Meyer1997dat Van de 2

P1: SDA/MKV P2: SDA/VKS QC: SDA
February 18, 1997 11:30 Annual Reviews AR027-11 AR27-011N
Annu. Rev. Pharmacol. Toxicol. 1997. 37:269–96

Copyright c 1997 by Annual Reviews Inc. All rights reserved
MOLECULAR MECHANISMS OF
GENETIC POLYMORPHISMS
OF DRUG METABOLISM
Annu. Rev. Pharmacol. Toxicol. 1997.37:269-296. Downloaded from www.annualreviews.org
Urs A. Meyer
Biozentrum of the University of Basel, CH-4056 Basel, Switzerland
by University of Sussex on 11/05/12. For personal use only.
Ulrich M. Zanger
Dr. Margarete Fischer-Bosch-Institute for Clinical Pharmacology,
D-70376 Stuttgart, Germany
KEY WORDS: Genetic polymorphism, CYP2D6, CYP2C19, N-acetyltransferases, drug meta-

bolism, pharmacogenetics
ABSTRACT
One of the major causes of interindividual variation of drug effects is genetic
variation of drug metabolism. Genetic polymorphisms of drug-metabolizing en-
zymes give rise to distinct subgroups in the population that differ in their ability to
perform certain drug biotransformation reactions. Polymorphisms are generated
by mutations in the genes for these enzymes, which cause decreased, increased, or
absent enzyme expression or activity by multiple molecular mechanisms. More-
over, the variant alleles exist in the population at relatively high frequency. Ge-
netic polymorphisms have been described for most drug metabolizing enzymes.
The molecular mechanisms of three polymorphisms are reviewed here.
The acetylation polymorphism concerns the metabolism of a variety of ary-
lamine and hydrazine drugs, as well as carcinogens by the cytosolic N-acetyltrans-
ferase NAT2. Seven mutations of the NAT2 gene that occur singly or in combi-
nation define numerous alleles associated with decreased function.
The debrisoquine-sparteine polymorphism of drug oxidation affects the meta-
bolism of more than 40 drugs. The poor metabolizer phenotype is caused by
several “loss of function” alleles of the cytochrome P450 CYP2D6 gene. On the
other hand, “ultrarapid” metabolizers are caused by duplication or amplification
of an active CYP2D6 gene. Intermediate metabolizers are often heterozygotes or
carry alleles with mutations that decrease enzyme activity only moderately.
The mephenytoin polymorphism affects the metabolism of mephenytoin and
several other drugs. Two mutant alleles of CYP2C19 have so far been identified
to cause this polymorphism.
269
0362-1642/97/0415-0269$08.00
270 MEYER & ZANGER
These polymorphisms show recessive transmission of the poor or slow metab-

olizer phenotype, i.e. two mutant alleles define the genotype in these individuals.
Simple DNA tests based on the primary mutations have been developed to predict
the phenotype. Analysis of allele frequencies in different populations revealed
major differences, thereby tracing the molecular history and evolution of these
polymorphisms.
INTRODUCTION
The term genetic polymorphism defines monogenic traits that exist in the normal
population in at least two phenotypes, neither of which is rare (1, 2). Genetic
polymorphisms in drug metabolism are common occurrences. Mutations in the
genes for numerous drug-metabolizing enzymes cause enzyme variants with
higher or lower activity or lead to the partial or total absence of an enzyme

protein.
Genetic polymorphisms have been described for a wide variety of drug and
xenobiotic metabolizing enzymes. Many of these variations were first iden-
tified by the occurrence of adverse reactions after normal doses of drugs in
patients or volunteers. The association between decreased drug clearance and
decreased activity of a drug-metabolizing enzyme, the inherited nature of the
deficiency, and its frequency and clinical importance was then evaluated. Phe-
notyping methods involving administration of probe drugs and measurement
of metabolites in body fluids or determination of enzyme activity in families
and populations were used for this purpose. During the past 10 years, several
of these polymorphisms have been studied at the protein and gene level, and
simple DNA tests have been developed to predict the phenotype.
In this review, we summarize studies in our laboratory and in others of the
molecular mechanisms of three genetic polymorphisms, the acetylation (or
NAT2) polymorphism, the debrisoquine (or CYP2D6) polymorphism, and the
mephenytoin (or CYP2C19) polymorphism. We restrict our review to these
polymorphisms because their occurrence in different large populations is well
established, they involve clinically important drugs, and they represent the best
studied examples of these variations in regard to mutations of the genes and
their functional and clinical consequences.
In addition to the three polymorphisms described here, genetic polymor-
phisms are known for several other human cytochrome P450 genes that code for
drug-metabolizing enzymes, including CYP1A1, CYP2A6, CYP2C9, CYP2E1,
and for a number of conjugating enzymes, such as glutathion S-transferases,
methyl-transferases, and sulfotransferases. However, the importance of these
polymorphisms for clinically used drugs is less obvious at this time, or their
GENETIC POLYMORPHISM 271
genes have not been studied in detail yet. Recent reviews and books have been
published that cover various aspects of genetic polymorphisms and their clinical
or toxicologic relevance (3–11). Because of space limitations, we sometimes
refer to these reviews, rather than citing original articles.
THE N-ACETYLTRANSFERASE POLYMORPHISM

Discovery and Clinical Relevance
Variability in drug acetylation was discovered nearly 40 years ago when the fate
of isoniazid was studied in patients who had tuberculosis. Marked interindi-

vidual variation in the elimination of isoniazid was first described in 1953 by
Bönicke & Reif (12). Family studies revealed that this variation was geneti-
cally controlled and that “slow acetylators” of isoniazid were homozygous for
a recessive gene, whereas “rapid acetylators” were either homozygous or het-

erozygous for the normal or wild-type gene. In the following years, a number
of sulfonamides (sulfadiazine, sulfamethazine, sulfapyridine, sulfameridine,
and sulfadoxine), numerous other drugs (aminoglutethimide, amonafide, am-
rinone, dapsone, dipyrone, endralazine, hydralazine, isoniazid, prizidilol, and
procainamide), and metabolites of clonazepam and caffeine were discovered to
be polymorphically acetylated. Recent reviews have discussed the acetylation
polymorphism (6, 13–18).
The proportions of rapid and slow acetylator phenotypes vary remarkably in
populations of different ethnic or geographic origin. Most populations in Europe
and North America have 40 to 70% slow acetylators, whereas populations
along the Pacific Asian littoral (e.g. Japanese, Chinese, Korean, and Thai) have
only 10 to 30% slow acetylators. The molecular aspect of interethnic and
geographic variation of the acetylation polymorphism is discussed later in this
review.
The association of the acetylation polymorphism with drug toxicity, such
as sulfonamide hypersensitivity reactions (19), and its role as a risk factor for
certain diseases, in particular bladder cancer, has received much attention and
has been extensively studied (6, 13, 20–22). A discussion of these associations
and their presumed mechanisms is beyond the scope of this review.
Molecular Mechanism
STUDIES IN HUMAN LIVER Early studies clearly established that the observed
variation in acetylation of isoniazid was caused by a difference in the activity
of a cytosolic N-acetyltransferase in the liver that involved transfer of acetate
from acetyl coenzyme A (CoASAc) to the substrate (CoASAc; NAT, EC2.3.1.5)
Some arylamines, such as p-aminobenzoate and (PABA) and p-aminosalicylate
(PAS), are equally well acetylated by rapid and slow acetylators and therefore
272 MEYER & ZANGER
were called “monomorphic” in contrast to the “polymorphic” substrates iso-

niazid, sulfamethazine, and others (13, 23). Jenne (24) performed the first
detailed biochemical study of drug acetylation in liver tissue of rapid and slow
acetylators and suggested that two different enzymes were responsible for the
metabolism of monomorphic and polymorphic arylamines, one being quanti-
tatively decreased in livers of slow acetylators.
Major progress in the purification of a human liver acetyltransferase that
can acetylate the substrate sulfamethazine and the development of polyclonal
antibodies that recognize this protein was achieved in 1989 (25). In liver biopsy
specimens from surgical patients who had been phenotyped with the use of
the caffeine test, slow acetylation was associated with markedly reduced but
still detectable levels of immunoreactive NAT protein (26), which suggests a
quantitative rather than a qualitative difference of the human enzyme protein,
and confirms the initial proposal of Jenne (24). Subsequent studies (23) revealed
that an early unstable peak of NAT-activity on anion-exchange chromatography

with low affinity for sulfamethazine, but high affinity for the monomorphic
substrates PABA and PAS, had been overlooked. This fraction corresponds to
what we now recognize as the distinct but related enzyme NAT1, whereas the
initially purified protein (25) corresponds to the polymorphic NAT2.
In New Zealand white rapid acetylator rabbits, NAT protein and its mRNA
are more abundant. This finding allowed Andres et al (27) to prepare a highly
purified enzyme. Information from peptide sequences of this enzyme were used
by Blum et al (28) to synthesize an oligonucleotide probe and, in combination
with antibodies prepared against purified rabbit NAT, to clone a rabbit cDNA
encoding an enzyme with high specific activity for sulfamethazine acetylation.
Immunochemical studies suggested complete absence of this protein in slow
acetylator rabbits, in contrast to the only partial reduction of NAT2 protein
in livers of human slow acetylators. The absence of the NAT protein in slow
acetylator rabbits has now been explained by a gene deletion mechanism (28).
N-ACETYLTRANSFERASE GENES NAT1 AND NAT2 Human genes for acetyltrans-

ferases were cloned by Blum et al (29), who used the rabbit cDNA probe from
genomic DNA of an obligate heterozygous rapid acetylator individual identi-
fied by a pedigree phenotyping study. The recombinant clones define three
different, but closely related, human NAT genes: NAT1, NAT2, and the pseudo-
gene NATP. According to a recently proposed nomenclature, NAT1 corresponds
to NAT1∗ 3, and NAT2 corresponds to NAT2∗ 4 (18). The product of the NAT2
(NAT2∗ 4) gene expressed in COS cells showed protein immunoreactivity and ki-
netic properties identical to those of human NAT2 (NAT2∗ 4) (23, 26, 29), whose
content in human liver varies with the acetylator phenotype. This established
the NAT2 locus as the site of the classic human acetylation polymorphism. The
NAT1 (NAT1∗ 3) gene gives rise to a NAT protein with different electrophoretic
and kinetic properties. This protein corresponds to the monomorphic NAT1
(NAT1 3) enzyme with high affinity for substrates such as PABA and PAS (23).
Each of the two genes NAT1 and NAT2 has a single, intronless protein-coding
exon with an open reading frame of 870 bp that produces a 290 amino acid (33–
34 kDa) protein. Whereas the NAT1 gene produces its entire transcript from a
single exon, the NAT2 mRNA is derived from both the protein-coding exon and
a second, untranslated exon of 100 bp that is located about 8 kb upstream of
the translation start site (29–30). The nucleotide homology between NAT1 and
NAT2 is 87% in the coding region. There are only 55 amino acid differences,
only 28 of which are nonconservative changes. The homologies to the rabbit
cDNA rnat are 82% for both NAT1 and NAT2 (29). The pseudogene NATP
has high sequence homology to NAT1 and NAT2 (79%) but contains multiple
frameshifts and stop codons. NAT1 and NAT2 were mapped to chromosome
8p21.3–23.1 (29, 31).

The discovery of 2 separate genes that encode NAT1 and NAT2 has re-
solved the old question of monomorphic and polymorphic substrates (23).
Although NAT2 and NAT1 also have overlapping arylamine substrate speci-
ficities, e.g. for 2-aminofluorene and sulfomethoxazole (32), they also have
unique activities. Only NAT1 can metabolize PABA and the folate catabolite p-
aminobenzoyl-glutamate (33, 34). There is a conserved cysteine residue across
all vertebrate NATs that corresponds to cysteine 68 (18, 35, 36), whereas the
different substrate specificity between NAT1 and NAT2 seems to be related
to differences in the C-terminal portion. Recent studies suggest that NAT1
function also may be genetically variable in human populations (see below).
GENETIC VARIABILITY OF NAT2 At the protein level, the slow acetylator pheno-
type is associated with a reduction of 10 to 20% of the quantity of NAT2 in liver
cytosol (26), whereas the mRNA content remains unchanged (37). Several “de-
creased function alleles” of NAT2 have been identified that cause this decrease
in NAT2 protein and, consequently, the recessively inherited slow acetylator
phenotype (37–41) reviewed by Vatsis et al (18).
Decreased function alleles Mutant alleles of NAT2 were first identified by re-
striction fragment analysis of genomic DNA samples from healthy individuals
whose acetylator phenotype had been established by caffeine phenotyping (37)
or isoniazid (42) and of DNA from human liver samples with known NAT2 en-
zyme activity (37). Restriction fragment length polymorphisms (RFLPs) with
NAT2 probes generated by Mspl, Kpnl, and Bam HI segregated with acetylator
phenotype and defined three mutant alleles of NAT2, initially called M1, M2,
and M3 (37). To explore the molecular mechanism of slow acetylation, NAT2
genes were cloned from genomic libraries (37) or characterized as cDNAs (38),
274 MEYER & ZANGER
Table 1 Mutations of the NAT2 gene
Consequence for
Amino acid N-acetylation activity
Mutation change in vivoa in vitrob Reference
G191A R64Q Decreased Decreased a 41, b 140
C282T Silent (Y94) Decreased Normal a 44, b 37, b 39, b 52
T341C I114T Decreased Decreased a 44, b 5, b 52
C481T Silent (L161) Unknown Normal b 37, b 51
G590A R197Q Unknown Decreased b 37, b 52

A803G K268R Normal Normal a 45, b 51, b 52
G857A G286E Unknown Controversial b Agundez and Meyer, unpublished;

b 39, b 52
a
The in vivo effect of the mutation is derived from the acetylator phenotype in homozygous carriers of this
mutation. No homozygous carriers have been reported for the C481T, G590A, and G857A mutation.
b
Data from expression of recombinant NAT2 alleles with site-specific mutations and chimeric genes in various
systems, COS-1 cells (37), CHO cells (39, 51) or in E. coli (52, 140).
and their sequence determined and compared with the wild-type gene sequence.
The M1 allele had mutations at positions 341 and 481 (Tables 1 and 2), M2 at
positions 282 and 590 (37), and M3 at position 857 (38). According to a recent
nomenclature (18), M1 corresponds to NAT2∗ 5A, M2 to NAT2∗ 6A, and M3 to
NAT2∗ 7A (Table 2). Initially, only one mutation was reported at position 857
for NAT2∗ 7A (38). More recent studies now reveal two mutations in most if
not all NAT ∗ 7 alleles, one at position 857, and the other at position 282 (39).
These data were confirmed and extended by Vatsis et al (43) and by Hickman
et al (40), who sequenced amplified DNA from livers and lymphocytes of slow
acetylators by using the sequence information of Blum et al (29). Vatsis and co-
workers (43) observed an allele with mutations at positions 341, 481, and 803
(NAT2∗ 5B). Hickman and co-workers (40) observed an allele with mutations at
positions 341 and 803 (NAT2∗ 5C). Moreover, Bell et al (41) described a muta-
tion at position 191 that occurs almost exclusively in blacks. The mutations of
NAT2 and their functional consequences are summarized in Table 1.
Of the seven point mutations in the coding region, five cause amino acid
changes, and two are silent. Three mutations (G191A, C282T, and T341C),
when present on both alleles (homozygosity), demonstrably cause the slow
acetylator phenotype in vivo (41, 44), whereas homozygous carriers of a sin-
gle mutation A803G are rapid acetylators (45). No homozygous carriers of
mutations C418T, G590A, and G857A have been identified, even in large epi-
demiologic studies totaling more than 2000 DNA samples (44, 46–48).
The common slow acetylator alleles carry multiple mutations, as summa-
rized in Table 2. The NAT2∗ 5 A, B, C alleles share mutation T341C, whereas
Table 2 Common NAT2 alleles and their frequencies in caucasian populationsa
Mutations Allele designationb Associated phenotype Frequency (whites)
None NAT2∗ 4 (wild-type) Rapid 22.7c /21.6d

T341C, C481T, A803G NAT2∗ 5B (r3 , S1a) Slow 38.3/41.6
C282T, G590A NAT2∗ 6A (M2) Slow 27.8/23.6
T341C, C481T NAT2∗ 5A (M1) Slow 4.2/1.5
T341C, A803G NAT2∗ 5C (S1c) Slow 4.0/0.8
C282T, G857A NAT2∗ 7B (M3) Slow 1.3/1.2
C282T NAT2∗ 13 (S4) Slow 1.5/1.9
G590A NAT2∗ 6B (R2) Unknown <0.1/2.0

G191A NAT2∗ 14A (M4) Slow <0.1/0.6e
A803G NAT2∗ 12A Rapid <0.1/2.5
a
Compiled from published genotyping/phenotyping results, the presumed inactivating mutation (Table 1) is
underlined.
b
The nomenclature of NAT2 alleles is summarized in Ref. 18.
c
From data on 1688 alleles (Germany) (44)
d
From data on 1080 alleles (Spain) (46)
e
Allele 14A is found in frequencies of 7 to 9% in US blacks (41) and 5 and 9% in 2 native African populations
(50).
NAT2∗ 6A, ∗ 7B, and ∗ 13 share mutation C282T. Together they account for more
than 99% of mutant alleles in white slow acetylators (16, 44, 46, 47). Addi-
tional rare alleles are NAT2∗ 12A and the extremely rare NAT2∗ 5D, ∗ 5E, ∗ 12B,
∗
12C, ∗ 14B, ∗ 14C, ∗ 14D, and ∗ 14E (44–48), at least in whites. New point
mutations (A434C, A845C) and additional alleles have been reported (40, 46,
48, 49). The knowledge of practically all mutations of NAT2 that occur with
frequencies of more than 0.1% and the information on their in vivo relevance in
causing the slow acetylator phenotype has led to the development of genotyp-
ing tests of high efficiency and accuracy, either by allele-specific amplification
by polymerase chain reaction (PCR), by restriction analysis, or ideally by a
combination of the 2 procedures (37, 43–47, 49, 50).
Functional characterization of mutant alleles It is not entirely clear yet how

these mutations cause a decreased amount of NAT2 protein in livers of slow
acetylators. Initial studies in liver tissue (37) indicated that the genotype NAT2
∗
5A/ ∗ 5A and ∗ 6A/ ∗ 6A produce normal levels of NAT2 mRNA in the liver,
whereas the quantity of NAT2 immunoreactive protein was markedly reduced
or absent. None of these alleles appears to result in a total absence of NAT2
protein in the liver (26). Expression of wild-type–variant chimeric genes in
COS cells (37) or in CHO cells (51), however, produced controversial results.
In the study of Blum et al (37), both mutations of ∗ 5A were required. In the study
of Abe et al (51), however, the T341C mutation alone reduced enzyme protein.
The mutation G590A causing Arg197Glu in NAT2∗ 6A and the mutation G857A
276 MEYER & ZANGER
causing Gly286Glu of NAT2∗ 7B apparently result in proteins with reduced

stability (37, 52). Additional studies by Hein et al (52) with recombinant NAT2
allotypic variants expressed in Escherichia coli provide additional in vitro data.
The expressed products of NAT2∗ 5A, ∗ 5B and ∗ 5C had lower activities than
∗
6A and ∗ 7B, whereas ∗ 13 (C282T) had normal activity. Similarly, in vitro
expressions in mammalian cell systems suggested normal activity of expressed
recombinant NAT2 with C282T (37, 39, 52). These in vitro results are in
contrast to recent in vivo data from comparisons of caffeine metabolic ratios
with genotypes by Cascorbi et al (44). NAT2∗ 13 was clearly a slow acetylator
allele in homozygous carriers, and ∗ 5B had an apparently higher activity than

∗
6A. Thus, heterologous expression of NAT2 alleles in E. coli, COS, or CHO
cells apparently does not consistently reflect the effects of mutations on stability
or affinity for substrates of the protein in human liver (Table 1).
ALLELES OF NAT1 Recent studies by Cribb et al (53), Ward et al (54), and

Vatsis & Weber (55) have provided evidence that NAT1 function is also widely
variable in human populations. PAS acetylation (by urinary metabolic ratio) in
130 individuals was highly variable and suggested a bimodal distribution (56).
The comparison of sequences of NAT1 genes from 13 individuals revealed
two variants: allele V2 (NAT1∗ 10), with two mutations in the 30 UTR, and
allele V3 (NAT1∗ 11), with five point mutations and a nonanucleotide deletion
in the 30 UTR. These alleles were present in 50% of the individuals and showed
Mendelian inheritance in a small family. Sequence differences were also noted
between the wild-type (V1 ) allele and a previously published NAT1 sequence
derived from a Japanese individual (38).
The functional significance of these variants has not been tested. It is there-
fore not known whether the described variants are responsible for the apparent
catalytic differences seen with NAT1 specific substrates. These recent findings,
however, far from being resolved, put the term “monomorphic” for the NAT1
locus in question.
Interethnic Variability and Evolution

The frequency of the slow acetylator phenotype varies considerably among
ethnic groups. Genotyping data of the common NAT2 alleles now allows us to
trace the molecular history of the acetylation polymorphism (Table 3). Two
groups can be distinguished by the incidence of mutations T341C/C481T and
G857A. Whites and Africans are very similar and have high frequencies of
T341C/C481T (>28%) and low frequencies of G857A (≤5%). Japanese,
Chinese, Korean, and other Asian populations have a low incidence of T341C/
C481T mutations (≤7%) and a higher incidence of G857A mutation (10 to
18%). The difference in the incidence of the slow acetylator phenotype be-
Table 3 Interethnic comparison of NAT2 allelesa
Rapid
No. of acetylator
individuals alleles (%)b Slow acetylator alleles (%)b
Ethnic group analyzed NAT2∗ 4 (wt) NAT2∗ 5A/B/C NAT2∗ 6A/B NAT2∗ 7A/B NAT2∗ 14A/B
Whites, US 421 24 43 31 2 0
Whites, Europe 434 26 46 26 2 0
Whites, Spain 504 22 44 26 1 1
African Americans 214 35 30 23 5 8
Native Africans 102 27 40 22 2 9

(Gabonese)
Hispanics 148 40 28 18 14 1
Japanese 224 67 1 22 10 0
Chinese 254 53 5 30 12 0
Koreans 85 68 2 18 11 1
Filipino 100 40 7 36 18 0
Aborigines (Australia) 49 41 2 17 40 —
Amerindians
Ngawbe 71 74 2 4 24 0
Embera 101 65 10 18 21 0
a
Compiled and modified from Refs. 41, 44, 46–48, 50, 57, 142.
b
The sum of all alleles is not necessarily 100% because rare alleles were not considered in all studies.
tween Whites/Africans (40 to 70%) and Asian populations (10 to 30%) is thus
mostly the result of the low incidence of the T341C/C481T alleles (NAT2∗ 5A,
B, C) in Asians. Amerindians appear to be similar to Asian populations regard-
ing their high proportion of rapid acetylators and the T341C/C481T to G857A
relationship (57). Hispanics appear as intermediate between the White/African
and the Asian groups (47, 48). The data are consistent with the concept that the
acetylation polymorphism has an ancient and common African origin, before
spreading of human populations (58). The practical absence of the NAT2∗ 12
and ∗ 14 alleles (A803G and G191A) in whites as compared with the >8% in-
cidence of G191A in Africans suggests that these mutations occurred after the
African/Non-African split ∼90,000 years ago.
THE DEBRISOQUINE-SPARTEINE POLYMORPHISM

In 1977, physicians at St. Mary’s Hospital Medical School in London made the
serendipitous observation that a volunteer’s hypotensive response to debriso-
quine, a sympatholytic antihypertensive drug, was markedly increased because
of impaired metabolism (59). At about the same time, a group of physicians
278 MEYER & ZANGER
in Bonn, Germany, independently observed increased side effects associated

with decreased oxidative metabolism of sparteine, an alkaloid drug with antiar-
rhythmic actions (60). Family studies revealed that both oxidative metabolic
reactions are under monogenic control and that poor metabolizers are homozy-
gous for a recessive allele. A simple test, the urinary metabolic ratio (MR), after
administration of a probe drug can identify the extensive metabolizer (EM) and
the poor metabolizer (PM) phenotype. This test, which has been applied to
thousands of individuals, has documented that 5 to 10% of individuals in white
populations are of the PM phenotype, compared with only 1 to 2% in Asian
populations (6, 61).

The clinical importance of this polymorphism was initially questioned be-
cause the drugs leading to its discovery were soon either obsolete or of small
distribution. It soon became known, however, that many other drugs are inef-
ficiently metabolized in PM individuals. More than 40 clinically used drugs
are now identified whose metabolism in vivo cosegregates with that of debriso-
quine and sparteine. They include antiarrhythmics, such as N-propylajmaline,
flecainide, propafenone, and mexiletine; antidepressants, such as amitriptyline,
clomipramine, desipramine, fluoxetine, imipramine, maprotiline, mianserin,
paroxetine, and nortriptyline; neuroleptics, such as haloperidol, perphenazine,
and thioridazine; antianginals, such as perhexiline; opioids, such as dextrome-
torphan and codeine; and amphetamines, such as methamphetamine and
methylenedioxymethamphetamine (ecstasy). Moreover, a large number of
β-adrenergic receptor antagonists are influenced in their elimination by this
polymorphism (6, 10, 62, 65, 66).
Numerous case reports and clinical studies have demonstrated that for some
of these drugs, the polymorphic oxidation has therapeutic consequences, which
lead either to a higher propensity to develop adverse reactions at conventional
doses (as demonstrated by its discovery) or to decreased drug effects, e.g. ab-
sence of the analgesic effect of codeine in PMs (63) or therapeutic failure at
normal antidepressant doses in ultrarapid metabolizers (64). Moreover, epi-
demiologic studies have indicated that PMs and EMs may be at higher risk
for certain diseases, including Parkinson’s disease and some forms of cancer,
although many of these associations are disputed. Detailed reviews of the clin-
ical and toxicologic implications of the debrisoquine polymorphism have been
published (4–6, 62, 65, 66).
Molecular Mechanism
STUDIES IN HUMAN LIVER The identification of the target P450 isozyme took
several years of research by different groups, as has been reviewed in detail
(67). Early studies in human liver microsomes had suggested the deficiency of
a minor P450 enzyme with debrisoquine 4-hydroxylase activity in the liver of
PM individuals. However, purification of the respective P450 from human liver

was extremely difficult, and antibodies obtained against these fractions were
not specific enough to allow conclusions regarding the mechanism of deficient
metabolism (68, 69). With improved enzyme assays of high specificity and sen-
sitivity, the deficient enzyme was characterized in liver biopsy specimens from
donors who had known in vivo phenotype as a stereoselective (+)-bufuralol 10 -
hydroxylase (70, 71). Polyclonal antibodies against rat P450db1, which defined
the 2D subfamily in rats (72), were then shown to cross-react specifically with
human CYP2D6, and the levels of CYP2D6 determined in a large collection of
human liver specimens were highly correlated to bufuralol 10 -hydroxylase ac-

tivity (71, 73). The lack of immunoreactivity observed in several liver samples
of PMs suggested absence of CYP2D6 protein as cause for the metabolic defi-
ciency (71, 73). This result was further confirmed with monoclonal antibodies
(71, 74) and human LKM1 autoantibodies (75).
By using the polyclonal antibody against rat P450db1, a full length human
cDNA was isolated. Sequence analysis revealed a distinct, novel member of
the CYP2D subfamily (73). In addition to the biochemical evidence, one other
observation strongly suggested CYP2D6 as the target gene of the debrisoquine-
sparteine polymorphism. Eichelbaum and colleagues (76) found strong linkage
of polymorphic sparteine oxidation to the P1 blood group, which had been
mapped to the long arm of chromosome 22. The CYP2D locus (22q13.1) could
subsequently be mapped by using the CYP2D6 cDNA (77, 78).
CYP2D GENES The CYP2D6 wild-type locus in human beings is comprised

of the three highly homologous genes, CYP2D8P, CYP2D7P, and CYP2D6,
which are located in this orientation (50 to 30 ) on a contiguous region of about
45 kb (79–81). Like other genes of the CYP2 family, CYP2D genes consist of
nine exons and eight introns. CYP2D8P is a pseudogene that contains multiple
deletions and insertions and causes a highly disrupted open reading frame (79,
81). The CYP2D7P gene is more similar to CYP2D6 than to CYP2D8P, and
its coding sequence indicates only a single inactivating mutation, an insertion
of T226 in the first exon. However, no specific mRNA product was detected
in RNA from eight human livers, which indicates that it also is a pseudogene
(79). Other sequenced CYP2D7 genes also contain the T226 mutation (81–
83). Hence, CYP2D6 is the only CYP2D protein product in human beings, in
contrast to rodents, which express most or all of their five functional CYP2D
genes.
GENETIC VARIABILITY Basic information on the structural variability of the

CYP2D6 locus was obtained in an extended RFLP analysis, in which 20 re-
striction enzymes were used to identify phenotype-associated polymorphisms
280 MEYER & ZANGER
in leukocyte DNA of PM and EM individuals and their families (84). The

fragmentation patterns obtained with Xba I initially defined four allelic forms
of the CYP2D6 gene, represented by fragments of 29-kb, 44-kb, 11.5-kb, or
16 + 9-kb length, respectively (84, 85). However, only the alleles represented
by 44-kb and 11.5-kb fragments were clearly associated with the PM pheno-
type, thereby accounting for about one half the alleles in the PM group. The
remaining half was of the noninformative 29-kb type, which was also the most
frequent haplotype in EMs and thus represented the major polymorphic allele
(84). The 16 + 9-kb pattern was later also shown to be associated with the PM
phenotype (85).
Null alleles Different approaches were taken in several parallel investigations

to uncover primary mutations at the DNA level that could explain the PM pheno-
type (80, 82, 86, 87). By complete sequence analysis of a CYP2D6 allele (44M)
isolated from a PM individual characterized as 44-kb/11.5-kb by Xba I RFLP

analysis, several mutations were identified in introns and in exons, four of which
cause amino acid substitutions (82). One intronic mutation, G1934A, appeared
to be the most consequential difference to the wild-type gene in that it affected
the consensus acceptor splice site of the third intron. The mechanism proposed
for this mutation is outlined in Figure 1. The G residue within the consensus AG
dinucleotide of the 30 acceptor splice site of the normal gene is changed into an A
in the mutant allele. As the first exon residue is a G, a new consensus site forms
Figure 1 Mechanism of the common CYP2D6 B-mutation present in CYP2D6∗ 4 alleles as pro-
posed by Hanioka et al (82) and Gough et al (87).
one base downstream, thereby predicting an mRNA with a single base deletion
to be the spliced gene product (82). Indeed, variant cDNAs with this deletion
were isolated from libraries prepared from low in vitro activity livers (73, 87).
At the protein level, this mechanism predicts premature translation termination
to occur at codon 182 (Figure 1), thus explaining the absence of immunoreactive
P450 protein and enzyme activity in PMs homozygous for this mutant allele.
The major polymorphic allele, characterized as a 29-kb Xba I fragment
(84), was studied by Kagimoto et al (86), who constructed genomic libraries
from leukocyte DNA of three different PM individuals with a homozygous
29-/29-kb Xba I RFLP genotype. Sequence analysis of all exons and exon/intron
junctions and comparison to the CYP2D6 wild-type sequence (79) of sev-
eral clones revealed that an allele with multiple mutations, termed 29-B (or a
very similar allele, 29-B0 ; Figure 2) was present in all three individuals. The
Figure 2 Distribution and effect of the most common mutations of the CYP2D6 gene. Some
rare alleles are not included (see Ref. 92 for a more complete list). Positions of mutations are
numbered according to Kimura et al (79). Mutations are represented by open diamonds (silent
mutations), closed diamonds (mutations causing amino acid changes), and closed circles (loss-of-
function mutations) arranged along vertical lines that represent the alleles indicated on top. The
dotted line for CYP2D6∗ 5 indicates that the CYP2D6 gene is deleted. Frequencies for the whole
population (white) and for PM populations are approximate values (in %) compiled and modified
from Refs. 84, 89, 91, 95, 96, 99, 102, 103, 137, 138, 141, 143.
282 MEYER & ZANGER
seven mutations found in the sequenced areas were the same as those of the
44M allele and included the G1934A transition (86). COS cell expression of
chimeric CYP2D6 genes and mutant cDNAs that contain individual and groups
of mutations by Kagimoto et al (86) demonstrated that the G1934A mutation
completely abolishes expression of protein and activity. In further agreement
with the mechanistic model, mutant CYP2D6 mRNA observed in transfected
COS cells had the same size as wild-type mRNA (86). Diagnostic DNA tests
then confirmed the high frequency of this mutation among PM individuals (10,
88–91).
The other mutations present in the “B-type” alleles do not severely affect
protein expression and activity, with the exception of C188T (P34S), which
dramatically decreased enzyme activity but not protein expression in transfected
COS cells (86). This mutation, as well as a mutation in exon 9 (4268), is also
present in several functional alleles (Figure 2) and is discussed separately below.
According to a recently proposed CYP2D6 nomenclature system, alleles with

multiple mutations are grouped according to common detrimental mutations
(92). Group ∗ 4 comprises all alleles with the G1934A mutation, to which the
29-B (∗ 4A) and 29-B0 (∗ 4B), as well as the 44M alleles (∗ 4A) belong (92)
(Figure 2).
The second most frequent nonfunctional or null allele in whites is represented
by the 11.5-kb Xba I fragment, which was shown to be the result of a large
deletion mutation at the CYP2D locus, designated D-allele (80). The structure
of this allele was analyzed in genomic clones from a PM individual homozygous
for the XbaI 11.5-kb haplotype and was shown to comprise the pseudogenes
CYP2D8P and CYP2D7P, whereas the entire CYP2D6 coding sequence is
deleted.
Figure 2 summarizes the currently known loss of function mutations, most of
which can be detected with simple DNA tests (88, 93, 94). The more recently
reported alleles (∗ 6 to ∗ 16) were usually detected in PM individuals who had
none of the known mutations of alleles A, B, and D. The two most frequent
of these (∗ 6 and ∗ 3) also result in premature translation termination because of
single base deletions. In contrast, the single mutation of allele ∗ 7 predicts an
amino acid substitution, H324P, in exon six (95). Expression of CYP2D6(324P)
in COS cells and in insect cells resulted in comparable amounts of mutant and
wild-type protein, but the 324P mutant lacked heme and was enzymatically
inactive (96).
Functional alleles Among EM individuals, the MR for debrisoquine varies

up to 1000-fold, from as low as 0.01 up to the antimode of 12.6 for debrisoquine
(64, 97) and to a similar extent for other substrates. The terms “intermediate
metabolizer” (for MR1-12.6) and “ultrarapid metabolizer” [for MR < 0.1, (64)]
have been used to describe these phenotypic subpopulations. Some recently

identified mutations can be correlated to increased or decreased metabolic ca-
pacity and thus appear to contribute to the extreme variability among EMs.
In two human liver samples with low CYP2D6 enzymatic activity, trace
amounts of a cross-reactive protein with slightly higher apparent Mr were de-
tected (74). Subsequent cDNA cloning revealed a CYP2D6-C variant (∗ 9) with
a deleted codon 281, coding for lysine (98). The kinetic parameters determined
with several substrates for the mutant enzyme expressed by recombinant vac-
cinia virus in HepG2 cells were comparable to those of the wild-type. Based
on the in vitro analyses of liver microsomes, it was initially thought that the
allele could be associated with the PM phenotype, but a family study with sev-
eral members of the C/D (∗ 5/∗ 9) genotype then allowed the correlation of this
allele to intermediate debrisoquine MRs (99). The overall C-allele frequency
in whites is about 1% (97). Interestingly, however, a higher frequency of this
mutation was reported in Spain (100).

The C188T mutation, which causes a Pro to Ser amino acid change at position
34, was first identified as one of several amino acid substitutions in the B-type
alleles (82, 86) and was shown by expression in COS cells to impair CYP2D6
enzyme function, but not expression, dramatically (86). Because of this effect,
alleles with this mutation but absent detrimental G1934A transition, as first
observed in Asian populations and termed J (for Japanese) or Ch (for Chinese)
(83, 101, 136), are grouped in family ∗ 10 (Figure 2). In whites, these alleles
are much less frequent than in Asians, but may contribute to the intermediate
metabolizer phenotype, as the presence of this mutation was correlated to higher
MR of EMs in several studies (83, 101, 102, 136). As noted earlier (82), Pro34
is part of a proline-rich region that is highly conserved among microsomal
P450s and may function as a hinge between the hydrophobic membrane anchor
and the globular heme-binding portion of the enzyme. Enzymatic activity
of COS cell–expressed P34S mutant CYP2D6 was not detectable (86). In
combination with a mutation in exon 9 (allele ∗ 10; Figure 2), it was estimated
to be about 5% of the wild-type activity (83). Nevertheless, individuals who
have a combined genotype of one ∗ 10 and one nonfunctional allele are always
EMs, which indicates that the low amounts of functional enzyme produced by
∗
10 still provide sufficient metabolic capacity to cause metabolic ratios within
the EM phenotype range.
At the opposite side of the spectrum of EMs, the “ultrarapid metabolizer phe-
notype” was also shown to have a genetic basis. It was first observed in a clinical
setting in which two patients required antidepressants at unusually high doses
(“megaprescribing”) to attain therapeutic plasma concentrations (64). Both
patients were subsequently shown to carry one normal and one novel allele
characterized by a 42-kb Xba I fragment, which was distinct from the 44-kb
284 MEYER & ZANGER
haplotype (64). The extremely rapid metabolism in the two patients was sug-
gested to be the consequence of a gene duplication, which led to two functional
copies of CYP2D6∗ 2 on the 42-kb fragment, thus causing more enzyme to be
expressed (64, 97, 103). In another family with ultrarapid metabolism of de-
brisoquine in three members (MR < 0.02), molecular analysis demonstrated
dominant inheritance of a 13-fold amplified CYP2D6∗ 2 gene from the father
to two children (103). The mechanism of this excessive gene amplification re-
mains speculative and restricted to a single observation, whereas the duplicated
allele (CYP2D6∗ 2 × 2) was shown to occur with allele frequencies of ∼3.5%
in Spanish (104) and ∼1% in Swedish populations, where alleles with tripli-
cated CYP2D6∗ 2 were also observed (97). As much as 13% of an Ethiopian
population seemed to carry this gene duplication (105).
The implication of these findings is that each of the multiple CYP2D6∗ 2
copies on the variant amplified alleles is expressed and contributes to expression
of a functional protein. Although it was not explicitly shown that each of the
multiple copies represents a complete functional gene, the inverse correlation
found between the number of copies and the MR for debrisoquine strongly
suggests such a mechanism (97, 103).
Multiple copies and amplification of CYP2D6 apparently involves only the
variant CYP2D6∗ 2, which is distinct from the wild-type gene (79) at several
positions, including a gene conversion with CYP2D7 in the first intron and
two point mutations that cause amino acid substitutions R296C and S486T
(103). Being present also in a significant proportion of the genes of the com-
mon 29-kb haplotype, it represents the second most frequent functional allele
in whites, with an overall frequency of approximately 30% (103, 106). Corre-
lation studies suggest that this variant codes for a protein with normal function,
although position 296 maps to a putative substrate recognition sequence of
cytochrome P450.

As recently reviewed (6, 9), significant ethnic differences exist in the frequency
of the PM phenotype, which appears to be rare (1% to 2%) in most popula-
tions with the exception of Caucasians (5% to 10%). Interethnic variability of
CYP2D6 alleles is less completely studied than the NAT2 polymorphism, but
the existing data allow some conclusions on the origin of the most common
alleles (Table 4).
The most extreme variabilities are observed for the two allele families,
CYP2D6 ∗ 4 and ∗ 10 (Figure 2). The practical absence of the common “white”
B-allele from East Asian populations explains the very low prevalence of the
PM trait in the latter. A similarly low frequency of this allele was described
for two African populations (105, 107). In most African population studies,
Table 4 Interethnic comparison of CYP2D6 allele frequencies
No. of Nonfunctional
individuals Functional alleles (%) alleles (%)
Ethnic group analyzed ∗1 ∗2 ∗2 × 2 ∗ 10 ∗3 ∗4 ∗5
Whitesa Compiled 34 33 1.5–3.5 3 1 20 5

Chineseb Compiled 23 20 1 50 0 <1 5
Japanesec Compiled 42 12 0 33 0 0 13
Koreand 21 33 24 2 36 0 0 5
Ngawbe 30 17 0
Amerindianse
Sinhalesef 77 0 9
African 127 2.4 8.5 5.5
Americansg
Ethiopiansh 115 13 10 0 1.3
Zimbabweani
114 0 1.8 3.9

Data compiled and modified from references. The sum of all alleles is not necessarily 100%.
a
89, 91, 97, 103, 104, 138.
b
83, 101, 102, 139.
c
136, 139.
d
139.
e
108.
f
144.
g
6.
h
105.
i
107.
PM frequencies were also found to be low, although dissociations have been

observed for some probe drugs (6, 9). A possible explanation for these findings
is that the B-allele appeared relatively recently in Caucasoids after their sepa-
ration from Asian/Amerindian groups about 35,000 years ago (58, 107). The
intermediate frequency of ∗ 4 alleles in American blacks is most likely caused by
penetrance of Caucasian-derived genes (6). However, a high frequency (17%)
was observed in an Amerindian tribes by Jorge et al (108), an observation that
challenges the above hypothesis. The ∗ 3 allele (A-mutation) also appears to
be rare except in whites, whereas the deletion-allele, CYP2D6∗ 5, occurs with
comparable frequency in most populations.
Another major interethnic difference is a shift in the metabolic ratio distri-
bution to higher values in Chinese populations as compared with whites (9).
The molecular basis for this phenotypic difference is a very high prevalence
in Asians of ∗ 10 alleles with lower enzyme activity due to the C188T (P34S)
mutation. Its frequency in whites may have been overestimated initially, as it
appears to be rather rare according to recent data (91).
The two major functional ∗ 1 and ∗ 2 alleles appear to be frequent throughout
all populations, but one should keep in mind that the frequencies for these al-
leles are determined by the absence of other mutations. Therefore, undiscovered
286 MEYER & ZANGER
mutations may change the picture considerably in the future. Compared with
whites, a considerably higher incidence of gene duplication and amplification
alleles was reported for Ethiopians (105). The authors discussed the interest-
ing possibility that duplicated and amplified L-alleles (CYP2D6∗ 2 × N) may
have originated in Africa and transmitted to whites rather recently during the
immigration of Arabs into Spain, where a higher frequency is observed than in
the rest of Europe (104). There are no data yet on CYP2D6 alleles in Pacific
populations.
THE MEPHENYTOIN POLYMORPHISM

A genetic polymorphism of another cytochrome P450 isozyme was revealed
by the discovery of deficient 40 -hydroxylation of mephenytoin (3-methyl-5-
phenyl-5 ethylhydantoin; Mesantoin), a now rarely used anticonvulsant drug

(109, 110). The deficiency is restricted to one of the two major metabolic
pathways of mephenytoin disposition, namely stereoselective hydroxylation of
S-mephenytoin in the p-phenyl position to 40 -OH-mephenytoin. The other main
reaction, N-demethylation of R-mephenytoin to 5-phenyl-5-ethylhydantoin
(PEH; Nirvanol) remains unaffected. This deficiency is inherited as an au-
tosomal recessive trait (111, 112) and occurs with a frequency of 2.5 to 6% in
whites, but with a much higher frequency in Japanese (18 to 23%) and Chinese
(15 to 17%) individuals (6, 113). Recent reviews of this polymorphism have
been published (6, 113, 114). Individuals who have the PM phenotype are
predisposed to have the central adverse effects of mephenytoin, eg., sedation
after administration of a single dose of 100 mg for phenotyping purposes (115,
116). Additional substrates for the enzyme metabolizing S-mephenytoin to 40 -
OH-mephenytoin have been tentatively identified by in vitro inhibition studies
and some of these have been confirmed by cosegregation in populations. Thus,
the metabolism of the N-demethylated metabolite of mephenytoin (nirvanol),
mephobarbital, and hexobarbital, the side-chain oxidation of propanolol, the
demethylation of imipramin, and the metabolism of diazepam and desmethyl-
diazepam cosegregate with the 20 -hydroxylation of S-mephenytoin (6, 113,
114). More recent studies also suggest that the metabolism of the antidepres-
sant citalopram, the proton-pump inhibitor omeprazol (117), and probably the
related drugs pantoprazole and lansoprazole (UA Meyer, unpublished data), as
well as the antimalarial prodrugs proguanyl and chlorproguanyl (118), coseg-
regate with polymorphic mephenytoin metabolism. The clinical consequences
of impaired mephenytoin hydroxylation have not been studied in detail. There
is some evidence that the frequency of adverse effects of mephenytoin, mepho-
barbital, and hexobarbital may be higher in poor metabolizers of mephenytoin
(115, 116). Of potential significance would be a decreased efficacy of pro-

phylaxis for chloroquine-resistant P. falciparum malaria in PMs because of
the decreased formation of the active antiplasmodial metabolite cycloguanil or
chlorocycloguanil. Initial studies in Tanzania, however, did not support such
a relationship (119). May et al (120) have described an association of the PM
phenotype with scleroderma, particularly when it occurs in combination with
slow dapsone hydroxylation.
Molecular Mechanism
STUDIES IN HUMAN LIVER Initial studies in microsomes of liver biopsy spec-

imens from EM and PM individuals suggested the deficiency of a specific
P450 isozyme (121). Studies in several laboratories during the next few years,
however, failed to identify unequivocally the gene and the enzyme affected
by this polymorphism. Proteins were purified from human liver by Gut et al
(122) and Shimada et al (123) by monitoring S-mephenytoin 40 hydroxyla-

tion activity. Antibodies against the purified fractions inhibited S-mephenytoin
40 -hydroxylation, but there was no correlation between immunochemically rec-
ognized levels of these proteins and catalytic activity (124, 125). The issue has
now been resolved in that a protein with the N-terminus of CYP2C19, identified
by cloning studies (126), was purified by Wrighton et al (127). Immunoblots of
this protein correlated with S-mephenytoin 40 -hydroxylase activity. These data
were confirmed by Goldstein et al (128) by expression of a CYP2C19 cDNA in
yeast and refuted previous assumptions that variants of CYP2C9 may contribute
significantly to hepatic S-mephenytoin 40 -hydroxylase activity. CYP2C19 ap-
pears to be virtually absent on immunoblots of liver microsomes of PMs of
mephenytoin (129).
CYP2C GENES At least seven genes or pseudogenes of the CYP2C subfamily
appear to be localized on chromosome 10 (124, 130). The complete CYP2C18
gene and part of the CYP2C9 gene have been isolated and both are ∼55 kb in
size and have nine exons, as have other genes of family 2 (131). The CYP2C19
gene has not been studied so far, but it is closely related to other genes of the
2C subfamily, e.g. CYP2C9, with 95% similarity in the upstream region and in
several exons (114). According to recent nomenclature concepts, the wild-type
CYP2C19 gene may also be designated as CYP2C19∗ 1.
ALLELES OF CYP2C19 Reverse transcription and amplification of mRNA from
liver samples with low microsomal S-mephenytoin 40 -hydroxylase activity re-
vealed a 2C19 cDNA fragment with a 40-bp deletion at the beginning of exon 5,
which included a SmaI restriction site. This results in the deletion of 13 amino
acids (215–227) and shifts the reading frame, thereby producing a stop codon
20 amino acids downstream and consequently a truncated protein (234 amino
288 MEYER & ZANGER
acids), which would be devoid of a heme binding site and therefore be inactive.
Sequencing of amplified genomic DNA fragments from PM livers revealed a G
to A substitution in the coding sequence of exon 5 that corresponded to bp 681
of the cDNA. This mutation, designated CYP2C19m1 (or CYP2C19∗ 2) creates
an aberrant splice site that is apparently preferred exclusively over the normal
splice site. The abolition of a SmaI site by this mutation led to the development
of a convenient genotyping test. This mutation accounted for 75% and 83% of
the defective allele in a small sample of Japanese and white PM individuals,
respectively (129). A second mutation, defined as CYP2C19m2 (CYP2C19∗ 3),
has recently been discovered in DNA of a Japanese PM who did not have the
m1 mutation. CYP2C19m2 involves a G → A transition at bp 636 in exon 4,
which also creates a premature stop codon and destroys a BamHI restriction
site. This change would result in a truncated 211 amino acid polypeptide (132).
A PCR test for this mutation revealed that CYP2C19m2 accounts for the re-
maining defective alleles in Japanese or Chinese PMs. PCR-based tests for

the CYP2C19m1 and m2 mutations have been described (129, 132) and have
been evaluated in population and family studies to be of high sensitivity and
selectivity. However, whereas virtually all PMs in Japan had the genotypes
m1/m1, m1/m2, or m2/m2, the m2 mutation appears to be exceedingly rare in
whites.
A large family study (43 relatives of families of 11 PM probands) in Denmark
(133) revealed only one m2 mutation, and random testing of 800 alleles from
whites revealed no m2 alleles (114). In conclusion, the two CYP2C19 mutations
m1 and m2 explain virtually all PMs in Japanese and Chinese populations, but
additional mutations of CYP2C19 must exist in whites.

The ethnic and geographic distribution of the mephenytoin polymorphism has
been less well studied than that of the acetylation and debrisoquine polymor-
phism. A summary of 18 phenotyping studies that has recently been published
(134) includes Saudi Arabians, Filipinos, and Indians. An interesting relation-
ship between longitude and the frequency of PMs was observed, which suggests
a cline containing a step that occurs between Riyadh and Bombay. The Saudi
population resembled Europeans in the frequency of PMs for mephenytoin but
was similar to Asians in the frequency of PMs for debrisoquine.
DIRECTIONS FOR FUTURE RESEARCH

The initial observations of unexpected toxicity and the large number of com-
pounds metabolized by NAT2, CYP2D6, and CYP2C19 have resulted in a
considerable research effort to understand the molecular mechanisms of these
polymorphisms. For example, the unraveling of the complex and highly poly-
morphic CYP2D gene cluster on chromosome 22 has thus resulted in the char-
acterization of a total of 48 mutations and 53 alleles of the CYP2D6 locus alone
(91). In contrast to the detailed knowledge of molecular mechanisms, how-
ever, the clinical relevance of these polymorphisms is poorly documented and
is deduced largely from anecdotal reports and epidemiologic studies in small
numbers of patients. There is an obvious discrepancy between detailed molec-
ular information and only rudimentary clinical evaluation of the consequences
of these polymorphisms. The availability of genotyping tests should help de-
sign prospective clinical studies of adverse drug reactions and drug toxicity and
should ultimately serve to help in the adjustment of drug doses to individual
metabolic capacity.
The associations of these polymorphisms with individual risk to develop cer-
tain diseases suggest that the polymorphic enzymes may activate or inactivate
environmental carcinogens, other xenobiotics, or endobiotics. With the ex-

ception of amine carcinogens as substrates for acetyltransferases, however, the
search for chemical carcinogens that may be activated or inactivated by CYP2D6
or CYP2C19 has not been successful. Finally, the potential role of the discussed
enzymes in the metabolism of endogenous substances, for instance endogenous
codeine and morphine by CYP2D6 (135) or hormones and neurotransmitters,
as inferred from indirect observations, has not been evaluated (62).
ACKNOWLEDGMENTS
We thank Drs. F. Broly, J. Agundez, and M. Ingelman-Sundberg for sending
us data and copies of articles that were in press. The research in the laboratory
of UAM is supported by the Swiss National Research Foundation. Ulrich M.
Zanger is supported by the Robert Bosch Foundation, Stuttgart, Germany.
Visit the Annual Reviews home page at
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P1: SDA/MKV P2: SDA/VKS QC: SDA

February 18, 1997 11:30 Annual Reviews AR027-11 AR27-011N

Annu. Rev. Pharmacol. Toxicol. 1997. 37:269–96

KEY WORDS: Genetic polymorphism, CYP2D6, CYP2C19, N-acetyltransferases, drug meta-

270 MEYER & ZANGER

These polymorphisms show recessive transmission of the poor or slow metab-

higher or lower activity or lead to the partial or total absence of an enzyme

GENETIC POLYMORPHISM 271

THE N-ACETYLTRANSFERASE POLYMORPHISM

of isoniazid was studied in patients who had tuberculosis. Marked interindi-

a recessive gene, whereas “rapid acetylators” were either homozygous or het-

272 MEYER & ZANGER

were called “monomorphic” in contrast to the “polymorphic” substrates iso-

that an early unstable peak of NAT-activity on anion-exchange chromatography

N-ACETYLTRANSFERASE GENES NAT1 AND NAT2 Human genes for acetyltrans-

GENETIC POLYMORPHISM 273

8p21.3–23.1 (29, 31).

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Table 1 Mutations of the NAT2 gene

G191A R64Q Decreased Decreased a 41, b 140

C282T Silent (Y94) Decreased Normal a 44, b 37, b 39, b 52

T341C I114T Decreased Decreased a 44, b 5, b 52

C481T Silent (L161) Unknown Normal b 37, b 51

G590A R197Q Unknown Decreased b 37, b 52

A803G K268R Normal Normal a 45, b 51, b 52

G857A G286E Unknown Controversial b Agundez and Meyer, unpublished;

GENETIC POLYMORPHISM 275

Table 2 Common NAT2 alleles and their frequencies in caucasian populationsa

Mutations Allele designationb Associated phenotype Frequency (whites)

None NAT2∗ 4 (wild-type) Rapid 22.7c /21.6d

G590A NAT2∗ 6B (R2) Unknown <0.1/2.0

Functional characterization of mutant alleles It is not entirely clear yet how

276 MEYER & ZANGER

causing Gly286Glu of NAT2∗ 7B apparently result in proteins with reduced

allele in homozygous carriers, and ∗ 5B had an apparently higher activity than

ALLELES OF NAT1 Recent studies by Cribb et al (53), Ward et al (54), and

Interethnic Variability and Evolution

GENETIC POLYMORPHISM 277

Table 3 Interethnic comparison of NAT2 allelesa

Native Africans 102 27 40 22 2 9

THE DEBRISOQUINE-SPARTEINE POLYMORPHISM

278 MEYER & ZANGER

in Bonn, Germany, independently observed increased side effects associated

populations (6, 61).

GENETIC POLYMORPHISM 279

PM individuals. However, purification of the respective P450 from human liver

human liver specimens were highly correlated to bufuralol 10 -hydroxylase ac-

CYP2D GENES The CYP2D6 wild-type locus in human beings is comprised

GENETIC VARIABILITY Basic information on the structural variability of the

280 MEYER & ZANGER

in leukocyte DNA of PM and EM individuals and their families (84). The

Null alleles Different approaches were taken in several parallel investigations

isolated from a PM individual characterized as 44-kb/11.5-kb by Xba I RFLP

GENETIC POLYMORPHISM 281

282 MEYER & ZANGER

According to a recently proposed CYP2D6 nomenclature system, alleles with

Functional alleles Among EM individuals, the MR for debrisoquine varies

GENETIC POLYMORPHISM 283

have been used to describe these phenotypic subpopulations. Some recently

mutation was reported in Spain (100).

284 MEYER & ZANGER

Interethnic Variability and Evolution

GENETIC POLYMORPHISM 285