G6PD Deficiency

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REVIEW ARTICLE
G6PD Deficiency
By Ernest Beutler
T HIRTY-FIVE YEARS ago Dr William Dameshek, the

first editor of the emerging journal Blood, invited me
to write a review on “The Hemolytic Effect of Prima-
leaflet.’*When hemolysis is severe, the urine turns dark
and the patient may complain of back pain. When G6PD
deficiency is relatively mild, as in the class 3 G6PD A-,*
quine.”’ At the time, primaquine sensitivity, which had just the hemolytic anemia is self-limited” because only the older
recently been shown to be caused by a deficiency of the RBCs are de~troyed’~ and young RBCs have normal or near-
enzyme glucose-6-phosphate dehydrogenase (G6PD); rep- normal enzyme activity. In patients with more severe forms
resented a unique example of an inherited deficiency of an of enzyme deficiency such as G6PD Mediterranean, young
enzyme that caused hemolytic anemia. cells are severely deficient in G6PD,I5 and as a consequence,
Although many other red blood cell (RBC) enzyme defi- hemolysis continues until well after the administration of
ciencies are now k n ~ w n , ~G6PD
“ deficiency still reigns as drug is stopped.I6*l7
the most common of all clinically significant enzyme defects, The fact that primaquine was only one of many drugs that
not only in hematology, but in human biology as a whole. precipitated hemolysis in G6PD-deficient individuals was
A variety of drugs and infections cause hemolytic anemia recognized early in our studies by in vivo challenge of ”Cr-
in persons with the deficiency, and nonhematologic sequelae labeled erythrocyte.” Therefore, in the 1950s, when aperson
have been claimed as well. Using classical biochemical tech- with G6PD deficiency developed hemolytic anemia, it was
niques, enormous apparent diversity of mutations causing generally assumed that hemolysis had been precipitated by
G6PD deficiency was documented in hundreds of publica- a drug, and whatever drug had been ingested was considered
tions. The distribution of the deficiency in different popula- to be culpable. As a result, a long list of drugs thought to
tions has been investigated exhaustively, and gene frequen- cause hemolysis evolved. On more careful study many of
cies of over 0.5 have been observed in some ethnic groups. them have been proven to be quite innocent with respect to
With the advances made possible by the cloning of G6PD the cause of hemolytic anemia in G6PD deficiency.”
cDNA and gene7.*has come a better understanding of the As a matter of fact, it is difficult to be certain in some
diversity that exists. In this review, I will attempt to put cases, whether a cause-and-effect relationship exists between
what we have learned in the past 35 years into perspective ingestion of a drug and hemolysis. The most robust data
and to touch upon what still needs to be learned. regarding the potential hemolytic effect of drugs and chemi-
cals comes from clinical investigations with ”Cr-labeled
CLINICAL MANIFESTATIONS erythrocytes. However, even results obtained using RBC sur-
vival studies can be misleading. Individual inherited differ-
Hemolytic Anemia ences in drug metabolism such as acetylator status play a
significant role in determining whether a drug will be hemo-
Drug-inducedhemolysis. G6PD deficiency was discov- lytic.’8~20*2’
Thus, if a recipient who efficiently catabolizes
ered as a result of a series of investigations performed to the active hemolytic metabolite of a drug is challenged, he-
understand why some persons were uniquely sensitive to the molysis will not be apparent, but the drug may be hemolytic
development of hemolytic anemia when they ingested the in a subset of individuals who metabolize the drug less effi-
8-aminoquinoline antimalarial drug primaquine? Thus, the ciently. Moreover, even when a drug does shorten RBC life
first and best-known morbid effect of G6PD deficiency was span, as shown by performing sensitive studies with ”Cr-
drug-induced hemolysis. Primaquine is but one of many labeled erythrocytes, the degree of hemolysis may be so
drugs that shortens RBC life span in G6PD-deficient persons modest as to be of no clinical significance.” Sulfamethoxa-
(see below). The administration of such drugs is followed, zole, a componentof the commonly used combinationSeptraa
after a 1- or 2-day delay, by a fall in the hemoglobin (Hb) and Bactrima, has been shown to produce shortening of the
concentration. Heinz bodies, particles of denatured protein
adherent to the RBC membrane, appear in the early stages of
drug administration and disappear as hemolysis progresses.” From the Department of Molecular and Experimental Medicine,
Another morphologic feature observed on the blood film is The Scripps Research Institute, La Jolla, CA.
the appearance of RBCs that have variously been designated Submitted June 15, 1994; accepted August 25, 1994.
“irregularly contracted RBCs,” “eccentrocytes,” “hemi- Supported by National Institutes of Health Grants No. HL25552
ghosts,” “double-colored RBC,” and “cross-bonded and RR00833 and the Sam Stein and Rose Stein Charitable Trust
cells.” The Hb of these cells is confined to one side of the Fund. This is manuscript 8667-MEM from The Scripps Research
erythrocyte, leaving the other part as a flat, Hb-free ghost. Institute.
In this portion of the cell, the inside surface of the membrane Address reprint requests to Ernest Beutler. MD, Department of
is tightly bonded.” Often Heinz bodies are included in the Molecular and Experimental Medicine, The Scripps Research Insti-
flattened region, where they may bulge visibly out of the tute, 10666 North Torrey Pines Rd, La Jolla, CA 92037.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
* G6PD variants have been ~lassified’~
as follows: class 1, heredi- indicate this fact.
tary nonspherocytic hemolytic anemia; class 2, severe deficiency; 0 1994 by The American Society of Hematology.
class 3, mild deficiency; class 4, not deficient variant. 0006-4971/94/8411-0044$3.00/0
Blood, Vol 84, No 1 1 (December l), 1994 pp 3613-3636 3613

361 4 ERNEST BEUTLER
Table 1. Drugs and Chemicals That Should Be Avoided hemolysis in favism may be more explosive than occurs as
by Persons With GGPD Deficiency a result of drug administration,"ingeneralthe course of
Acetanilid Primaquine hemolysis in favism is very similar to that occurring after
Furazolidone ( F u r o ~ o n e ) ~ ~ ~ , ~ ~ 'Sulfacetamide drug ingestion. Hemolysisdoes notusually begin for 24
Methylene Blue Sulfamethoxazole (Gantanol) hours after ingestion of the beans and hemoglobinuria may
Nalidixic acid (NegGram) Sulfanilamide continue for several days."
Naphthalene Sulfapyridine Mechanism of hemolysis. Themechanism by which
Niridazole (Ambilhar) Thiazolesulfone drugs and fava beans produce hemolytic anemia is not well
Isobutyl nitrite358 Toluidine blue understood. Such drugs donot lyse RBCs in vitro.37 Instead,
Na~hthalene~~'.~~' Trinitrotoluene (TNT)
they appear to inflict oxidative injury on the erythrocytes
Nitrofurantoin (Furadantin) Urate oxidase36z
and, therefore, are often designated as oxidative drugs. Be-
Phenazopyridine ( P y r i d i ~ m ) ~ ~ ' Phenylhydrazine
cause of its relatively high frequency in some areas in the
Unless otherwise indicated, references given in reference 19. Mediterranean region, the mechanism by which fava beans
produce hemolysis has received special attention, with the
suggestion that the pathogenesis of favism and drug-induced
RBC lifespan23 in Asiansubjectswith G6PD deficiency, hemolytic anemia may be essentially the same."Vicine,
but no significant hemolysis could be shownwhen this com- convice, ascorbate, and L-DOPA are abundantin fava beans
bination was usedclinically in patientswith G6PD A-.24 and have been considered candidate toxins. The most likely
RBCs from subjects withsevereclass 2 variants such as offenders are vicine and convicine, ,&glucosides of pyrimi-
G6PD Mediterranean may be sensitive to drugs when those
with milderdefectssuchasG6PDA-are not. Thedata
obtained from"Cr survival must be supplemented with less Table 2. Some Common Drugs That CanSafely Be Administered in
reliable information gained from clinical observations. Clini- Therapeutic Doses to GGPD-Deficient Subjects Without
Nonspherocytic Hemolytic Anemia
cal studiesare confounded by the effect of intercurrent infec-
tions which may be responsible for hemolysis rather than Acetaminophen (paracetamol, Tylenol, Tralgon, hydroxyacetanilid)
the drug that has been administered. For example, the clinical Acetophenetidin (phenacetin)
observations that hemolytic anemia is caused by acetamino- Acetylsalicylic acid (aspirin)
phenhave been madeduring the concurrent presence of Aminopyrine (Pyramidon, amidopyrine)
infectionz5; investigations of the putative hemolytic effect of Actazoline (Antistine)
Antipyrine
this drug with "Cr-labeled erythrocytes fail to show shorten-
Ascorbic acid (vitamin C)*
ing of RBC life spar^.^"^^ Reports of single cases implicating Benzhexol (Artane)
agentssuchas melphalan,** dimer~aprol,'~ Chloramphenicol
and sodium metasophan noramidipyrine3' aredifficult to in- Chlorguanidine (Proguanil, Paludrine)
terpret. When more than a decade has passed without any Chloroquine
confirmingreport, one is inclined to regard the originally Colchicine
reported episode as being coincidental rather than etiologic. Diphenylhydramine (Benadryl)
Detailed analysis of the evidence regardingthe hemolytic Isoniazid
potential of a large number of drugs and chemicals has been L-Dopa
Menadione sodium bisulfite (Hykinone)
publishedpreviously.'' Table 1 listsdrugsandchemicals
Menapthone
that appear, on the basis of the available evidence, to cause
pArninobenzoic acid
clinicallysignificanthemolytic anemia. Drugs that can be Phenylbutazone
givensafely to G6PD-deficientpersons are listed in Ta- Phenytoin
ble 2. Probenecid (Benemid)
Favism. A clinical manifestation of G6PD deficiency Procainarnide hydrochloride (Pronestyl)
closely related to drug-induced hemolysis is the hemolytic Pyrimethamine (Daraprim)
anemia induced by ingestion of the fava bean, Vicia fabu. Quinidine
Favism, this hemolytic anemia, hasbeen known since antiq- Quinine
uity. Indeed, the demise of Pythagoras has been attributed Streptomycin
Sulfacytine
to unwillingness to enter a bean field, possibly because of
Sulfadiazine
favism," although the evidence supporting this interpretation
Sulfaguanidine
is feeble. Patients with favism are always G6PD deficient, Sulfamerazine
but not allG6PD-deficient individualsdevelop hemolysis Sulfamethoxypyridazine (Kynex)
whentheyingest fava beans. Thus, G6PD deficiency is a Sulfisoxazole (Gantrisin)
necessary but notsufficient cause of favism.Presumably Tiaprofenic acid''
some other factor,probably also geneti?and very likely Trimethoprim
related to metabolism of the activeingredients in the beans, Tripelennamine (Pyribenzamine)
is involved. The vast majority of cases of favism occurs in Vitamin K
individualswithseverelydeficient (class 2) variants of Unless otherwise indicated, references given in reference 19.
G6PD, but occasionally favism has been observed in a pa- *Very high "therapeutic" doses (-80 g administered intrave-
tient with
G6PDAlthough at times
the
onset of nously) have precipitated severe, even fatal, h e m ~ l y s i s . ~ " ~ ~
G6PDDEFICIENCY 3615
dine compounds that are converted by @glucosidases to LOCATION OF POINT MUTATIONS

their aglycones, vicine and isouramil, respectively. These
compounds form reactive semiquinoid-free radicals and can IN G6PD DEFICIENCY
generate active oxygen species. This results in the formation
of ferrylhemoglobin, methemoglobin, and inactivation of
15001 a
various enzymes. The reactions that occur are complex and
varied and, therefore, largely unpredictable.",384
New drugs continue to be introduced into medical prac-
tice, and it would be extremely useful to be able to predict
which of these cannot safely be given to patients with G6PD
deficiency. Unfortunately those drugs that do produce hemo-
lysis have no clearly understood common denominator either
in structure or in chemical properties. Moreover, in some
(perhaps in most) instances the injury to the enzyme-defi-
cient erythrocyte is not mediated by the chemical compound
that is administered, but rather by a metabolic product. In
vitro systems have been devised in an attempt to mimic what
occurs in the body?548 The RBCs of some animal species,
notably sheep,"' regularly have low RBC G6PD levels.
Fig 1. The distribution along the G6PD sequence of point muta-
Moreover, hereditary deficiencies occurring within species tions causing G6PD deficiencyis shown. Class 1 (0, nonspherocytic
are d o c ~ m e n t e d ; ~but
- ~ ~these have limited appeal with re- hemolytic anemia), class 2 (A,severedeficiency),andclass3 (W,
spect to attempts to predict the hemolytic potential of drugs moderate to mild deficiency) tend to have different distributions.
because of species differences in drug metabolism and of Class 1 mutants, in particular, tend to be clustered in the region of
the putative glucose-6-phosphate bindingsite (amino acid 205; cDNA
RBC metabolism. nucleotides 613-6151 and putative NADP-binding site (amino acids
Infection-induced hemolysis. Although, for historical 386 and 387; nucleotides 1,1561,161).
reasons, drug-induced hemolysis has attracted the most at-
tention, it is likely that hemolysis induced by infection may
be a more common cause of clinically significant hemolysis.
Numerous reports attest to the importance of infection in Presumably class 1 variants produce chronic hemolysis
causing hemolytic anemia.25,55-88 It is clear that many differ- because the functional severity of the defect is so great that
ent types of infections may trigger hemolysis in the G6PD- the erythrocyte cannot even withstand the normal stresses
deficient patient. The mechanism by which this occurs is not that it encounters in the circulation. The functional severity
clear, butan imaginative suggestion has been that during in these patients is not usually reflected by the level of the
phagocytosis, leukocytes damage erythrocytes in their envi- enzyme asit is measured in the laboratory. The RBCs of
ronment by discharging active oxygen species during phago- patients with class 1 variants may have residual G6PD activ-
cytosis.'* Perhaps nitric oxide might also play such a role.89 ity as high as 35% of normal9' when measured under stan-
It is unlikely that such a mechanism is operative in the case dard conditions. The functional impairment that leads to the
of viral infections such as hepatitis, but it may play a role shortening of the RBC life span in these patients may include
in some infections. such factors as susceptibility to inhibition by NADPH" and
Diabetes mellitus-inducedhemolysis. It has been sug- in vivo lability." Possibly the most consistent common fea-
gested that episodes of diabetic acidosis61.wmay precipitate ture of class l variants is the location of the mutation. In
hemolytic episodes in persons with G6PD deficiency, but in the great majority of cases, it is in the region of the putative
one study, no evidence was found that such an effect ex- NADP-binding or glucose-6-phosphate binding site of the
isted."It has also been reported that hypoglycemia may molecule. (see below and Fig l )
precipitate hemolysis in G6PD d e f i ~ i e n c y . ~ ~
Hereditarynonspherocytichemolyticanemia. It was in Neonatal Jaundice
1958,93not long after G6PD deficiency was identified as the Neonatal jaundice is one of the most life- and health-
cause of primaquine sensitivity, that it was recognized that threatening consequences of G6PD deficiency, and kemic-
the enzyme deficiency could cause chronic hemolysis as terus may occur in these It is often erroneously
well. The syndrome of hereditary nonspherocytic hemolytic assumed that the jaundice is the result of hemolysis. How-
anemia did not occur in persons who inherited the common, ever, this is apparently not usually the case. Anemia is not
polymorphic variants of G6PD such as G6PD A- or G6PD present in G6PD-deficient infants that develop neonatal ic-
Mediterranean, but rather in patients who had inherited rare terus.'@' Instead of the icterus being a manifestation of accel-
mutations, designated class 1 because of their association erated RBC destruction, it now seems likely that it is largely
with chronic hemolysis. (Exceptional instances in which the result of the impairment of liver function, presumably
class 2 variants have seemed to be associated with chronic because of a deficiency of the enzyme in the liver. It is
hemolytic anemiay4.95 are discussed below). The severity of entirely possible that some shortening of RBC life span also
hemolysis varies greatly. Although it is usuallymild,the plays a r01e.Io5Neonatal jaundice has occurred primarily in
patient with"G6PD C a m p i n a ~ ' ~ ~has ' ~ 'transfusion-de-
'~~ ~~i~I06-lO and
S Medit"ranean'",'Og-''' infants. In one
pendent hemolysis resembling thalassemia major, study,lo3G6PD A ~ r e s ' ~has
~ ' been associated with a particu-
361 6 ERNEST BEUTLER
larly highincidence of jaundice.Early reports from the more severely deficient variants indicated that other tissues
United States suggested that African-American infants did were indeedinvolved in G6PD deficiency.Mediterranean
not have a significantly increasedincidence of neonatal jaun- subjects were found to have leukocyte enzyme activity that
dice.l12-114 However, anecdotal observations from the United was only 22%'" and 39%'" of normal, platelet activity that
States"' andsurveys in Jamaica""' and in Africa"h"'X all was 19% of normal,'2" saliva activity that was 7% of nor-
suggest that an increased incidence of neonatal icterus may mal,'" and liver activity that was 60% of normal."' In one
occur also in infants with G6PD A-. study of liver biopsy samples, marked variability in the level
of enzyme was found,but the activity was consistently lower
Transfusion With G6PD-Dejcient Blood in G6PD A- subjects than in most subjects whodid not
have RBC G6PD deficiency."' G6PD activity could not be
There is evidence that G6PD-deficient RBCs maintain via-
detected in the breast milk of severely deficient mothers.'"
bility less well than do normal cellseven without being
Cultured fibroblasts from a deficient male from Ferrara, Italy
subjected to oxidative stress."' However, the consequences
hadonly 10% of normal G6PD activity.''s In Chinese pa-
of transfusing a single unit of G6PD-deficient RBCs into an
tients with severeRBCenzyme deficiency, the leukocyte
adult are probably minor. It has been pointed out that in the
G6PD activity was 25% of normal, platelets 28% of normal,
case of G6PD A-, the number of cells that would be de- liver 49% of normal,adrenal13% of normal and kidneys
stroyed if a hemolytic stress occurred would be no greater
13% of normal.'" Lensesfrom patients with G6PD A-
than the number of nonviable cells in a unit of blood nearing were found to contain about 40% of normal activity"' and
its expiration date.'2" Transfusion of G6PD-deficient blood
cataractous lenses of Mediterranean subjects were devoid of
may be an issue of greater potential importance in parts of
detectable enzyme when cataracts of patients with normal
the world in which the incidence of the defect is very high
erythrocyte G6PD levels averaged S mU/mg protein.''x
and where more severely deficient class 2 variants such as Hemcrtologic effects. In our original studies of G6PD
G6PD Mediterraneanare prevalent. In such areas, it is possi-
deficiency, 5'Crerythrocyte survival was determined on
ble for a patient to receive, by chance, several units of defi-
many primaquine sensitive subjects, who now would be des-
cient blood. In one instance, it has been suggested that fatal ignated as being hemizygotes for G6PD A-. The baseline
hemolysis occurred in a young woman as a result of receiv- RBC survivals of these subjects Subse-
was
ing G6PD-deficient blood,I2' but the reports of such severe
quently,studies of '2DFP and "Cr RBC survival in such
consequences arenot themselves convincingof a cause-and-
G6PD A- subjects wereclaimed to show marked shortening
effectrelationship. In acontrolledstudy,onlyminorin- of the erythrocyte life span, with a mean "Cr half-life of
creases in bilirubin levels were found in individuals receiv- only 20.2 in three subjects with a control mean of 28.7.'"'
ingaunit of severelyG6PD-deficient blood,'22 but the The average 32DFP-labeled RBC half-life of G6PD-deficient
changes might be greater if a hemolytic stress were present. subjects was reported to be 48 days with a mean normal of
In general, it has notbeenthepractice to screen blood 66.1 days."" Such shortening of RBC life-span has not been
bank blood for G6PD deficiency, even in areas in which the observed either before or after this one investigation, even
gene frequency is very high.'" However, caution is justified with much more severe forms of G6PD deficiency, and the
in the exchange transfusion of newborninfants.Here,in finding of marked shortening of RBC life span in G6PD A-
contrast to adults, the proportion of deficient cells could be subjects in the absence of astress cannotbe regardedas
very high, and the products of Hb catabolism disposed of valid. However, some minimal shortening of RBC life span
inefficiently by the immature liver."4"2h has been observed in some studiesof Mediterranean subjects
with G6PD deficiency: mean "Cr half-lives of 22.9 days,"'
Other Manifestations of G6PD Dejciencv 26 days,I4* and 28.9 days.'" Interestingly, even though the
It is reasonabletoassumethat agenetictraitthathas RBC life span of these subjects is nearly normal. three re-
reached such high frequencies in many populations that it is ports document a slight decrease in the Hb concentration of
carried by some200,000,000persons wouldnot have a the blood of normal subjects with the Mediterranean form
readily apparent effect onfitness. For this reason, if no other, of G6PD defi~iency.'~"'~' In one of these studies,'" the mean
it has been generally assumed that those who carry polymor- difference betweenthe Hb levels of deficient and nonnal
phic genes for G6PD deficiency would not suffer from any subjectswas nearly 2 g/dLandwasaccompanied by an
morbidity. Nonetheless, a number of studies have suggested increase in the average reticulocyte count of 0.28%. and of
that G6PD-deficient individuals might, even in the absence the mean corpuscular volume of 3.9 fL, all of these differ-
of any stress, have some clinical abnormalities. ences beingstatisticallysignificant. There have also been
Tissuedistribution of the deficiency. Early studies indi- occasional cases of apparent G6PD Mediterranean with low-
cated that the deficiency of G6PD activity was limited to grade h e m ~ l y s i s , ' j although
~.~~ these studies were performed
the RBCs; liver and leukocyte activity was reported to be before verification of the genotype by DNA analysis was
normal, and it was even suggested that the defect might not possible. Thus, it appears that under certain circumstances,
be in the G6PD gene itself, but rather some other gene that either genetic orenvironmental, low-gradehemolysis can
influenced the stability of the enzyme in the erythro~yte.'~' occur in persons with G6PD Mediterranean. It is doubtful
Plateletactivity was found to average about 40% of nor- whether this actually occurs in the milder, class 3 variants
mal.'*' such as G6PD A-.
These studies were probably performed on patients with Lqe expectancy. Large-scale studies haveassessedthe
G6PD A-, and subsequent investigationsinpatientswith effect of G6PD A- on the overall health of Afro-American
G6PD DEFICIENCY 3617
Table 3. Putative Abnormalities Suggested to Occur in Persons W

* GGPD Deficiency
Contradictory
Proposed Effect Evidence Class* Reference Reference
Platelet abnormalities 2 A 366, 367 368

Skin pedicle flap loss 3 A 369
Athletic performance, impairment 3 C 370
Seizure disorders A 1, 2 371. 312
Cataracts, increased incidence 1 A 3 11, 373-375 311
2 C 316 378.319
depressionof or
Manifestations schizophrenia C 3 192,380 381
C Renin release, impairment 382
383,384
tolerance
Glucose
3 C diabetes
tests, abnormalities
or 385-387
C Insulin release, abnormalities
production, Cortisol 388 2. 3 389
Survival, decrease 3 C 147
C Cardiovascular
factor disease, risk 391
Cholelithiasis 392
Myoglobuinuria
to Susceptibility 21 395
396 A 397
artery
Coronary disease,
C incidence
decreased 398 3. 4
ality function, Leukocyte 396, 12 399 400
2 jaundice Increased
A in hepatitis 401
Mental 402
Dehydroepiandrostrone
2 C levels
sulfate,
serum
increased 403
Abbreviations: A, anectodal; C, controlled series.
* Class 1, hereditary nonspherocytic hemolytic anemia; class 2. severe deficiency: class 3, mild deficiency; class 4, not deficient (eg, GGPD
A+'3). Classification based o n racial origin if data not given.
veterans. In an investigation of 1,413 black males Petrakis THE ENZYME

et all4' found that the incidence of G6PD deficiency was
12.1% in the 5- to 20-year age group, 5.6% in the 21- to Structure
49-year age group, and only 3.8% of those above the age of The G6PD monomer consists of 515 amino acid subunits
49. While acknowledging that there might be a number of with a calculated molecular weight of 59,256 daltons. The
explanations for this, they concluded that G6PD-deficient active enzyme exists as a dimer15h,'57 and contains tightly
subjects had a reduced life span. However, this seems unbound Aggregation of the inactive monomers
likely in view of the fact that it would require a very high into catalytically active dimers and higher forms requires the
excess mortality rate among persons with G6PD deficiency. presence of NADP@ '. ' Thus, NADP appears to be bound to
Indeed, a study of 65,154 black male patients admitted to the enzyme both as a structural component and as one of
US Veteran's Administration hospitals showed no increased the substrates of the r e a c t i ~ n . ~ The
~ ~ .binding
' ~ ~ . ~sites
~ ~ for
mortality among patients who were G6PD deficient and no this coenzyme have not been identifiedat the structural level,
significant difference in the mean ages of G6PD-deficient but examination of mutants has suggested that amino acids
and -nondeficient patients.14' 386 and 387, the basic amino acids lysine and arginine,
Cancer. Epidemiologic studies suggested to some that respectively, seem to bind one of the phosphates of NADP.'63
the incidence of cancer maybe lower in G6PD-deficient The evidence that this site is involved in the binding of
person^.^^^"^^ However, these investigations were generally NADP is as follows: (1) all mutants that rapidly lose activity
based on screening methods that do not efficiently ascertain at a 10 pmol/L NADP concentration, but are reactivated
G6PD deficiency in heterozygotes, and even in hemizygotes at high concentrations of NADP have been shown to have
who have a disorder that might decrease lU3C life span. mutations in this region; (2) mutations in this region result
Indeed, in one was
it shown that the RBC G6PD in paradoxical electrophoretic migration of the enzyme as if
activity of cancer patients is higher than that of controls. it hadbecome more positively charged, even when the amino
More recent studies tend not to show any differences be- acid change adds a negative charge, suggesting failure of
tween the incidence of cancer in G6PD-deficient and normal binding of negatively charged NADP. It has also been sug-
subjects.'s4.'55 gested, on the basis of the deduced conformation of the
Otherputativeclinical and laboratoryabnormulities. peptide chain of the yeast enzyme, that the NADP binding
The many other possible clinical and laboratory conse- site may be elsewhere," but the data on the human enzyme
quences of G6PD deficiency that have been proposed, either seems much more compelling to me. The glucose-6-phos-
on the basis of anecdotal observations or more detailed stud- phate binding site has been identified at amino acid 205 by
ies are summarized in Table 3. In many instances, contradic- locating a lysine at this position that is reactive with pyri-
tory data have also been presented and it is difficult to judge doxal phosphate in competition with glucose-6-phos-
the validity of the many claims that have been made. phate.165"68
3618 ERNEST
Table 4. GGPD Variants That Have Been Characterized at the DNA Level
~~ ~~ ~ ~~
-
Variant Nucleotide Substitution WHO Class Amino Acid Substitution References
Gaohe 95A-G 2 32 His Arg 404
Gaozhou
"Sunderland" 105-107 del 35 Ile - del 405
"Aures" 143T-C 48 Ile - Thr 406
Metaponto 172 G - A 58 Asp - Asn 407
A- 204
Distrito Federal 408
"Matera" 407
Castilla 202 G +
A 3 408
Alabama [376 A +
G] 409
Betica 242
Tepic 408
Ferrara 410
Ube
Konan
241 C - T 3 81 Arg - Cys 41 1
"Lagosanto" 242 G + A 3 81 Arg - His 412
"Vancouver" 317 C + G 1 106 Ser

182 Arg
198 Arg
-
+
+
Cys
Trp
Cys
413
Stio Borga 337 G + A 113 Asp - Asn 414
A 376 A G 126 Asn -Asp 41 5
-
--t
"Chinese-4" 392 G T 131 Gly -Val 416
466 G - A 156 Glu - Lys 407
-
"llesha"
Mahidol 487 G A 163 Gly + Ser 41 7
Plymouth 488 G A 163 Gly +Asp 418
- -
+
165 Asn Asp 218
-
"Chinese-3" 493 A G
"Shinshu" 527 A + G 176 Asp Gly 419
Santamaria 181 Asp +Val

126 Asn + A s p 1 420
Mediterranean 407
Dallas 268
Birmingham 563 C + T 2 188 Ser + Phe 268
"Sassari" 42 1
"Cagliari" 42 1
Panama 409
"Coimbra" 592 C T 2 198 Arg Cys 422
-
+ +
593 G C l 198 Arg Pro 423
-
"Santiago"
-
+
Sibari 634 A G 3 212 Met Val 251
Minnesota
Marion
Gastonia
637 G - T 1 213 Val - Leu 216
648 "Harilaou G 216 Phe - 217, 420
-
+ 1 Leu
City" "Mexico 3 680 G A 227 Arg + Gln 423

(Continued on following page)
GGPD DEFICIENCY 3619
Table 4. GGPD Variants That Have Been Characterized at the DNA Level (Cont'dl
Substitution
Nucleotide Variant References
WHOSubstitution
Class Acid Amino
A-
3
[ 227 Arg
126 Asn
IFP] 204
"Stonybrook" 724-729 1 242-243 219

GGC del Gly & Thr
Wayne 769 G - C 1 257 Arg - Gly 424
"Cleveland" 820 G -.A 1 274 Glu + Lys 219
"Chinese-l" 835A-rT 2 279 Thr + Ser 250
Seattle 421
Lodi 844 G + C 2 282 Asp + His 393
"Modena" 425
854 G - A 3 285 Arg - His 426
-
"Montalbano"
Viangchan 871 G - A 2 291 Val Met 424
-
Jammu
"West Virginia" 910 G - T 1 303 Val Phe 219
Kalyam 949G-A 3 317 Glu + Lys 427

Kerala
A-
Betica
Selma
968T-C
[376 A G +
1 3 323 Leu -, Pro
126 Asn -, Asp 1 204
"Nara" 953-976 del 1 319-326 del 428
Chatham 1003 G - A 3 335 Ala -,Thr 407
"Fushan" 1004 C -t A 2 335 Ala Asp
-
219
-
+
"Chinese-5' 1024 C T ? 342 Leu Phe 416
"lerepetra" 1057 C - T 2 353 Pro - Ser 423
Loma Linda 1089 C - A 1 363 Asn - Lys 216
"Olornouc" 1141 T - C 1 381 Phe -. Leu 219
Tomah 1153T-C 1 385 Cys - Arg 407
Iowa
Walter Reed
Iowa City
1156 A - G 1 386 Lys - Glu 163
Springfield
Guadalajara 1159C-T 387 Arg - Gys
--
423
"Mt. Sinai" 1159C-T 381 Arg Cys 248

376 A + G 126 Asn Asp
Beverly Hills
Genova
Worcester
1160 G - A 1 387 Arg - His
163
429
409
"Praba" 1166A-G 389 Glu - Gly 219
Nashville
Anaheim
"Calgary"
1178 G - A 393 Arg - His 216
430
"Portici"
Alhambra 1180G-C 1 394 Val - Leu
-
423
"Puerto Limon" 1192 G - A 1 398 Glu
~~ Lvs 420
(Continued on following page]
3620 ERNEST
Table 4. GGPD Variants That Have Been Characterized

at the DNA Level (Cont'dl
Variant Substitution
Nucleotide WHO
Substitution
Class Acid Amino References
Riverside 1228 G - T 1 410 Gly + Cys 163
"Japan"
"Shinagawa"
1229 G + A 1 410 Gly - Asp 423
419
Tokyo 1246 G - A 1 416 Glu - Lys 431
"Georgia" 1284 C - A 1 428 Tyr -+ End 219
"Varnsdorf" NIA 219

10 splice site del
Pawnee 1316 G - C 2 439 Arg - Pro 423
Telti
Kobe
1318 C - T 1 440 Leu - Phe 418
432
de "Santiago Cuba" 1339 G - A 1 447 Gly -t Arg 407
"Cassano" 1347 G + C 2 +His 449 Gln 251
Unlon
Maewo
1360 C - T 2 454 Arg - Cys 250 249,
247, 251
Andalus 1361 G - A 1 454 Arg - His 433
Cosenza 1376 G - C 2 459 Arg - Pro 251
Taiwan-Hakka
Gifu-like 2 1376 G + T 459 Arg + Leu 434
435
Kaiping
Anant 1388 G - A 2 463 Arg + His 434
Dhon
Petrich
Sapporo
"Campinas" 1 1463 G - T 488 Gly - Val

Class 1, nonspherocytic hemolytic anemia; class 2. severe deficiency; class 3, moderate deficiency; class 4, not deficient.
96
Enzymology backup mechanisms for each other. Which is more active at

G6PD catalyzes the first step in the hexose monophos- any one time may depend on the particular conditions under
phate pathway (HMP). It oxidizes glucose-6-phosphate to 6- which the measurements are being made and very likely the
phosphogluconolactone, reducing NADP to NADPH. The particular peroxide substrate that is being catabolized.
HMP is the only source of NADPH in the erythrocytes and The K, of G6PD for NADP is very low, roughly 2 to 4
it also serves to produce the ribose needed for synthesis of pmol/L, and the enzyme is strongly inhibited competitively
nucleotides in the salvage pathways. The main function of by NADPH. Thus, the NADPWNADP ratio within the RBC
the pathway seems to beto protect the RBC against oxidative controls the rate of the reaction in an autoregulatory manner.
damage. Glutathione peroxidase (GSHPx) removes peroxide In the quiescent state, the NADPWNADP ratio is very
from the e r y t h r ~ c y t e .Reduced
'~~ glutathione (GSH) serves high,'77~'78and G6PD is nearly completely inhibited. When
as a substrate for this enzyme, and because NADPH is re- NADPH is oxidized, as when oxidized glutathione is reduced
quired for the reduction of oxidized glutathione and protein in the glutathione reductase reaction, NADPH is converted
sulfhydryl group^,'^" it is an essential factor in the chain of to NADP and G6PD becomes active, reducing NADP to
reactions that defends the RBC against peroxide. RBCs are NADPH. G6PD-deficient cells are unable to respond ade-
a particularlyrich source of catalase, but this enzyme is quately to such an oxidative stress. When the susceptibility
relatively inefficient at removing low levels of peroxide lev- of a mutant enzyme to inhibition by NADPH is greater than
normal, this compounds the metabolic difficulty of the cell.'*
els. Moreover, catalase has the ability to bindNADPH
tightly'71,'7Z and the inactive form, compound 11, is reacti- GENETICS
vated by NADPH. Thus, the activity of the HMP serves to
remove peroxide not only through the action of GSHPx,
but also by activating catalase.I7' The long-standing con- X-Linkage and X-Inactivation
troversy about which is the more important, catalase or Even beforethe basic defect was known, X-linkage of
~ ~ ~ p ~ , 1 6 9 , I 7 3 -seems
176 to me tobe largely irrelevant and primaquine sensitivity was established by application of the
futile. Clearly, both enzymes may play a role and serve as glutathione stability test,179the earliest reliable means for
the detection of the defect of primaquine sensitivity. Pedi- had been published. Because most mutants of G6PD had
grees of Afro-American families showed"' that glutathione abnormal properties (either electrophoretic, kinetic, or both),
instability was most frequently transmitted from mother to it wasto be expected that the mutations affecting this enzyme
son, although there were instances in which the defect could would be found in the coding region. This has indeed proven
not be detected in the mother and even where apparent fa- to be the case. Facile polymerase chain reaction (PCR)-based
ther-to-son transmission occurred. It was correctly presumed methods for the detection of mutations have been devel-
that these anomalies were caused by inadequate ascertain- OPed,2'6-2'8 and these have made it possible to define the
ment of heterozygous females with the relatively crude tech- mutations in many individuals (Table 4).
nology then available. With the recognition that the basic Distribution and nature of mutations. As of this writing,
defect was a deficiency of G6PD, X-linkage was confirmed 60 mutations or combination of mutations have been docu-
by estimation of enzyme activity,'" studies of electropho- mented in G6PD (Table 4); all but one of these are associated
retic rnobility,IE2and study of linkage with color blindness.Is3with enzyme deficiency. The types of mutations found are
Still, there were families in which genetic transmission aber- more restricted than is the case with many other genes. It
rant for X-linkage was observed.IE4These aberrations led us appears that total G6PD deficiency is not compatible with
to suggest that one of the two X-chromosomes mightbe life. Thus, most mutations are missense point mutations and
inactive in human fern ale^,"^.''^ at the same time and quite deletions (of which three are known) and are found in multi-
independently of the proposal made by Lyon on the basis ples of three nucleotides so that a frameshift does not occur.
of X-linked traits in mice.'87 Only one splicing mutation has been found and no promoter
More recently, it has been appreciated that G6PD is one mutations have been identified. There is only one exception
of a cluster of genes on the distal long arm of the X chromo- to the rule that mutations found in patients do not preclude
some (q28). Included in this group of genes are those for the synthesis of enzyme; a mutation that we have designated
"Georgia" changes Tyr42' to a stop
the fragile X,188hemophilia A,189color v i s i ~ n , ' ~ a~putative
.'~' Eighty-three
gene for bipolar affective illness,Iy2the ABP-280 filamin percent of the peptide chain would have been synthesized
gene Bornholm eye disease,'94 clasped-thumb by the time the stop codon were encountered. Perhaps the
mental retardation (MASA) syndrome,'95 and dyskeratosis truncated protein made is partially functional. It is also note-
congenita."' worthy thatthe mutation was found in a female heterozygote,
and it is conceivable that unbalanced X-inactivation helped
The G6PD Gene to prevent the dire consequences that might otherwise have
been expected from a null mutation.
G6PD was cloned and sequenced by Persico et a173197-'yy The distribution of mutations along the length of the
and thenindependently by Takizawa and Yoshida.' The gene cDNA is also not random, as shown in Fig 1. Point mutations
contains 13 exons and is over 20 Kb in length. The first that cause the formation of class 1 variants, which are those
exon contains no coding sequence and the intron between associated with nonspherocytic hemolytic anemia, are
exons 2 and 3 is extraordinarily long, extending for 9,857 largely confined to two areas, one of which approximates
bp. The sequence of the entire gene is known.200At the 5' the NADP or NADPH binding site of the enzyme and the
end of the gene is a cytidine-guanine dinucleotide (CpG)- other of which is in the region of the glucose-6-P binding
rich island. Differential demethylation of some of the CpG's site. As shown in Table 4, of the 23 point mutations that are
is associated with expression of the gene on the active X associated with class 1 variants, 5 cause substitutions in the
chromosome2" and this island appears to be preserved be-
amino acid range 198 to 257 and 15 in the range of 363 to
tween man and mouse."' A 2,850-bp segment of the 5' end 447. Thus, 87% of these mutations are found in two areas
has been fused to a reporter and deletional analysis showed
that comprise only 28%of the polypeptide. There are, of
that a 436-bp domain was sufficient for full expression.203
course, exceptions and cases in which changing a single
Some heterogeneity of G6PD mRNA has been found, but
codon produces different clinical syndromes. Thus, changing
its functional significance is doubtful. The existence ofan
Met"'to Val produces a class I variant, G6PD Santiago,
alternatively spliced form has butbeen
whereas changing the same amino acid to Cys produces the
the amount of this mRNA, which contains 138 nucleotides
class 2 variant Coimbra. Similarly, the common class 2 vari-
of whatisusually the 3' end of intron 7 without losing
ant G6PD Union is the result of a mutation of Arg454to Cys,
frame, is always very small. Production of the enzyme has
whereas a change of the same amino acid to His produces
been accomplished in vitro in Cos cells2" and in Escerichia
coli,208-210 a variant, G6PD Andalus, associated withmild hemolytic
A suggestion2" that G6PD was, in reality, a trans-
anemia.
lation product made from two separate mRNAs has proved
to be based on an a r t i f a ~ t . ~ ' ~ - ~ ' ~ Frequency of mutations in various populations. The fre-
quency of G6PD deficiency differs markedly among differ-
ent populations. Among black Americans, the gene fre-
Mutations quency of enzyme deficiency is 0.10 to 0.1 l.'48.220The
Biochemical characterization has led to the description of frequency of G6PD Mediterranean56iTis 0.70 among Kurdish
no less than 442 variants of G6PD believed to be distinct. Jews?2' probably the highest incidence of G6PD deficiency
Two hundred ninety nine of these were characterized by in any population. In a Greek survey of over 1,200,000 in-
methods agreed uponby a World Health Organization fants, a gene frequency of 0.045 was documented.222In Asia
(WHO) expert gr0up13 and were considered, at least by those too, high frequencies are e n c o ~ n t e r e d . ~Detailed
~ " ~ ~ ~popula-
who described them, as being different from the others that tion frequency data may be found in a number of comprehen-
3622 ERNESTBEUTLER
sive reviews,",'26~227butthese data were compiled before

mutation analysis was possible.
In the era before mutation identification was possible at Table 5. Population Distribution of Common GGPD Mutations
the DNA level, it was generally recognized that certain types
Mutation Population Reference
of mutations were characteristic of certain populations. The
electrophoretic mobility of deficient samples from Africans G a ~ h e ' ~ ~ : G a o ~ h o u ' China
~~ 219, 249, 404
was almost always rapid, and the variant characteristic of A~res'~~' Algeria 406
this ethnic group was designated G6PD A-,182 in contradis- Saudi Arabia 103
tinction to the electrophoretically rapid, normally active Spain 2 47
G6PD A+ enzyme found in the same population. The more ,
ube24lT. KonanZ4"
Japan 41 1
severe deficiency, found in Mediterranean populations, in
which the residual enzyme had a normal electrophoretic mo- ~-202N376G~
Africa 245, 266, 436
bility was designated G6PD Meditemanean.228However, on Italy 410, 412
the basis of biochemical characterization it was believed that Spain 242, 241
there was great heterogeneity among the different variants Canary Islands 219
found in this region of the ~ o r l d . In~ Asia ~ ~ too,- ~ many
~ ~ Mexico 408
different variants were c h a r a c t e r i ~ e d . ~ ~ " ~ ~ ' China 336, 416
With development of the ability to define the mutations
Philippines 249
in the G6PD gene, some aspects of the situation became
more complicated, but others were simplified. G6PD A-, Southeast Asia 249, 417
which had generally been regarded as a distinct, homoge- Chinairaiwan 218,336
neous mutation, proved to be the result of the superimposi-
Costa Rica 420
tion of several point substitutions onthe background of Canary Islands 219
G6PD A+37hC. The mutation always found in G6PD A-'7h" Italy 412
is characteristic of G6PD A+. Inmost cases, the second
mutation is 202A, but it can also be 968C or 680T (Table Italy 407, 412
Sardinia 219, 421
4). The fact that African deficiency mutations of the G6PD
Greece 219
A- type appear to occur only in the context of the 376G
Saudi Arabia 103, 335
mutation of G6PD A+ suggested to us at one time that the Iran 335
primordial human G6PD may have been G6PD A+.242.243 It Iraq 335
is now clear on the basis of haplotype analysis that the A+ Israel 335
mutation is more recent in origin than the prototypal G6PD Egypt 335
~.244.24-5
A more attractive explanation for the association of Jews, Kurdish 221
the 3766 mutation with other African deficiency mutations Jews, Ashkenazi 409
is provided by the finding of Town et a1246that the 202A Seattle8"'* Italy 250
mutation produced by site-directed mutagenesis alone is not Spain 247
enough to produce enzyme deficiency; the 3766 mutation, Sardinia 219
which ordinarily does not produce enzyme deficiency, is Canary Islands 219
required. Thus, it is possible that the mutations at nt 202,
India 424
680, and 968 would have had no selective advantage against China 219
malaria whenthey occurred in a G6PD gene that did not Laos 249, 250, 424
have the 3766 mutation. However, two mutations that have Philippines 249, 250
been found to produce hemolytic anemia when present to- A-376GM8C Africa 204
gether with the 376G mutation also result in deficiency when
Spain 242, 247
found alone. These are 542TZ4' and 1 159T.248
Canary Islands 219
The data regarding the incidence of G6PD deficiency in
various populations223~225can now begin to be viewed from India 427
the perspective of which mutations are actually found. The Philippines 249
emerging data regarding the distribution of different muta-
tions in various populations is summarized in Table 5. In Chinaraiwan 336
general, mutations are limited to contiguous geographical Philippines/Laos 249, 250
areas or to areas where population migrations are well docu- China 219,336
mented. Thus, G6PD A- has a broad distribution that in- Japan 249
cludes all of Africa, Southern Europe, and wherever the Spain 247
slave trade brought Africans to the New World. G6PD Medi- Italy 251
terranean isfound in Southern Europe, throughout the middle China 219, 336, 434, 435
East and in India, and G6PD Canton is found in Asia. An
China 219,336, 434
interesting exception to this rule is provided by G6PD
unionl36OT. This mutationwas described originally in the Laos 250
Philippines and has indeedbeen documented at the DNA * See Table 4 for additional designations for the same variant.
level among Filipinos in H a ~ a i i . ~Surprisingly,

~ ~ , ~ ' ~ this vari- it, together with the panethnic intron 11 site, can be used to
ant has also been detected in Spain,z47Italy:" and in the investigate the origin of non-African mutations. Because it
Vanuatu archipelago in the Southwestern Pacific.252 is a part of the mature mRNA, the 1311 mutation serves as
When mutations are as widely dispersed as is G6PD a marker of gene expression. Nucleotide 1311 of the cDNA
Union'", the question arises of whether they represent re- is normally a T. However, in some individuals it is a C. We
current, independent mutations at a susceptible site in the found the mutant (C) genotype in 9/54 X-chromosomes from
gene, on the one hand, or whether they have a single origin Europeans of mixed origins, 9/41 X-chromosomes of Ash-
and have been spread through population flow. Study of kenazi Jewish subjects, 3/18 X-chromosomes of Sicilians,
other mutations in the G6PD gene that do not cause enzyme 5/20 African X-chromosomes and 9/20 Asian Indian X-chro-
deficiency, but represent polymorphisms that together consti- mosomes. In contrast, the mutation was found in only 3/59
tute various haplotypes, is a useful tool for the study of this Asian X-chromosomes and 3/30 CentraYSouth American X-
question. chromosomes?6' Because it is in the coding region, one may
G6PDpolymorphisms that do not causeenzymedeji- assess expression of the gene by reverse transcribing cellular
ciency. Although principal attention has been paid to muta- mRNA and examining the cDNA for the mutation. We have
tions of the G6PD molecule that produce enzyme deficiency, done this in the case of a patient with X-linked chronic
and therefore, may cause anemia, other mutations in the gene granulomatous disease, establishing the clonal nature of the
have been of considerable interest in studies of populations mutation269and it has also been adapted to study hematopoie-
and of genetic linkage. G6PD A+ is the polymorphism of sis in normal subjects and in a patient with polycythemia
this type that has been known for the longest period of time. vera.27o
Now recognized as a A+ mutation at cDNA nt 376, this
mutation was first discovered because of the faster electro- G6PD Dejiciency as a Balanced Polymorphism
phoretic migration of the enzyme.253It was used by Linder When a gene that has some potential for decreasing fitness
and Ga~tIe?'~to show that uterine myomas arose from single achieves a high frequency in some populations, it is neces-
cell precursors, by our group to show that lymphomas have sary to assume thatin those populations it also confers a
a single cell origin but that colon carcinoma may arise from survival advantage. Thus, a balance has been achieved be-
many and subsequently by others to study the patho- tween the advantage and the disadvantage conferred by a
genesis of many other neoplasms.256-262 gene, and this is designated a balanced polymorphism. One
Study of the DNA sequence has shown a number of addi- of the most studied of such polymorphisms is the mutation
tional polymorphic sites that are "silent" in the sense that for sickle Hb, and evidence from a variety of sources has
they do not change the sequence of the protein. Among led to the conclusion that the advantage conferred by this
Africans, there is a polymorphism in intron 5 creating a Pvu gene is resistance to falciparum malaria. The mortality
I1 site263and at nucleotide 1116 of the coding region creating caused by malaria in some parts of the worldis so high
a Pst I site.264Intron 7 contains a C-T substitution in some that a large number of genetic traits that defend against this
Africans. A Sca I site can be created with a mismatched infection have evolved in mankind, and many polymor-
primer and the polymorphic site has been designated phisms affecting the RBC seem to have reached high fre-
S ssca 9.245 Intron 11 also contains a polymorphism, wide-
9
quencies for this reason.z71
spread in many populations, that produces an Nla I11 site.265 Malaria. The geographic distribution of G6PD defi-
Another polymorphism in the coding region that does not ciency led M o t ~ l s k y , ~Siniscalco
~* et al,273Allison,'s' and
produce an amino acid substitution at nt 13 11is widespread Allison and Clyde274to suggest nearly 35 years ago that
in all populations. These polymorphic sites create haplotypes G6PD deficiency is also one of the polymorphisms that con-
that have been useful in establishing the order in which the fers resistance to infection with falciparum malaria. The evi-
various mutations of the G6PD gene arose. The G6PD dence for this, recently reviewed in detail by Greene,275
~-20ZAf376G
mutation is of quite recent origin and may have comes from several types of studies:
had a single origin.2u*24s~2M Based on the distance of these (1) Epidemiologic investigations indicate that the highest
mutations from the G6PD A- mutation at nt 202 we have gene frequencies are present among populations living in
calculated that it is extremely unlikely that G6PD A- arose low-lying areas in which the incidence of malaria is
more than 80,000 years ago, although its origin might have high.227.276 These relationships have been q ~ e s t i o n e d , ~ ~ ~ - ~ ~ ~
been much more recent.2u In contrast, G6PD Mediterra- and it has been suggested that an additional factor, perhaps
is found in the context of two different haplotypes. oxidative stress,275~27'may be required for G6PD deficiency
In most European patients with this mutation, a T is present to confer immunity to malaria. The malaria parasite appears
at nt 1311, whereas on the Indian subcontinent, most subjects to be sensitive to oxidative ~ t r e ~ ~and , ~
it has
~ ~been
- ~ sug-
' ~
with this mutation have a C at nt 1311.267,26' This finding gested that the eating of fava beans protects synergistically
is consistent with recurrent independent mutational events with G6PD deficiency against malaria,275~2'2~2'3itbut is diffi-
producing the G6PD Mediterranean563T mutation in different cuit to explain protection against malaria on this basis in
populations, but it is also possible that this mutation is very sub-Sahara Africa where fava beans are not cultivated.
old and thatcrossovers occurred in the gene. Similarly G6PD ( 2 ) Decreased parasitemia among patients with G6PD de-
J a r n t n ~ ' ~and
' ~ G6PD V i a n g ~ h a n ' ~occur
' ~ in different hap- ficiency when compared with normal individuals was origi-
lotypes and may have had separate origin^.'^' nally reported by and has been confirmed in a
The nt 1311 is of greater potential value than the other number of s t ~ d i e s . ~ ~A~ number
. ~ ~ . ~ of
' ~negative studies
polymorphic sites. Because this polymorphism is panethnic, have also been rep0rted,2'~*~'~ but are considered tobe
3624 ERNEST BEUTLER
flawed.’75 Although one study indicated that protection ex- Quuntitution of G6PD uctivity in erythrocytes. The sim-
tended only to heterozygous females,2xxthis conclusion has plest type of quantitative assaymeasuresthereduction of
not been borne outin other investigations, and it seems likely NADP to NADPHin the presenceof glucose-6-P andhemo-
that hemizygous males are also protected.275However, based lysate. In reality,thistype of assaymeasures both G6PD
on the now-disputed finding that it is heterozygotes that are and 6-phosphogluconate dehydrogenase(6-PGD) activity. In
resistant to malaria, an interesting explanation was devised. the reaction mixture, as in the cell, the immediate product
It was suggested that when deficient cells areparasitized that of the G6PDreaction, 6-phosphogluconolactone is converted
theparasite G6PD is eventuallyinduced, but thatthis re- to 6-phosphogluconate which serves as substrate for the 6-
quires several cycles in deficient host cells. Heterozygotes, PGD reaction. Thus,2 moles of NADP are reduced for each
who have a mixture of normal and deficient cells would host mole of glucose-6-Pconsumed in the mixture.Although
the parasite in normal cells sufficiently often to prevent the methods that measure G6PD activityindependently of 6-
induction of enzyme.253.’sYHowever, subsequent data from PGD deficiency have beenavailable for many years,”““”
the same group of investigators indicated that in reality, the such methods have little additional utility in diagnosing the
G6PD activity of the host cells did not influence the expres- deficiency state, because 6-PGDdoes not usually limit
sion of parasite enzyme.”” the rate of the reaction, particularly in G6PD-deficient indi-
(3) Studies in heterozygotes forG6PD deficiency, in viduals.
whomtwo populationsof RBCs coexist, show thatmore Screening ,for G6PD deficiency. In hemizygous males
parasites are present in the cells with normal enzyme activity who are not undergoing hemolysis, as will be found in popu-
than in the deficient cells. In an elegant investigation of the lation surveys, semi-quantitative or nonquantitative screen-
number of parasites in the RBCs of patients heterozygous ing methods are entirely adequate. Dye reduction tests, tirst
forG6PD deficiency, Luzzattoet al”’ showed that more introduced by Motulsky and Campbell-Kraut”’ as the bril-
parasites could be found in G6PD-sufficient than in G6PD- liant cresyl blue decolorization test, have been widely used.
deficient cells. Other receptors for the electrons from NADPHgenerated in
(4) In vitro studies show that malaria parasites grow less the G6PD and 6-PGDreactions include methyleneblue,’14~’lc
well in G6PD-deficient than normal cells.’5r,2X’.ZXLJ.2y2 MTT tetrazolium,”’ and methemoglobin.‘” A test in which
Sickling. Thecoexistence of thegenefor sicklingand protection against denaturation of Hb under oxidative stress
that for G6PD deficiency in the African population has led serves as an endpoint hasalso been d e ~ e l o p e d . ~ ”Although
”~
to many investigations regarding the possible relationships all of thesetests are still sometimes used,particularly in
betweenthese twodisorders.Insomestudies, apositive population surveys, they have largely been replaced by the
association
has
been found betweenthese
and
it fluorescent spot test, in which the generation of NADPH is
was suggested that the gene for G6PDdeficiency might con- detected directly visually under ultraviolet light.3y.”7~’”
fer an advantage on patients with sickle celldisease, pro- Detection of G6PD deficiency in patients undergoing he-
longing their survival. However, it was shown that sibs of molysis. Whilethediagnosis of deficientmalesordinarily
patient with sickle cell (SS) disease also had an equally high poses nospecialdifficulties, thesamecannot be written
incidence of G6PD deficiency and suggested that concor- about the detectionofG6PD deficiency in patients with some
dance betweenthese genes was not caused by a selective of the milder G6PD-deficient variants (class 3) undergoing
advantage, but rather by dilution of genes of African origin, a hemolytic episode. Because the oldermembers of the RBC
so that individuals with many African genes would have a population are selectively removed in patients with variants
higher probability of carrying both of these defects than such as G6PD A-,I4 leaving the younger cells with near-
individuals in whom the proportion of African genes was normal activity in the circulation,’ a screening test may give
lower.’‘’ Indeed, ithasbeen shown that G6PD deficiency quite normalresults, at least for aweek or twoafterthe
does notaffect the clinical course of sickle disease,""^"" hemolytic episode. The same problem in diagnosis does not
neitherincreasing its severityas had been suggested’”’ or exist in the case of severe (class 2) variants because in these
decreasing it as had also been propo~ed.”~ Moreover, most variants, even the very youngcells areseverely enzyme
studies of fairly homogeneous populations show theinci- deticient.lF.lh
dence of hemoglobin S and G6PD deficiency are quite inde- Severaldifferent approaches may be used todiagnose
pendent.’”3~’‘17 patients who have just undergone hemolysis. The simplest
i s merely to wait for a week or two or to perform family
DIAGNOSIS studies.Alternatively, one may deplete the sample being
studied of reticulocytes by centrifugation. The denser cells.
although not truly old as has sometimes been believed, are
Detection of G6PD Deficiency depleted of very young RBCS.”’ Accordingly, it has been
Before the underlying defect, G6PD deficiency, had been found that even during hemolysis, the dense fraction of cells
uncovered, two methods for detecting individuals sensitive is G6PD deticient.”’-’24 Another approach is to compare the
to the hemolytic effect of primaquine had been developed, activity of G6PD with that of another age-dependent RBC
the Heinzbody test”* and the GSH stability test.179Although enzyme suchas hexokinase or glutamic oxaloacetic transam-
still occasionally used, these surrogate tests are obsolete and ”’
inase. This approachhas been usedalso to detect the G6PD
no longer have a role in the diagnosis of G6PD deficiency. A - genotype in patients with sickle cell disease, in which
Instead, quantitative assays or screening tests that detect se- the mean RBC age is greatly decreased.Zy9
vere deficiency should be used to diagnose the disorder. The most powerful approach for establishing the diagnosis
in the context of hemolysis is analysis of genomic DNA to be distinct. Variants that were believed to be likely to
obtained from circulating leukocytes (see below). Neither be different, specifically, G6PD Cornel1 and Chicago, were
the presence of young erythrocytes nor, for that matter, of shown to be from members of the same extended family.334
transfused cells confounds the results obtained from such an The development of a number of PCR-based methods for
analysis. the detection of known mutations in G6PD has made it possi-
Heterozygotedetection. Detection of heterozygotes for ble to detect G6PD deficiency and to identify the specific
G6PD deficiency poses special problems. Because of X- mutation responsible with relative ease. The advantage of
inactivation, heterozygotes have two RBC populations.’s5,326 the use of this type of technology is that DNA samples are
One of these populations consists of normal RBCs and the much more stable than the enzyme in blood samples, and
other of RBCs that are as deficient as those of a hemizygous that very small volumes suffice for diagnosis. Methods of
male with the same deficient variant. On the average, half detection include the use of restriction endonucleases to
of the cells are normal and half are deficient. However, in cleave naturally occurring restriction sites’” or restriction
some heterozygous women most of the cells are deficient; sites produced by making mismatched oligonucleotides335~336
in others most are normal. The result of assaying the activity and allele-specific oligonucleotide h y b r i d i z a t i ~ n .These
~~~
of enzyme per gram Hb reflects the proportion of normal methods are sufficiently facile for population screening and
and abnormal cells in the individual being studied, and some require so small a sample that they can be used for prenatal
heterozygous women will have normal RBC enzyme activity diagno~is.~~’
whereas others will be grossly deficient in enzyme activity.
Thus, the usual RBC enzyme activity measurements cannot
TREATMENT
be relied upon for the detection of heterozygotes.
A more acceptable approach is to use techniques in which When hemolytic episodes occur in G6PD-deficient indi-
each RBC acts as an independent metabolic unit. Methemo- viduals, the inciting agent, drug or infection, should be re-
globin reduction can be used for this purpose, but only if moved whenever possible. However, in patients who have
the dye that links methemoglobin reduction to NADP re- class 3 variants such as G6PD A-, it may be possible to
duction does not result in cell-to-cell interaction. Nile blue continue essential drug therapy with careful monitoring of
sulfate can be used for this purpose, butnot methylene the blood count. Blood transfusion is only occasionally re-
blUe.327-329Reduction of a tetrazolium dye can also serve as quired to support patients who have undergone severe hemo-
an e n d p ~ i n t . ’ ~ Although
~~”~ such methods may be able to lytic episodes, usually in patients with favism.
identify heterozygotes with as few as 5% to 10% normal or It has been suggested3” that attacks of favism maybe
abnormal cells, some heterozygotes will escape detection ameliorated by the administration of desfemoxamine. In one
because virtually no normal or no abnormal cells are present patients with favism who received a single 500-mg
in the circulation. dose of desfemoxamine and packed RBC transfusions had
The most accurate method for heterozygote detection is a shorter duration of hemoglobinuria, greater rise in Hb level
to detect the mutation in genomic DNA. Although X-inacti- and more rapid drop in reticulocyte count than control pa-
vation may alter the methylation pattern on the inactive X- tients who received packed cells alone. However, it was not
c h r o m ~ s o m eand ~ ~prevent
~ ~ ~ ~transcription
~ of the inactive clear that both groups received the same volume of transfu-
gene,269it does not prevent the detection of the difference sion.
in the nucleotide sequence of the gene. Thus, heterozygote To permit NADPH to be produced by a different route,
detection by DNA analysis is entirely reliable, provided that xylitol administration has also been proposed as a way to
the mutation to be detected is known. prevent or treat hemolysis of G6PD deficiency.340Clinical
Ident$cation of G6PD variants. It became apparent studies in which two severely G6PD-deficient volunteers
early in the study of G6PD deficiency that there were differ- were pretreated with 10 g xylitol per day and then given
ences in the characteristics of the residual enzyme in differ- primaquine and 20 g xylitol per day showed no protection
ent deficient individuals. Fortunately, a WHO expert com- against hemolysis.341
mittee standardized the methods for the purification and It has been suggested thatvitamin E, by virtue of its
characterization of G6PD variants in 1967,13and most inves- antioxidant effect, might protect against chronic hemolysis
tigators subsequently used the same techniques for the exam- in G6PD deficiency causing chronic hemolytic anemia.
ination of different variants. The technology that was agreed Some studies have shown a favorable response to this vita-
upon consists of partially purifying the enzyme by absorption min141,342-344. , others have not.345,346
on and elution from diethylaminoethyl cellulose, followed The most dangerous consequence of G6PD deficiency is
by ammonium sulfate fractionation. The partially purified neonatal icterus. Kernicterus has been documented re-
enzyme is then examined kinetically, electrophoretically, peatedly in populations in which class 2 variants are com-
mon,100-103.107,’47”’2
and by measuring its thermal stability. This technology andit has been pointed out this is an
proved to be useful in obtaining a general impression of the important preventable form of mental retardation.”’ Photo-
degree of diversity of G6PD in various populations. How- therapy [ I1.3’3.354 has been used to reduce bilirubin levels, and
ever, the volumes of blood required were large, and it was phenobarbital has been used prophylactically with some suc-
often difficult to be certain whether relatively minor differ- cess.” Agar, given to reduce bilirubin reabsorption, was
ences in properties were caused by the existence of new found to be ineffective.”’ Exchange transfusion is required
variants or whether the observed variation was methodo- if the bilirubin exceeds 20 mg/dL,”’ but G6PD-deficient
logic. As pointed out above, 442 variants have been claimed blood should not be used for this purpose.
3626 ERNEST BEUTLER
5. Valentine WN, Paglia DE: Erythroenzymopathies and hemo-

lytic anemia: The many faces of inherited variant enzymes. J Lab
Clin Med115:12, 1990
6.ValentineWN,Tanaka KR, Paglia DE: Hemolyticanemias
and erythrocyte enzymopathies. Ann Intern Med 103:245, 1985
7. Persico MC, Viglietto G, Martino G, Toniolo D, Paonessa G,
Moscatelli C, Dono R, VulliamyT, Luzzatto L, D’Urso M: Isolation
of
human glucose-6-phosphatedehydrogenase (G6PD) cDNA
clones: Primary structure of the protein and unusual 5’ non-coding
region. Nucleic Acids Res 14:251 1. 1986
8. Takizawa T, Huang IY, lkuta T, Yoshida A: Human glucose-
6-phosphate dehydrogenase: Primary structure andcDNA cloning.
Proc NatlAcad Sci USA83:4157.1986
9. Beutler E: The red cell: A tiny dynamo, in Wintrobe MM (ed):
Fig 2. Crystals of human RBC G6PD [magnification x 120) (Cour- BloodPureandEloquent, New York, NY, McGraw-Hill.l980, p
tesy of Drs Enrico A. Stura, Jairo Aievalo, and Ian A. Wilson, The 141
Scripps Research Institute. Original magnification in Kodachrome x
IO. Dern RJ, Beutler E, Alving AS: The hemolytic effect of pri-
601.
maquine. 11. The natural course ofthe hemolytic anemia andthe
mechanismof itsself-limitedcharacter. J LabClinMed44:171,
Future Prospects 1954
I I . Arese P, De Flora A: Denaturation of normal and abnormal
It has now been almost 40 years since G6PD deficiency erythrocytes 11. Pathophysiology of hemolysis in glucose-6-phos-
was identified as the cause of primaquine sensitivity, and phate dehydrogenase deficiency. Semin Hematol 27: I, 1990
many thousands of papers documenting clinical events. pop- 12. Fischer TM, Meloni T, Pescarmona GP, Arese P Membrane
ulation distribution, biochemical characteristics, and molecu- cross bonding in red cells in faviccrisis: A missing link in the
lar biology have been published. Are there any questions mechanism of extravascular haemolysis. Br J HaematolS9: 159, 1985
that remain to be answered? The natural occurrence of many 13. BetkeK, Beutler E, BrewerGJ,KirkmanHN,LuzzattoL,
mutations and documentation of their biochemical effects is MotulskyAG,RamotB,Siniscalco M: Standardization of proce-
a rich resource for furthering our understanding of structure- dures for the study of glucose-6-phosphate dehydrogenase. Report
of a WHO scientific group. WHO Technical Report, Serial No. 366.
function relationships of enzymes. For this reason, both our
I967
group and Luzzatto’s, working together with crystallogra-
14. Beutler E, Dern RJ, Alving AS: The hemolytic effect of pri-
phers, have invested considerable effort in attempting to maquine. IV. The relationship of cell age to hemolysis. J Lab Clin
solve the three-dimensional structure of the enzyme. AI- Med 44:439, 1954
though we have succeeded in crystallizing the enzyme (Fig 15. Piomelli S, Corash LM, Davenport DD, Miraglia J, Amorosi
2), it is apparently too inhomogeneous to allow useful infor- EL: In vivo lability of glucose-6-phosphate dehydrogenasein GdA-
mation to be obtained. Thus, our understanding of the func- and Gd Mediterranean deficiency. J Clin Invest 47:940, 1968
tional sites has been limited to inferences drawn from the 16. Pannacciulli I, Tizianello A, Ajmar F. Salvidio E: The course
location of mutations that havewell-defined biochemical of experimentally-induced hemolytic anemiain a primaquine- sensi-
defects. Answers to other questions are needed as well. We tive Caucasian. A case study. Blood 25:92. 1965
would like to understand the difference between individuals 17. George JN, Sears DA, McCurdy P, Conrad M E Primaquine
who develop favism and those who do not. Are divicine and sensitivity in Caucasians: Hemolytic reactions induced by primaquine
isouramil really the active principles of the beans? What in G-6-PD deficient subjects. J Lab Clin Med 70:80. 1967
18. Dern RJ, Beutler E, Alving AS: The hemolytic effect of pri-
are the actual mechanisms of drug-induced and infection-
maquine. V. Primaquine sensitivity as a manifestation of a multiple
induced hemolysis? What is the mechanism of neonatal ic- drug sensitivity. J Lab Clin Med 4530. 1955
terus? Why is it the RBCs that are primarily affected in the 19. Beutler E: Hemolytic Anemia in Disorders of Red Cell Me-
deficiency state? tabolism. New York, NY, Plenum, 1978
It is of the nature of science that as we solve problems 20. Woolhouse NM, Atu-Taylor LC: Influence of double genetic
new problems arise. G6PD deficiency is no exception to this polymorphism on response to sulfamethazine. Clin Pharmacol Ther
rule. 3 I :377, 1982
21. Magon AM, Leipzig RM, Zannoni VG, Brewer GJ: Interac-
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1994 84: 3613-3636
G6PD deficiency
E Beutler
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From www.bloodjournal.org by guest on December 27, 2015. For personal use only.

T HIRTY-FIVE YEARS ago Dr William Dameshek, the

Blood, Vol 84, No 1 1 (December l), 1994 pp 3613-3636 3613

361 4 ERNEST BEUTLER

dine compounds that are converted by @glucosidases to LOCATION OF POINT MUTATIONS

361 6 ERNEST BEUTLER

G6PD DEFICIENCY 3617

Table 3. Putative Abnormalities Suggested to Occur in Persons W

Platelet abnormalities 2 A 366, 367 368

veterans. In an investigation of 1,413 black males Petrakis THE ENZYME

Gaohe 95A-G 2 32 His Arg 404

"Sunderland" 105-107 del 35 Ile - del 405

"Aures" 143T-C 48 Ile - Thr 406

Metaponto 172 G - A 58 Asp - Asn 407

"Lagosanto" 242 G + A 3 81 Arg - His 412

"Vancouver" 317 C + G 1 106 Ser

Stio Borga 337 G + A 113 Asp - Asn 414

A 376 A G 126 Asn -Asp 41 5

"Chinese-4" 392 G T 131 Gly -Val 416

466 G - A 156 Glu - Lys 407

Mahidol 487 G A 163 Gly + Ser 41 7

Plymouth 488 G A 163 Gly +Asp 418

165 Asn Asp 218

"Shinshu" 527 A + G 176 Asp Gly 419

Santamaria 181 Asp +Val

"Coimbra" 592 C T 2 198 Arg Cys 422

593 G C l 198 Arg Pro 423

Sibari 634 A G 3 212 Met Val 251

648 "Harilaou G 216 Phe - 217, 420

City" "Mexico 3 680 G A 227 Arg + Gln 423

GGPD DEFICIENCY 3619

"Stonybrook" 724-729 1 242-243 219

Wayne 769 G - C 1 257 Arg - Gly 424

"Cleveland" 820 G -.A 1 274 Glu + Lys 219

"Chinese-l" 835A-rT 2 279 Thr + Ser 250

854 G - A 3 285 Arg - His 426

Viangchan 871 G - A 2 291 Val Met 424

"West Virginia" 910 G - T 1 303 Val Phe 219

Kalyam 949G-A 3 317 Glu + Lys 427

"Nara" 953-976 del 1 319-326 del 428

Chatham 1003 G - A 3 335 Ala -,Thr 407

"Fushan" 1004 C -t A 2 335 Ala Asp

"Chinese-5' 1024 C T ? 342 Leu Phe 416

"lerepetra" 1057 C - T 2 353 Pro - Ser 423

Loma Linda 1089 C - A 1 363 Asn - Lys 216

"Olornouc" 1141 T - C 1 381 Phe -. Leu 219

Tomah 1153T-C 1 385 Cys - Arg 407

Guadalajara 1159C-T 387 Arg - Gys

"Mt. Sinai" 1159C-T 381 Arg Cys 248

Alhambra 1180G-C 1 394 Val - Leu

Table 4. GGPD Variants That Have Been Characterized

Riverside 1228 G - T 1 410 Gly + Cys 163

Tokyo 1246 G - A 1 416 Glu - Lys 431

"Georgia" 1284 C - A 1 428 Tyr -+ End 219

"Varnsdorf" NIA 219

Pawnee 1316 G - C 2 439 Arg - Pro 423

de "Santiago Cuba" 1339 G - A 1 447 Gly -t Arg 407

"Cassano" 1347 G + C 2 +His 449 Gln 251

Andalus 1361 G - A 1 454 Arg - His 433

Cosenza 1376 G - C 2 459 Arg - Pro 251