate treatment.

A useful hallmark of severe oxidative injury to red cells, such as in G6PD deficiency, is the presence of "hemighost" erythrocytes (Figure).²⁴ Besides indicating a correct diagnosis, the percentage of hemighost cells will indicate the amount of red cells which will haemolyse within the next 24-48 hours. As discussed above, prompt awareness of such acute episode would alert the doctor to implement prophylactic measures to avert renal shutdown.

The list of drugs causing haemolysis in G6PD deficient subjects are shown in Table III. Two types of data are listed:

1. Controlled Studies where the offending drug was given to G6PD deficient volunteers (as in studies by Alving and coworkers in the 1950's) or in cross transfusion of radioactive (51 Cr)-labelled G6PD deficient red cells to compatible normal subjects who then took the drugs.²⁵

2. Case Reports where one of the drugs given for an illness was blamed as the trigger. Such data are more difficult to intepret. For example, a patient with typhoid fever was given aspirin and chloramphenicol and haemolysis could be due to any or all of these factors. These data however, should be taken seriously because although a drug may be exonerated by controlled studies, individual variation in metabolism or illness-altered metabolism of a drug might still be responsible for the haemolytic trigger. For example, in subjects given a sulphonamide e.g. sulphamethoxazole, only high dose of 90 mg/kg/day cause haemolysis in four out of six subjects which indicates that individual variation in catabolism can result in higher level of those metabolites that are responsible for the haemolysis.²⁵

Haemolysis complicating illnesses

A well studied disease is typhoid fever, where severe haemolysis occurred in the second to third week of illness especially in patients with coexisting G6PD deficiency and can be prevented by prompt treatment of the infection early on in the disease. This was elegantly demonstrated by using G6PD deficient cells cross transfused into normal subjects in the early stage of typhoid fever. Chloramphenicol was exonerated as the culprit; in fact, when used early on as treatment, chloramphenicol would avert this severe complication in typhoid fever.²⁶

Viral hepatitis patients with G6PD deficiency may be complicated by severe haemolysis and renal failure and we have observed a few patients who died from this complication. Controlled studies have shown that both G6PD normal and G6PD deficient red cells showed moderate haemolysis when transfused into patients with hepatitis.²⁷ When the records of those with massive haemolysis were scrutinized, most of them had received drugs including paracetamol, aspirin, amidopyrine and vitamin K analogue before the episode. We have tested all these agents in controlled studies using G6PD deficient red cells transfused into normal subjects and could not demonstrate increased haemolysis. Drug metabolism may have been altered by the hepatitis, resulting in increased amount of metabolites which cause haemolysis. In practice, one should avoid the use of these agents during an episode of hepatitis or use a smaller dose with caution.

Fig Erythrocyte Hemighosts (Eccentrocytes); Hallmark of severe oxidative injury in vitro. Peripheral Blood from a patient with massive intravascular haemolysis due to G6PD deficiency. (A) Blood film stained with Wright stain (x 1000). 50% of red cells showed condensed haemoglobin on one side of the cell (eccentrocytes, hemighost) and a clear apronlike membrane on the other side. Two red cells are completely devoid of haemoglobin (ghost). (B) Methyl violet stain (wet preparation) (x 1000). One large and many small Heinz bodies (purple stained) in each erythrocyte hemighost). (C) Scanning electron micrograph (x 3,000) of one normal erythrocyte, two hemighosts and one erythrocyte with the thin out part removed ("bite" cell). (D) Transmission electron micrograph (x 11,900). Erythrocyte hemighost showing a thin out part with near apposition of the membrane and a spheroidal part containing most of the haemoglobin. Heinz bodies are present in both parts of the cell.

Favism, herb- and chemical-induced haemolysis

Favism, haemolysis after the ingestion of beans e.g. broad beans, has been known from antiquity and described at the time of Pythagorus and his disciples (6th Century, BC). Favism was described in Szechwan, China after the ingestion of broad beans.²⁸ Szeinberg et al,²⁹ demonstrated that this only occurs in G6PD deficient subjects. The exact mechaniam for the haemolysis has not been fully explained. While L dopa which has a high concentration in broad beans is not responsible for the hemolysis,²⁵ other components, e.g. divicine and isouramil, have been incriminated.³⁰ It seemed from anecdotal evidence that dried broad bean, usually eaten in less amount, was safe. On the other hand, there is record that walking in a field of bean crops with possible exposure to pollens, could cause haemolysis in some individuals.

Herbs that provoke haemolysis have not been systematically studied. Coptis sinesis used in neonatal period to remove "placental toxin" has been incriminated and so has Calculus bovis, used as an anti-inflammatory agent.

Naphthaleine³¹ used for storing of clothes and bedding for the winter has been incriminated in small children and it is wise to air such garments before use. Antifungal spray used in the fields has been reported to caused hemolysid in G6PD deficient subjects.

In any clinical situation, a detailed history of drugs and/or herbs taken as well as exposure to food should be recorded. Further detailed information into such exposures would often yield important clue as to the trigger of haemolysis. In fact, this had led to the discovery that food-colouring agents, which are aniline dyes approved by health regulations, were the culprit for the recent series of cases of massive haemolysis in G6PD deficient subjects admitted to University Department of Medicine, Queen Mary Hospital in Hong Kong. These episodes included ingestion of coloured-rice in two Muslim brothers after the month of Ramadan; two cases of ingestion of highly-coloured curry dishes in Indian restaurant and one case of ingestion of coloured streaded -pork noodle from a fast food store.^32-34

Neonatal jaundice (NNJ) with kernicterus

In areas where G6PD deficiency is common, there is a higher incidence of severe NNJ.^35-37 This typically occurs on Day 4 to 7, which is later than the haemolytic jaundice of ABO or Rh incompatibility. In G6PD deficient subjects, severe haemolytic jaundice resulting in kernicterus can occur beyond the first week of birth, sometimes even as late as three to four weeks of life, a situation not reported in the Causasian population.^38,39 While the additional stress of oxidant drugs or agents used in the newborn was responsible for some of the severe cases, e.g. exposure to Chinese herbs³⁸ like Coptis chinensis ("chuen lien"), Calculus bovis ("neu huang") arid thorn roses ("leh mei hua"); naphthaleine in moth balls³⁸ or sulphonamide power used in newborn umbilical wound,⁴⁰ the majority of the G6PD deficient infants have exaggerated jaundice without haemolysis similar to the non-G6PD deficient subjects. Infection and liver conjugation defect were thought to be important causative factors. The problem of neonatal jaundice and kernicterus has been recently reviewed by Yeung.³⁹

Clinically, it is important to screen newborns for G6PD deficiency and followed such infant more carefully for rising bilirubin at end of first week. With the prompt institution of phototherapy⁴¹ or oral phenobarbital,⁴² much of the severe jaundice requiring exchange transfusion can be averted. Such screening programme was successfully implemented in Hong Kong and Singapore and more recently, in Guangzhou.⁴³

Congenital nonspherocytic haemolytic anaemia (CNSHA)

Dacie⁴⁴ separated CNSHA into two types depending on the result of autoincubation of red cells: type I when the increased haemolysis is correctible by the addition of adenosine triphosphate and type II when it is not. Type II CNSHA is now shown to be mostly due to pyruvate kinase (PK) deficiency. G6PD deficient patients with chronic haemolytic anaemia usually showed Type I result and is more common than PK deficiency.

From 1962-1982, 85 patients with G6PD and haemolytic anaemia were admitted to the University Department of Medicine, Queen Mary Hospital, in Hong Kong, four died of massive intravascular hemolysis with renal failure and the rest recovered. Only one had chronic haemolytic anamia on follow up. As discussed above, these rare variant of G6PD deficiency result from mutation that affect the function of the enzyme more severely (vide supra).

Extra-erythrocytic effects

In G6PD deficient individuals, their tissue G6PD enzyme levels are also lower than normal in the leukocytes, platelets, liver, kidneys and adrenals.⁴⁵ However, since the deficiency is not complete and there are alternate pathways in nucleated cells for the generation of NADPH, most of these subjects do not suffer from any other cellular dysfunction nor disease. We have tested the production of cortisol by the adrenals and the metabolism of cortisol as well as the bromsulphalein excretion by the liver and found these to be normal.^34,46

In some variants of G6PD deficiency with absent leucocyte G6PD level, there may be abnormal leucocyte function and such subjects present with proneness to infection, similar to chronic granulomatous disease.^47-49

There are many reports of disease association in G6PD deficient subjects. Some may be due to studies in heterogenous population where the association with G6PD deficiency is spurious. In our analysis of admission diagnosis and G6PD screened patients in the Hong Kong population, which is virtually homogenous in ethnic origin, only viral hepatitis and thyrotoxic periodic paralysis (TPP) patients have a disproportionally high incidence of deficiency.³³ The former can be explained by more severe jaundice in G6PD subjects with viral hepatitis and therefore an increased admission rate into hospital. The latter association has not been explained, although TPP patients are usually males and there has been reports of familial tendency.⁵⁰ It would seem that the genetic predisposition to TPP is linked to the G6PD deficient gene in the Southern Chinese.

EPILOGUE

G6PD Deficiency has been extensively studied since its discovery 40 years ago. It has taught us many lessons which can be summarized as follows:

1. It has helped to establish the discipline of PHARMACOGENETICS which is the study of the effect of individual genetic makeup that affect the efficacy, metabolism and side effects of drugs. Since some genetic trait are associated with ethnic groups, like G6PD deficiency, new drugs should be tested in more than one ethnic groups for safety parameters before approval. Furthermore, Continued Drug Monitoring after release should be in place, especially with Worldwide links, to spot unusual drug reactions early and to allow for prompt control.

2. G6PD gene showed marked polymorphism in many populations. It has illustrated the selective advantage of certain genetic trait by the environment and served also as a marker for origin of peoples, their migrations and genetic drift. It has also been used as a marker for the clonality of cancers.

3. The molecular basis of many of the enzyme variants has been determined and shed light onto the understanding of the structure-function relationship of enzymes in general.

4. Early detection of genetic trait, like G6PD deficiency, by neonatal screening is feasible and cost-effective. It allows the early preventive measures against severe jaundice and kernicterus to be implemented in neonatal life, as well as other preventive strategies later on in life including education of patient and doctors and the provision of a list of agents to be avoided (Table IV). Such G6PD deficient individuals can lead a full and normal healthy life. This serves as a prototype for other genetic defects where preventive and preemptive treatment are available. Obviously, such genetic trait does not warrant prenatal diagnosis and advice for termination of pregnancy. We are beginning to learn how to deal with individualized preventive and predictive health strategies instead of being draconian in our approach to health.

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