10
GENOMICS 14, 49-58 (1992) Evolution of a Highly Polymorphic Human Cytochrome P450 Gene Cluster: CYP2D6 MARKUS H. HElM AND URS A. MEYER Department of Pharmacology, Biocenter of the University of Basel, CH.4056 Basel, Switzerland Received February 17, 1992; revisedMay 13, 1992 The CYP2D gene cluster on human chromosome 22 containing the functional cytochrome P450 gene CYP2D6 and two or three highly homologous pseudo- genes is involved in a clinically important variation in the inactivation of drugs and environmental chemicals. Several mutant haplotypes of CYP2D6 have been iden- tified by restriction analysis and by PCR-based allele- specific amplification. To understand the evolutionary sequence of mutational events as well as recently dis- covered interracial differences, we analyzed the ar- rangement of the CYP2D haplotype containing a com- mon mutant allele of CYP2D6 associated with a XbaI 44-kb fragment. This haplotype contains four CYP2D genes instead of three. Comparison of the sequences of these genes with those of previously characterized hap- lotypes suggests that an early point mutation was fol- lowed by a crossover and a gene conversion event, the latter found preferentially in Caucasians. These data are consistent with the rapid evolution of this locus during "plant-animal warfare" with practical conse- quences for present-day defense of the organism against environmental adversity. © 1992 Academic Press, Inc. INTRODUCTION Cytochromes P450 (P450) are enzymes involved in the oxidative metabolism of numerous endogenous and exogenous molecules, including steroids, fatty acids, prostaglandins, leukotrienes, biogenic amines, phero- mones, plant metabolites, drugs, and chemical carcino- gens. These enzymes are present in all eucaryotes exam- ined and in at least some procaryotes and probably have existed for more than 1.5 billion years (Nebert and Gon- zales, 1987). In mammals the P450 superfamily consists of at least 10 families and a total of over 100 individual genes (Nebert et al., 1991). An interesting feature of these enzymes is their overlapping substrate specificity; a single P450 protein can metabolize numerous struc- turally diverse chemicals or one chemical can be metabo- lized by several P450s, providing the organism's capacity to metabolize and detoxify countless substances in the diet and environment. P450s with predominantly exoge- nous substrates presumably evolved to detoxify plant toxins (Gonzalez and Nebert, 1990). As plants and habi- tats changed, a particular P450 in a given species may not have been required for survival and its presence was no longer selected for. Mutant alleles of its gene could have spread in some populations. This may be the basis for the marked inherited variations in drug metabolism detected in rodents and man (Gonzalez, 1989; Meyer et al., 1990). Similar mechanisms also may explain inter- ethnic differences in drug metabolism (Kalow, 1991). A genetic deficiency of P450 CYP2D6 causes the de- brisoquine/sparteine polymorphism, an extensively studied genetic variation in oxidative drug metabolism in man. Five to 10% of the Caucasian populations of Europe and North America are "poor metabolizers," i.e., inefficient in the metabolism of the antihypertensive drug debrisoquine and over 25 other drugs, many of them derived from plant alkaloids (Meyer et al., 1990). The debrisoquine/sparteine polymorphism is caused by mutations of the CYP2D6 gene. This gene is part of a gene cluster on chromosome 22 that includes related pseudogenes, two of which have been sequenced, namely CYP2D7P and CYP2D8P (Kimura et al., 1989; Gonzalez et al., 1988a). Restriction fragment length polymor- phisms (RFLPs) after digestion with the restriction en- donuclease XbaI identify several haplotypes of this gene cluster. The three most frequent XbaI fragments were found to be 29, 44, and 11.5 kb, respectively (Skoda et al., 1988). They represent the following CYP2D gene clus- ters: The XbaI 29-kb fragment contains the two pseudo- genes CYP2D7P and CYP2D8P and the CYP2D6 gene. Four allelic variants of the C YP2D6 gene in this arrange- ment have been identified: the functional CYP2D6. WT wildtype allele and the defective CYP2D6.A, CYP2D6.B, and CYP2D6.C alleles (Kagimoto et al., 1990; Tyndale et al., 1991). These mutant alleles contain one or more than one inactivating point mutation and if present in the homozygous situation or combined with another defective allele result in the poor metabolizer phenotype. The XbaI 11.5-kb fragment lacks the entire CYP2D6 gene and consists of only the two pseudogenes CYP2D7P and CYP2D8P (Gaedigk et al., 1991). In the present study, we have analyzed the structure of the gene cluster variant that is characterized by a XbaI 49 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction iil any form reserved.

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Page 1: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

GENOMICS 14, 49-58 (1992)

Evolution of a Highly Polymorphic Human Cytochrome P450 Gene Cluster: CYP2D6

MARKUS H. HElM AND URS A. MEYER

Department of Pharmacology, Biocenter of the University of Basel, CH.4056 Basel, Switzerland

Received February 17, 1992; revised May 13, 1992

T h e C Y P 2 D g e n e c l u s t e r on h u m a n c h r o m o s o m e 2 2 c o n t a i n i n g the f u n c t i o n a l c y t o c h r o m e P 4 5 0 g e n e C Y P 2 D 6 and t w o or t h r e e h i g h l y h o m o l o g o u s p seu d o- g e n e s is i n v o l v e d in a c l i n i c a l l y i m p o r t a n t v a r i a t i o n in t he i n a c t i v a t i o n o f d r u g s a nd e n v i r o n m e n t a l c h e m i c a l s . S e v e r a l m u t a n t h a p l o t y p e s o f C Y P 2 D 6 h a v e b e e n id en - t i f i ed b y r e s t r i c t i o n a n a l y s i s a nd b y P C R - b a s e d a l l e l e - spec i f i c a m p l i f i c a t i o n . To u n d e r s t a n d t h e e v o l u t i o n a r y s e q u e n c e o f m u t a t i o n a l e v e n t s as w e l l as r e c e n t l y dis- c o v e r e d i n t e r r a c i a l d i f f e r e n c e s , w e a n a l y z e d t h e ar- r a n g e m e n t o f th e C Y P 2 D h a p l o t y p e c o n t a i n i n g a com- m o n m u t a n t a l l e l e o f C Y P 2 D 6 a s s o c i a t e d w i t h a XbaI 4 4 - k b f r a g m e n t . T h i s h a p l o t y p e c o n t a i n s four C Y P 2 D g e n e s i n s t e a d o f three . C o m p a r i s o n o f the s e q u e n c e s o f t h e s e g e n e s w i t h t h o s e o f p r e v i o u s l y c h a r a c t e r i z e d hap- l o t y p e s s u g g e s t s t h a t an e a r l y p o i n t m u t a t i o n w a s fol- l o w e d b y a c r o s s o v e r a nd a g e n e c o n v e r s i o n e v e n t , th e la t ter f o u n d p r e f e r e n t i a l l y in C a u c a s i a n s . T h e s e da ta are c o n s i s t e n t w i t h the r a p i d e v o l u t i o n o f th i s l ocu s d u r i n g " p l a n t - a n i m a l w a r f a r e " w i t h p r a c t i c a l c o n s e - q u e n c e s for p r e s e n t - d a y d e f e n s e o f the o r g a n i s m a g a i n s t e n v i r o n m e n t a l a d v e r s i t y . © 1992 Academic Press, Inc.

INTRODUCTION

Cytochromes P450 (P450) are enzymes involved in the oxidative metabolism of numerous endogenous and exogenous molecules, including steroids, fatty acids, prostaglandins, leukotrienes, biogenic amines, phero- mones, plant metabolites, drugs, and chemical carcino- gens. These enzymes are present in all eucaryotes exam- ined and in at least some procaryotes and probably have existed for more than 1.5 billion years (Nebert and Gon- zales, 1987). In mammals the P450 superfamily consists of at least 10 families and a total of over 100 individual genes (Nebert et al., 1991). An interesting feature of these enzymes is their overlapping substrate specificity; a single P450 protein can metabolize numerous struc- turally diverse chemicals or one chemical can be metabo- lized by several P450s, providing the organism's capacity to metabolize and detoxify countless substances in the diet and environment. P450s with predominantly exoge-

nous substrates presumably evolved to detoxify plant toxins (Gonzalez and Nebert, 1990). As plants and habi- tats changed, a particular P450 in a given species may not have been required for survival and its presence was no longer selected for. Mutant alleles of its gene could have spread in some populations. This may be the basis for the marked inherited variations in drug metabolism detected in rodents and man (Gonzalez, 1989; Meyer et al., 1990). Similar mechanisms also may explain inter- ethnic differences in drug metabolism (Kalow, 1991).

A genetic deficiency of P450 CYP2D6 causes the de- brisoquine/sparteine polymorphism, an extensively studied genetic variation in oxidative drug metabolism in man. Five to 10% of the Caucasian populations of Europe and North America are "poor metabolizers," i.e., inefficient in the metabolism of the antihypertensive drug debrisoquine and over 25 other drugs, many of them derived from plant alkaloids (Meyer et al., 1990). The debrisoquine/sparteine polymorphism is caused by mutations of the CYP2D6 gene. This gene is part of a gene cluster on chromosome 22 that includes related pseudogenes, two of which have been sequenced, namely C Y P 2 D 7 P and C Y P 2 D 8 P (Kimura et al., 1989; Gonzalez et al., 1988a). Restriction fragment length polymor- phisms (RFLPs) after digestion with the restriction en- donuclease XbaI identify several haplotypes of this gene cluster. The three most frequent XbaI fragments were found to be 29, 44, and 11.5 kb, respectively (Skoda et al., 1988). They represent the following CYP2D gene clus- ters: The XbaI 29-kb fragment contains the two pseudo- genes CYP2 D7 P and CYP2 D8 P and the CYP2D6 gene. Four allelic variants of the C YP2D6 gene in this arrange- ment have been identified: the functional C Y P 2 D 6 . WT wildtype allele and the defective C Y P 2 D 6 . A , C Y P 2 D 6 . B , and C Y P 2 D 6 . C alleles (Kagimoto et al., 1990; Tyndale et al., 1991). These mutant alleles contain one or more than one inactivating point mutation and if present in the homozygous situation or combined with another defective allele result in the poor metabolizer phenotype. The XbaI 11.5-kb fragment lacks the entire CYP2D6 gene and consists of only the two pseudogenes CYP2 D7 P and CYP2 D8 P (Gaedigk et al., 1991).

In the present study, we have analyzed the structure of the gene cluster variant that is characterized by a XbaI

49 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc.

All rights of reproduction iil any form reserved.

Page 2: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

50 HEIM AND MEYER

44 -k b f r a g m e n t , prev ious ly d e s i g n a t e d the "44-kb al lele ." A p p r o x i m a t e l y 30% of the m u t a n t al leles in poor m e t a b - ol izers are assoc ia ted w i th a 44-kb f r a g m e n t , a n d the e l u c i d a t i o n of i ts gene i n a c t i v a t i n g m e c h a n i s m wil l in- crease the p e r c e n t a g e of s e q u e n c e d m u t a n t al leles to

over 90%. Our resu l t s i nd ica t e t h a t the 44-kb f r a g m e n t c o n t a i n s

four C Y P 2 D genes ( compa red to th ree in the wi ld type c lus ter ) i n a reg ion s p a n n i n g more t h a n 40,000 bp, n a m e l y , t h ree p seudogenes a n d the m u t a n t C Y P 2 D 6 . B gene. T h e c o m p a r i s o n of t h i s a r r a n g e m e n t w i th the pre- v ious ly a n a l y z e d gene c lus te r s ref lected b y the 29- a n d 11.5-kb X b a I f r a g m e n t s (des igna ted 29wt, 29A, 29B, a n d 11.5 kb) c an exp l a in how a n d in w h a t s equence the dif- f e r en t m u t a n t c lus te r s evolved. A n u n e q u a l c rossover e v e n t b e t w e e n a f u n c t i o n a l h a p l o t y p e w i th a 29-kb frag- m e n t (hap lo type 2 9 W T or wi ld type) a n d a def ic ien t hap- lo type c o n t a i n i n g t he C Y P 2 D 6 . B allele a n d a 29-kb f r a g m e n t (hap lo type 29B) g e n e r a t e d a new def ic ien t hap- lo type w i th a n 11.5-kb f r a g m e n t (hap lo type 11.5) a n d a f u n c t i o n a l hap lo type w i th a 44-kb f r a g m e n t (hap lo type 44E, E for ex tens ive me tabo l i ze r ) a b o u t 1 m i l l i o n years ago. A f u n c t i o n a l 44E h a p l o t y p e has b e e n obse rved wi th a f r equency of 38% in the Ch inese p o p u l a t i o n ( J o h a n s - son et al., 1991). I n a s econd step, a gene c o n v e r s i o n e v e n t r e su l t ed in a m u t a n t hap lo type w i th a 44-kb frag- m e n t (hap lo type 44P, P for poor me tabo l i ze r ) f o u n d pref- e r e n t i a l l y in C a u c a s i a n s a n d c o n f e r r i n g def ic ien t m e t a b - o l i sm of d e b r i s o q u o n e a n d o the r drugs.

MATERIALS AND METHODS

DNA source and isolation of clones. Leukocyte DNA from a poor metabolizer phenotyped with debrisoquine and genotyped by RFLP analysis as Xba144/44 kb (Skoda et al., 1988) was completely digested with EcoRI and used for construction of a library in XEMBL4 (Fris- chauf et al., 1983). The library was screened with [a-32P]dATP-labeled CYP2D6 cDNA (Gonzalez et al., 1988b). Phage were analyzed by di- gestion with EcoRI and BamHI followed by Southern blotting and hybridization to the radiolabeled CYP2D6 cDNA.

Sequencing. The EcoRI inserts of the four phage 34, 41, 42, and 45 were digested with BamHI, and the resulting fragments were sub- cloned into the pBluescript vector (pBS, Stratagene). Subclones were either further subcloned or processed by the exonuclease III method (Henikoff, 1984) and sequenced by the dideoxy chain-termination procedure (Sanger et al., 1977) using universal and reverse primers. For exonic sequences, 18 synthesized oligonucleotides corresponding to the 5' and 3' parts of each of the nine exons were used (Kagimoto et al., 1990). Data were analyzed with the sequence analysis software package from the University of Wisconsin Group (Devereux et al., 1984).

Genomic Southern blots. DNA from individuals with the extensive or poor metabolizer phenotype and the defined XbaI genotype was used for genomic Southern blots after digestion with BamHI. After alkaline blotting overnight, the Biotrace RP (Gelman) membranes were prehybridized in 1 M sodium chloride, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), 100 #g/ml denatured salmon sperm DNA for at least 6 h at 65°C and hybridized to a [a-a2P]dATP-labeled CYP2D6 cDNA (Gonzalez et al., 1988b) overnight in the same solu- tion at 70°C. Membranes were washed 2× for 5 rain at room tempera- ture in 0.15 M sodium chloride, 0.015 M sodium citrate, and 1% SDS; once for 30 min at 72°C in 0.15 M sodium chloride, 0.015 M sodium citrate, and 1% SDS; and once for 30 rain at 72°C in 0.015 M sodium chloride, 0.0015 M sodium citrate, and 1% SDS.

E c o R [ d i g e s t i o n

34 41 42 45

15.1 kb ---. 13.7 kb.~.

9.4 k b - ~ 8.8 kb - - *

Barn HI d i g e s t i o n

34 41 42 45

6.7 kb--~

4.9 kb--*

3.2 kb---~

2.2 kb--~

1.9 k b--.~ 1.7 kb----*

FIG. 1. Southern blots of phage DNAs each containing an EcoRI insert representing a different C YP2D gene or pseudogene of the XbaI 44P haplotype. Genomic DNA from a poor metabolizer with the XbaI genotype 44/44 kb was completely digested with EcoRI and used for construction of a library in XEMBL4. The isolated phages contained one of the four EcoRI inserts hybridizing to the [a-a2P]dATP-labeled CYP2D6 cDNA shown here with the examples of phage 34, 41, 42, and 45. The lengths of these inserts (9.4, 8.8, 15.1, and 13.7 kb) match the lengths of the four fragments detected by the same probe on genomic Southern blots with DNA from poor metabolizers with an Xba144/44 kb genotype. Phage 34 contains CYP2D6B, phage 41 CYP2D8P, phage 42 CYP2D7AP, and phage 45 CYP2D7BP (see also Fig. 3). The four different inserts can also be identified by their characteristic BamHI pattern.

PCR amplifications of junction fragments. A 604-bp fragment was amplified from 0.5 ttg of genomic DNA already used for construction of the library with primer 1 (5' CCCCAGCGGACTTATCAACC 3', complementary to position 7517 to 7536 downstream of CYP2D7BP and to position 7497 to 7516 downstream of CYP2D7AP) andprimer 2 (5' CCTCCATTGTGCAATGATGC 3', complementary to position -1230 to -1211 of CYP2D6 and to position -1232 to -1213 of CYP2D7BP). The reaction was carried out in a total volume of 100 #1 in the presence of 1.5 mM magnesium chloride, 10 mM Tris-hydro- chloric acid, pH 8.3, 50 mM potassium chloride, 0.01% (w/v) gelatine, 200 ttM each dNTP, 0.5 #M each primer, 0.5 t~g of genomic DNA, and 1.5 U Taq polymerase (Perkin-Elmer). After an initial denaturation at 94°C for 90 s, 35 cycles of 60 s at 94°C, 90 s at 48°C, and 180 s at 72°C and a final extension period of 7 rain at 72°C were performed. The amplified fragment was gel-purified and sequenced directly using the same primers and the dideoxy chain-termination method (Sanger et al, 1977).

Sequence comparison. Sequence comparison was performed using the software package from the University of Wisconsin Group (Deve- reux et al., 1984) and the computer program NAG, kindly supplied by T. Ota, based on the method of Nei and Gojobori (1986). The new sequences reported herein have the EMBL Database Accession num- bers and names X58467 and HSCP2D7AP for CYP2D7AP, and X58468 and HSCP2D7BP for CYP2D7BP.

RESULTS

I so la t i on of C Y P 2 D Genes

D N A comple t e ly d iges ted w i th E c o R I f rom a poor me- t abo l i ze r (PM) wi th a X b a I 44/44 kb geno type c o n t a i n s four E c o R I f r a g m e n t s of 8.5, 9.5, 16, a n d 18 kb, whe reas the X b a I 29-kb f r a g m e n t lacks the 16-kb D N A , a n d t he 11.5-kb f r a g m e n t obv ious ly lacks b o t h the 16- a n d t he 9 .5-kb D N A s (Skoda e t al., 1988). T h e d i f fe ren t sizes of

Page 3: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

CYP2D6 POLYMORPHISM 51

the EcoRI inserts and the characteristic BamHI frag- ments therefore allowed us to classify the 22 phages iso- lated from the genomic library into four groups (Fig. 1). One phage from each group was processed for further analysis.

Sequences of the Isolated CYP2D Genes

Phage 41 contains the pseudogene CYP2D8P (Ki- mura et al., 1989). It was characterized by restriction site mapping and partial sequencing. Phage 34 contains the mutant CYP2D6*B. All exons and exon-intron junc- tions were sequenced, and the mutations found were the same as those previously described by us (Kagimoto et al., 1990). Phage 42 contains a pseudogene we designated CYP2D7AP. Except for about 1470 bp in the 5' region of this pseudogene, the complete 15,070-bp insert was se- quenced. CYP2D7AP is nearly identical to a pseudogene designated CYP2D7 (44/11.5) recently isolated from a PM with the XbaI 11.5/44 kb genotype (Hanioka et al., 1990). The deduced amino acid sequences of CYP2D7AP and of CYP2D7P (Kimura et al., 1989) dis- play 97.4% identity. The nucleotide differences in a re- gion from 1150 bp upstream to 1489 bp downstream be- tween the two pseudogenes are listed in Table 1. The T insertion in exon 1 of CYP2D7P (T226) causes a prema- ture termination of translation at amino acid position 253. This mutation is also present in CYP2D7AP, but an additional frameshift mutation in exon 4 (G2o35) leads to premature termination of translation at amino acid 225. Phage 45 contains a pseudogene designated CYP2- D7BP. The complete 13677-bp insert was sequenced. CYP2D7BP is a "chimeric" gene: In a 4853~bp region spanning from the second exon to 1489 bp downstream, only nine base changes from the CYP2D7AP sequence were found. From 5' of exon 2, however, including intron 1, exon 1, and 776 bp of upstream sequence, CYP2D7BP displays 42 base changes when compared to the sequence of CYP2D7AP, but only 5 when compared to that of CYP2D6. Table 1 therefore lists the differences from CYP2D6 for this 5' part of CYP2D7BP until the end of intron I and then uses CYP2D7P as the published refer- ence sequence for exon 2 to 1489 bp downstream. CYP2D7BP lacks the above-mentioned T insertion in exon 1, but the G insertion in exon 4 (G2o35) causes a premature termination of translation at amino acid 253.

The sequences downstream of CYP2D7AP and CYP2D7BP are nearly identical (Fig. 2). The sequence CCCACCCTTC is repeated four times in CYP2D7BP between position 2396 and position 2435 downstream, whereas in CYP2D7AP it is repeated twice between po- sition 2395 and position 2414 downstream (position 1 downstream corresponds to the published CYP2D7P). These repeats are flanked by direct repeats of the se- quence ACCCCGGG. In addition to this difference, nine base changes were found when the 7789 bp of down- stream sequence from CYP2D7AP were compared with the 7809 bp of CYP2D7BP. The downstream sequences of both CYP2D7AP and CYP2D7BP have an insertion

of about 1600 bp at position 540 of the CYP2D6 down- stream sequence. In these insertion sequences, we found several copies of 41- and 24-bp repeats that have been described in the 3' region of the human ~-globin cluster near the breakpoint of a deletion (Henthorn et al., 1986) and in the 370-bp sequence of clone SP-0.3-16 that was shown to be hypomethylated in sperm cell DNA (Zhang et al., 1987).

Gene Arrangement on the 44P Haplotype

The arrangement of the four genes on the 44-kb frag- ment found with the 44P haplotype is depicted in Fig. 3. Southern blots with BamHI-digested genomic DNA from one individual homozygous for the l l .5-kb frag- ment (Fig. 3C, lane A), one individual homozygous for the 29-kb fragment (lane B), and three poor metabolizer individuals with the 44/44 kb genotype (lanes C, D, E) were hybridized to the [a-32P]dATP-labeled probes S1- $6, the positions of which are indicated in Fig. 3B. S1 is 2.1-kb EcoRI-BamHI fragment isolated from phage 41 (CYP2D8P), $2 is a 1.45-kb EcoRI-KpnI fragment from the same phage, $3 is a 2.15-kb EcoRI-BamHI fragment from phage 42 (CYP2D7AP), $4 is a 4.25-kb EcoRI- BamHI fragment from the same phage that is nearly identical to the corresponding fragment from phage 45 (CYP2D7BP), $5 is a 0.55-kb EcoRI-BamHI fragment from phage 34 (CYP2D6.B) that is identical to the corresponding fragment of phage 45 (CYP2D7BP), and $6 is a 1.5-kb EcoRI-BamHI fragment from phage 34. Digestion of genomic DNA with BamHI yields frag- ments spanning the EcoRI boundaries of the phage in- serts. If two probes from different phages hybridize to the same fragment, we conclude that the inserts of these phages are adjacent to each other on the chromosome. S1 and $6 hybridized to two different fragments of 3.7 and 9.5 kb, respectively, not detected by any other probe. CYP2D8P therefore must be 5' of all the other genes, and CYP2D6*B must be at the 3' end of the gene cluster in the orientations shown in Fig. 3. Consistent with the presumed arrangement of CYP2D8 and CYP2D7P (Ki- mura et al., 1989), $2 and $3 both hybridized to an 8.8-kb fragment, showing that CYP2D7AP lies immediately downstream of CYP2D8P. Only one additional frag- ment is detected by both $4 and $5. It corresponds to the 4.8-kb B a m H I - B a m H I fragment between CYP2D7AP and CYP2D7BP as well as the identical fragment be- tween CYP2D7BP and CYP2D6.B . Any different alignment of the genes would yield different fragments detected by these probes. Since l l .5-kb fragments have only a CYP2D8P and a CYP2D7P, the DNA of the indi- vidual homozygous for the l l .5-kb fragment (Fig. 3C, lane A) lacks the 4.8-kb fragment detected by $4 and $5. Our data also document for the first time that CYP2D6 lies downstream of CYP2D7 on the 29-kb fragment (Fig. 3C, lane B) and that the intergenic sequence between these two genes apparently is identical to the intergenic sequences between C YP2D 7AP and C YP2D 7BP, and be-

Page 4: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

52 H E I M AND M E Y E R

TABLE 1

Comparison of Upstream, Downstream, Intron, and Exon Sequences o f C Y P 2 D T P , C Y P 2 D T A P , and C Y P 2 D T B P

C YP2D 7P C YP2D 7AP C YP2D 7BP C YP2D6

Upst ream

Exon 1

In t ron 1

Exon 2

In t ron 2

Exon 3

In t ron 3

Exon 4

In t ron 4

Exon 5

In t ron 5

Exon 6

In t ron 6

Exon 7

In t ron 7

Exon 8

In t ron 8

1150 bp 1149 bp

A_955 ~ C C-7v8 --~ T C-785 -~ T T_346 -~ C G-813 --~ C C-132 --~ G G-117 -~ A C-lo4 --~ T CAC-lo1~-96 Deleted G_67G_66 "-> CA G-so -~ A

None

398/399 G inserted

G1336 1406/1407

61626 61667

T1676 G1682

C191561916 2035/2036

C2o43T2044

62323 62468 62481

T2523 62632 62663 T2667

None

Deleted

Deleted --~ A

-~ A --~ T

None

-~ GC G inserted

--~ TC

--~ T --~ A --~ G

-~ C --~ A -~ A --~ A

None

None C2974

C3148 -~ G

C3300 -~ T G3311 --~ A A6862 Deleted A3625 ---> G A3664 --~ G A3~56 --* G

T~425 --~ C 3432/3433 T inserted G6437 --.-> A G3523 -...-> A A659o T6666 -~ C

None Aaa99

None

1531 bp 1531 bp

T " - C-1368 AA inserted - 1 1 4 9 / - 1 1 4 8

G "-- A-114v A ~- 6_912

T

T Deleted G G

None

Deleted C inserted Deleted A

A T

None

GC G inserted TC

T A G

C A A A

None

Deleted

G

T A

G G G

C T inserted A A Deleted C

G

None

~'- 6188

6396 C66s

~- C8~4 ~'- T931

Page 5: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

CYP2D6 POLYMORPHISM

T A B L E 1--Continued

53

C YP2D 7P C YP2D 7A P C YP2D 7BP C YP2D6

Exon 9

Downstream

C4239 --~ T T C42s9 -~ T T

1489 bp 1491 bp 1489 bp C151 -~ A A152 --~ G G C580 --~ T T G~84 Deleted C5s6 Deleted G755 -~ A T12Go --~ C C A1317 --~ G G 1373/1374 G inserted G inserted 1461/1462 G inserted G inserted

tween CYP2D7BP and CYP2D6B on the 44-kb frag- ment.

Additional evidence for this arrangement is obtained from the comparison of calculated restriction fragments for digestion with EcoRV, NcoI, and HindIII with our previous Southern blot data (Skoda et al., 1988): DNA from poor metabolizers with the XbaI polymorphic frag- ment was shown to contain EcoRI fragments of 8.5, 9.5, 16, and 18 kb, EcoRV fragments of 14.5 and 44 kb, NcoI

2D7AP RH B

II I NIV ~

2DTBP RB

II m

N B B B N H R

IJl II I I I I I I I E:~l~l::i:]:il I [ / t I / • I H I I l l l B . . / " "-,. u N

1 2 34 5 6 7 .8'9" "'"'-, . --. . , J " "%..

. ,.,

B " B B"" N H R ""% / -

• I I I n a b H I u JJ~ mm~ 1 2 34 567 89

LEGEND:

I E X O N [ZZ]CCCACCCTTC REPEAT ~ SP-0 3-16

~ A L U REPEAT ~ 41BP REPEAT

NOT SEQUENCED ~ M I S S I N G IN 2D6 4,,. 24BP REPEAT

FIG. 2. Features of CYP2D7AP and CYP2D7BP. The sequenced inserts of phage 42 and 45 of Fig. 1 are shown. The 13,677-bp insert of phage 45 was sequenced completely. It contains the nine exons of a pseudogene designated CYP2D7BP. The 15.1-kb insert of phage 42 was sequenced except for about 1470 bp. It contains the nine exons of a pseudogene designated CYP2D7AP. Both sequences are highly simi- lar from exon 2 to the 3' EcoRI site, but display marked differences 5' of exon 2, where CYP2D7BP is homologous to CYP2D6B. Five Alu repeats were found on the insert containing CYP2D7AP, whereas four are present 5' and 3' of CYP2D7BP. The sequence CCCACCCTTC is repeated four times 3' of CYP2D7BP and two times 3' of CYP2D7AP at the positions indicated. A 1600-bp sequence that is missing in the 3' region of CYP2D6 is present 3' of both pseudogenes and contains 41-b and 24-bp repeats found near the breakpoint of a large deletion in the human fl-globin gene cluster (Henthorn et al., 1986) as indicated by the small arrows. A 370-bp segment of this sequence also displays marked homology to a 370-bp insert of clone SP-0.3-16 that is specifi- cally hypomethylated (Zhang et al., 1987).

fragments of 6.3, 8, 9.9, and 12.5 kb, and only three HindIII fragments of 10.5, 14, and 16 kb with the CYP2D6 cDNA. All these fragments are consistent with the calculated restriction map shown in Fig. 3A. The large EcoRV fragment can be obtained only if CYP2D8P and C Y P 2 D 6 , B are on opposite ends of the cluster be- cause the other genes have no EcoRV sites. The calcu- lated length of the fragment of 38.5 kb is well within the range of the Southern blot data. The arrangement now also explains the lack of an additional HindIII fragment, since two of the HindIiI fragments were calculated to have exactly the same length of 13.6 kb, i.e., will have the same mobility on gel electrophoresis.

The possibility of a small EcoRI-EcoRI fragment between CYP2D7AP and CYP2D7BP or between CYP2D7BP and CYP2D6B was excluded by PCR am- plification of a fragment spanning the EcoRI site be- tween these genes using primers hybridizing to the 3' end of the EcoRI inserts of phages 42 and 45 and to the 5' end of phages 45 and 41, respectively (primer 1, 5' CCCCAGCGGACTTATCAACC 3'; primer 2, 5' CCTC- CATTGTGCAATGATGC 3'). The sequences of the ob- tained 604-bp fragments proved that the phage insert sequences connect with each other directly as indicated (data not shown, available on request).

Evolutionary Distances between Human CYP2D Genes

The results of pairwise comparisons of deduced AA sequences after correction for insertions and deletions are summarized in Table 2. Three classes of CYP2D genes can be distinguished: the CYP2D6 class with three alleles (CYP2D6, WT, CYP2D6, A, and CYP2D6,B), the CYP2D7P class with three variant forms (CYP2D7P, CYP2D7AP, and CYP2D7BP), and the CYP2D8P class. CYP2D7BP and CP2D7AP are not inherited in a strictly allelic way, since they can be transmitted to- gether as in the case of the 44P haplotype here described.

The deduced AA sequence of human CYP2D6 dis- plays between 69 and 72% identity to the deduced AA sequences of the five rat genes of the CYP2D family

Page 6: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

54 HEIM AND MEYER

A

B

C

ECO RV 14.5KB 38.5KB

NCOI 6 1KB

I

HIND III

8.0KB 13.7KB ; 9.8KB I i

13.6KB I 13.6KB

JUNCTION 7AP/7BP B

JUNCTION 7BPf6B @

i I < #41 >< #42 ! I I

l 5' 2D8P 2DTAP I nNNB NVRH BNB

I I ~ S~ $2 $3 I

l

>< #45

2D7BP B BN HRB B B

54 $5

BN It"

$4

>< #34 >

21~B HRB B BNN BNVR 3'

$5 $6

J

$ 2

A B C D E

$ 3 $ 4

A B C D E A B C D E

%:

~- 4.8kb.*"

$ 5

A B C D E

FIG. 3. Arrangement of the four genes on the XbaI 44P haplotype. Phage inserts are depicted by number of the phage (34, 41, 42, 45). Restriction sites are R, EcoRI; B, BamHI; H, HindIII; N, NcoI; V, EcoRV. The arrangement is based on three lines of evidence: (1) Genomic Southern blots with DNA from poor metabolizers with XbaI 44/44 kb genotype have EcoRI, EcoRV, NcoI, and HindIII fragments consistent with the calculated restriction map in A. (2) Sequences of DNA fragments amplified with primers complementary to sequences 5' and 3' of the EcoRI sites between CYP2D7AP, CYP2D7BP, and CYP2D6B show that the EcoRI inserts of phage 42, 45, and 41 are adjacent to each other (B). (3) Southern blots with the specific probes $1-$6 (B, C). Genomic DNA from a poor metabolizer with XbaI genotype 11.5/11.5 kb (lane A), an extensive metabolizer with genotype 29/29 kb (lane B), and three poor metabolizers with genotype 44/44 kb (lanes C, D, E) was digested with BamHI and analyzed by Southern blotting and hybridization to [a-~2P]dATP-labeled fragments $1-$6. Both $2 and $3 detect an 8.8-kb fragment corresponding to the segment from the BarnHI site in phage 41 to the first BamHI site in phage 42. $4 and $5, which are homologous to the 3' ends of phage 42 and 45 and the 5' ends of phage 45 and 34, respectively, detect a 4.8-kb fragment corresponding to the identical segments from the last BamHI site of phage 42 and 45 and the first BamHI site of phage 45 and 34. S1 detects a 3.7-kb fragment not detected by any other probe, and $6 a 9.5-kb fragment not detected by any other probe (data not shown). The corresponding segments on phage 41 and 34 are situated, therefore, at the 5' and 3' ends of the gene cluster.

Page 7: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

CYP2D6 POLYMORPHISM

TABLE 2

N u m b e r of Amino Acid Differences and Percentage of Identity of Deduced Amino Acid Sequences be tween Human C Y P 2 D Genes

55

2D6 2D6B 2D 7P 2D 7AP 2D 7BP 2D8P

2D6 - - 99.0% 94.2% 93.3% 93.8% 87.9% 2D6B 4 -- 94.4% 93.4% 94.4% 87.8% 2D7P 29 28 - - 97.4% 96.8% 87.3% 2D7AP 34 33 13 - - 98.6% 86.7% 2D7BP 31 28 16 7 - - 86.7% 2D8P 60 61 63 66 66 - -

(Ma t sunaga et al., 1990) and be tween 66 and 69% to three mouse C Y P 2 D genes Cyp2d-9, Cyp2d-lO, and Cyp2d6-11 (Wong et al., 1989). Pai rwise compar i son of all these genes suggests app rox ima te ly cons tan t ra tes of amino acid subs t i tu t ions since the divergence of rodents and pr imates . Assuming t h a t the roden t and p r ima t e lineage separa ted in evolut ion abou t 75 mil l ion years ago, the rate of AA subs t i tu t ions per site per 109 years (~ × 109; Nei, 1987) is be tween 2.19 ( s tandard deviat ion = 0.03) and 2.77 (0.03) for the C Y P 2 D genes.

T h e rate of nucleot ide subs t i tu t ion per site per 109 years was calculated separa te ly for s y n o n y m o u s (silent) and n o n s y n o n y m o u s (AA altering) subs t i tu t ions using a compu te r p r o g r a m based on the m e t hod of Nei and Go- jobori (1986). C o m p a r i n g the coding sequences of hu- m a n C Y P 2 D 6 and ra t CYP2D1, bo th known to me tabo- lize debrisoquine, and assuming again a divergence t ime of 75 mill ion years, X × 109 has a value of 3.91 ( s t andard deviat ion 0.37) and 1.25 (0.09) for s y n o n y m o u s and non- synonymous subst i tu t ions , respectively. T h e rat io be- tween these two types of subs t i tu t ions is 3.13. The rat ios be tween synonymous and n o n s y n o n y m o u s subst i tu- t ions compar ing C Y P 2 D 8 P with C Y P 2 D 6 and C Y P 2 - D 7 A P with C Y P 2 D 6 are 1.72 and 1.03, respectively. T h e overall ra te of nucleotide subs t i tu t ions per site per 109 years for synonymous and n o n s y n o n y m o u s nucleotide a l te ra t ions calculated by the same compu te r p rog ram (Nei and Gojobori, 1986) is 1.8. I t was used to es t imate the t ime of divergence of different h u m a n CYP2D genes and pseudogenes by dividing the p ropor t ion of different nucleotides be tween any two of t h e m by 2 t imes th is ra te (Nei, 1987).

DISCUSSION

Wi th the e lucidat ion of the molecular s t ruc ture of the m u t a n t 44P hap lo type of the C Y P 2 D locus charac te r - ized by a XbaI 44-kb f r agm en t descr ibed here, the de- br isoquine p o l y m o r p h i s m p robab ly is the mechanis t i - cally bes t unders tood inher i ted var ia t ion in h u m a n drug metabol i sm. Over 90% of the m u t a n t hap lo types of the C Y P 2 D 6 are now character ized: The 11.5 hap lo type ( l l . 5 - k b f ragment ) lacks the ent i re CYP2D6; the 29A hap lo type conta ins a m u t a t e d CYP2D6, namely , C Y P 2 D 6 . A ; and the 29B hap lo type ha rbors the mu- t a ted C Y P 2 D 6 . B , which is also p resen t on the 44P hap- lotype (Fig. 4). Geno typ ing individuals by a P C R - b a s e d

m e t h o d (He im and Meyer , 1990; Broley et al., 1991) can ident i fy C Y P 2 D 6 * A and C Y P 2 D 6 * B , and R F L P analy- sis adds no in fo rmat ion with regard to the p h e n o t y p e for these m u t a t e d 29-kb hap lo types as well as for the mu- t a t ed 44P haplotypes . I t is no tewor thy t ha t of the six m u t a t i o n s p resen t in exons of C Y P 2 D 6 * B , th ree were also found in the pseudogene C Y P 2 D 8 P , four of t h e m in C Y P 2 D 7 A P and C Y P 2 D 7 P , and five of t h e m in C Y P 2 D 7 B P . P C R genotyping me thods therefore should be control led for false posi t ive ident i f icat ions of C Y P 2 D 6 . B obta ined th rough ampl i f ica t ion f rom C Y P - 2D7 genes.

The incidence of poor metabol izers of debr isoquine is very low among the Chinese and Japanese , approxi- ma te ly 1%, bu t a surpr is ingly high n u m b e r of Chinese extensive metabol izers are homozygous for the "44E hap- lo type" ( Johansson et al., 1991). Al though individuals homozygous for th is hap lo type have a slightly lower ca- pac i ty for me tabo l i sm of debrisoquine, they canno t be classified as poor metabol izers . T h e molecular basis of the differences be tween the Chinese and the Caucas ian XbaI 44E and 44P haplo types is not yet unders tood, bu t the s tudy of the Chinese 44E hap lo type should benef i t subs tan t ia l ly f rom the knowledge provided here on the Caucas ian counte rpar t . In addit ion, the unde r s t and ing of this in te re thn ic difference can provide insights into the evolut ion of cy tochrome P450 CYP2D6. T h e impor- t ance of in te re thn ic differences for drug deve lopmen t and tes t ing is increasingly recognized (Kalow, 1991).

S T R U C T U R E F R E Q U E N C Y

4 4 . 8P R 7AP R 7BP R 6B R ~ ~ ~ ~ 0 . 3 0

2 9 A " 8P . 7(A)P R 6 A R ~::::::::::~ ~ ~ 0 . 0 5

R 8P R 7(A)P . 6B R 2 9 B ~:::::::~ ~ ~ 0 . 4 2

R 8P R 7 (A)PR 1 1 . 5 ~ ~ 0 . 1 5

FIG. 4. Schematic representation of four mutant haplotypes of CYP2D6 representing over 90% of the mutant haplotypes associated with the debrisoquine polymorphism. 8P, 7AP, 7BP, 6A, and 6B stand for CYP2D8P, CYP2D7AP, CYP2D7BP, CYP2D6*A, and CYP2D6.B, respectively. 7(A)P stands for either CYP2D7P or CYP2D7AP. The frequencies of these haplotypes in poor metabolizers are from Helm and Meyer (1990) and Broly et al. (1991).

Page 8: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

56 HEIM AND MEYER

A 29WT R 2 ~ P ~ 2DTAP 2D6 R

R 2D8PR 2 0 7 ~ ~'~"

11.5 ~ R 2D8P R 2DZAP

29B

44E R R 2DSP R 2DTAP R 2D6 , ~ l ~ j ~ .

B 44E R l ~ 2DSPR 2D7AP ~ 2D6

[:::] , ~ ,

1

~ 2D6B R

44P R 2DSPR 2DTAP R2DTBP R 2D6B R

FIG. 5. Hypothesis of the generation of the 44P haplotype by two conservative gene rearrangement events: (A) An unequal crossover between a haplotype 29WT with a XbaI 29-kb fragment with a func- tional CYP2D6* WT allele and a haplotype 29B with a XbaI 29-kb fragment with a mutant CYP2D6*B allele results in an ll.5-kb XbaI fragment (haplotype 11.5) and a 44-kb XbaI fragment containing both a functional CYP2D6* WT and a mutant CYP2D6*B allele (haplo- type 44E). (B) A gene conversion event inactivates the CYP2D6 on the "44E haplotype" from exon 2 to about 500 bp 3' of this gene into the chimeric pseudogene CYP2D7BP. CYP2D7AP not only delivered this region, but also introduced the 1600-bp segment 3' of the gene missing in CYP2D6 (for details, see Fig. 3 and text). The resulting mutant allele is designated 44P.

Four C Y P 2 D genes are a r ranged head- to- ta i l on the 44P hap lo type (Fig. 3). T h e a r r a n g e m e n t of C Y P 2 D 8 P and C Y P 2 D 7 P on the 29-kb f r agm en t was deduced f rom the isolat ion of over lapping clones. T h e pos i t ion of C Y P 2 D 6 on this f ragment , however, r ema ined contro- versial. Our findings provide direct evidence t h a t it is s i tua ted about 9.3 kb downs t r eam of C Y P 2 D 7 P (Fig. 3).

A possible mechanis t i c exp lana t ion for the genera t ion of the c o m m o n m u t a n t haplo types reflected by XbaI 11.5- and 44-kb f r agmen t s is suggested in Fig. 5. In a first step, unequal crossover be tween a 29 wildtype and a 29B hap lo type genera ted the 11.5- and the 44-kb f ragments conta in ing a "wi ld type" C Y P 2 D 6 and a m u t a t e d C Y P 2 D 6 , B . This cluster would thus car ry a funct ional "ex tens ive metabol izer" gene and is des ignated "44E" in Fig. 5. EcoRI digestion of this "44E" a r r a n g e m e n t would yield f r agments of 8.8 kb (CYP2D8P) , 9.4 kb ( C Y P 2 D 6 * B ) , 15.1 kb ( C Y P 2 D 7 A P ) , and abou t 12.1 kb (CYP2D6* W T ) ins tead of 13.7 kb as observed in mu- t an t 44P haplotypes . One rare Caucas ian "44-kb haplo- t ype" associated with the E M pheno type (44E) recent ly was repor ted (Roots et al., 1992), and R F L P studies in fact revealed an EcoRI f r agm en t 2 kb smal ler t h a n its coun te rpa r t of the m u t a n t 44-kb f ragment . In a second

step, a gene convers ion event be tween a C Y P 2 D 7 A P and the C Y P 2 D 6 gene on this 44E f r agmen t spann ing the region f rom the second exon to about 2000 bp down- s t r e am of the C Y P 2 D 7 A P gene would generate the "chi- mer ic" C Y P 2 D 7 B P gene. T h e resul t ing hap lo type (44P) would conta in only nonfunc t iona l C Y P 2 D 6 genes and would yield the addi t ional EcoRI 13.7-kb f r ag men t ( C Y P 2 D 7 B P ) detec ted with all m u t a n t 44P haplotypes . The following cons idera t ions suppor t this hypothesis : (a) Gene convers ions in P450s t h a t ex tend over long re- gions of the gene have been repor ted (Higashi et al., 1988). (b) T h e repet i t ive e lements in the 1600-bp inser- t ion d o w n s t r e a m of C Y P 2 D 7 A P and C Y P 2 D 7 B P (Fig. 2) have been descr ibed in o ther genes with D N A rea r rangments , i.e., in the genera t ion of a large delet ion in the fl-globin gene cluster ( H e n t h o r n et al., 1986).

Kinet ic studies wi th expressed cDNAs of the ra t C Y P 2 D 1 and the h u m a n C Y P 2 D 6 indicated t ha t these genes indeed are or thologous (Gonzalez et al., 1988b; M a t s u n a g a et al., 1989). Compar i son of nucleotide se- quences be tween these genes therefore was used to cal- culate ra tes of nucleotide subs t i tu t ions per site per 109 years. In general, low rates are found in genes t h a t code for p ro te ins with s t rong funct ional cons t ra in t s (Nei, 1987). H i s tone H4 with ra tes of 0.004 for AA al ter ing (nonsynonymous) and 1.43 for s i lent ( synonymous) sub- s t i tut ions, for example , is one of the more conserved pro te ins during evolution, whereas in te r fe ron-a 1 wi th ra tes of 1.41 (nonsynonymous ) and 3.53 ( synonymous) is a ra ther fast evolving pro te in (Li et al., 1985). Wi th a rate of 1.25 for n o n s y n o n y m o u s and 3.91 for synony- mous nucleotide subs t i tu t ion per site per 109 years, the C Y P 2 D genes also are re la t ively fas t evolving genes.

In funct ional genes, s y n o n y m o u s mu ta t i ons are found to be more f requent t h a n n o n s y n o n y m o u s muta t ions . Th i s can be expla ined by the neu t ra l theory of molecular evolut ion (Kimura , 1968). Accordingly, mu ta t i ons occur at random, bu t mos t amino acid a l ter ing mu ta t i ons are deleter ious and are quickly e l imina ted f rom the popula- tion, whereas si lent mu ta t i on under lay no "pur i fy ing" selection. Pseudogenes are no longer exposed to selec- t ive pressures , and therefore synonymous and nonsyn- onymous subs t i tu t ions occur a t the same rate. In C Y P 2 D 7 A P the rat io of s y n o n y m o u s to n o n s y n o n y m o u s mu ta t i ons indeed is 1.03. C Y P 2 D 7 A P therefore proba- bly was inac t iva ted shor t ly af ter its genera t ion th rough a gene dupl icat ion event in CYP2D6. Compar ing C Y P 2 D 6 with CYP2D8P, synonymous subs t i tu t ions are still ob- served 1.7 t imes more f requent ly t h a n AA al ter ing m u t a - t ions. Thus , for some mil l ion years C Y P 2 D 8 P m a y have been a funct ional gene.

T h e to ta l n u m b e r of subs t i tu t ions per site per 109 years for the C Y P 2 D genes is 1.8 ( s tandard deviat ion = 0.1). Th i s value and the to ta l n u m b e r of different nu- cleotides be tween any two C Y P 2 D genes can be used to calculate the t ime of divergence be tween these genes (Nei, 1987). Figure 6 displays a phylogenet ic t ree for the h u m a n C Y P 2 D genes. I t is realized t h a t some b r a n c h lengths m a y have been overes t ima ted because of the

Page 9: Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6

CYP2D6 POLYMORPHISM 57

6 10 years

20

15'

10.

5.

0

FIG. 6. Phylogenetic tree of the human CYP2D genes and pseu- dogenes. Time since divergence is displayed by the length of the branches.

higher overall nucleotide substitution rate in pseudo- genes, but the principal scenario of the evolution of these genes should be correct: A first gene duplication of a CYP2D6-1ike gene occurred about 18 million years ago and generated a CYP2D8, which probably was a func- tional gene for some million years. Once the gene was duplicated into two, a further increase in the number of genes was facilitated by unequal crossover events. About 9 million years ago, a second duplication of CYP2D6 leads to the CYP2D7, which probably became a pseudo- gene soon after. The resulting gene cluster is the proto- type of the XbaI 29-kb fragment. An unequal crossover as proposed in Fig. 5 produced both the 11.5 and 44E haplotypes about 1 to 2 million years ago. At about the same time, gene conversion events may have introduced some of the mutations of CYP2D7P pseudogenes into CYP2D6, resulting in the mutant CYP2D6.B, which is found in over 70% of mutant alleles (Fig. 4). CYP2D6.A, finally, is the result of a relatively recent point mutation in CYP2D6.

Poor metabolizers of debrisoquine have no apparent serious illnesses and according to present knowledge have no reproductive disadvantage. Does this mean that the CYP2D6 gene is on its way to being a pseudogene and that possibly it is predestined for extinction? It is likely that the ancestors of drug metabolizing enzymes evolved during so-called plant-animal warfare: As plants and animals diverged, animals began to ingest plants and plants defended themselves by developing new toxins (alkaloids, terpenes, etc.). In response, ani- mals developed new P450s able to detoxify these "phy- toalexins" (Gonzalez and Nebert, 1990). It is of interest that the preferred drug substrates and competitive inhib- itors of CYP2D6 are plant alkaloids (sparteine, N-pro- pylajmaline, codeine, quinidine) or are derived from plant components, as most drugs are (Fonne-Pfister and Meyer, 1988). Obviously, the present nutrition of West- ern societies no longer requires CYP2D6 activity. Could the low incidence of poor metabolizers in Chinese popu- lations reflect variant dietary pressures and explain the interethnic difference in the CYP2D6 polymorphism?

In any case, the clinical observation of inherited varia- tion of drug metabolism has spurred the investigation of these highly polymorphic genes in the human genome and their evolution with regard to their function of de- fending the organism against exogenous adversity.

A C K N O W L E D G M E N T S

We thank Markus Beer for his expert technical assistance, Masaaki Kagimoto for stimulating discussions, and Roger Jenni and Reinhard Doelz for help with computer analysis of sequences. This work was supported by the Swiss National Science Foundation.

R E F E R E N C E S

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Gonzalez, F. J., Vilbois, F., Hardwick, J. P., McBride, O. W., Nebert, D. W., Gelboin, H. V., and Meyer, U. A. (1988a). Human debriso- quine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22. Genomics 2: 174-179.

Gonzalez, F. J., Skoda, R. C., Kimura, S., Umeno, M., Zanger, U. M., Nebert, D. W., Gelboin, H. V., Hardwick, J. P., and Meyer, U. A. (1988b). Characterization of the common genetic defect in humans deficient in debrisoquine metabolism. Nature 331: 442-446.

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58 HEIM AND MEYER

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Kagimoto, M., Heim, M. H., Kagimoto, K., Zeugin, T., and Meyer, U.A. (1990). Multiple mutations of the human cytochrome P450IID6 gene (CYP2D6) in poor metabolizers of debrisoquine: Study of the functional significance of individual mutations by ex- pression of chimeric genes. J. Biol. Chem. 265: 17209-17214.

Kalow, W. (1991). Interethnic variation of drug metabolism. Trends Pharmacol. Sci. 12: 102-107.

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