sparteine metabolism—molecular mechanisms

Pharmac. Ther. Vol. 46, pp. 297-308, 1990 Printed in Great Britain. All rights reserved

0163-7258/90 $0.00+ 0.50 © 1990 Pergamon Press pie

Specialist Subject Editor: W. KALOW

THE GENETIC POLYMORPHISM OF DEBRISOQUINE/SPARTEINE METABOLISM--MOLECULAR MECHANISMS URS A. MEYER, RADEK C. SKODA an d ULRICH M. ZANGER Department t~f Pharmacology, Biocenter of the University of Basel, CH-4056 Basel, Switzerland Abstract--The genetic polymorphism of debrisoquine/sparteine metabolism is one of the best studied examples of a genetic variability in drug response. 5--10% of individuals in Caucasian populations are 'poor metabolizers' of debrisoquine, sparteine and over 20 other drugs. The discovery and the inheritance of deficient debrisoquine/sparteine metabolism are briefly described, followed by a detailed account of the studies leading to the characterization of the deficient reaction and the purification of cytochrome P-45011D1, the target enzyme of this polymorphism. It is demonstrated by immunological methods that deficient debrisoquine hydroxylation is due to the absence of P-450IIDI protein in the livers of poor metabolizers. The cloning and sequencing of the P-450IID1 cDNA and of lID1 related genes are summarized. The P-45011D1 cDNA has subsequently led to the discovery of aberrant splicing of P-450IID1 pre-mRNA as the cause of absent P-450IIDI protein. Finally, the identification of mutant alleles of the P-450IID1 gene (CYP2D) by restriction fragment length polymorphisms in lymphocyte DNA of poor metabolizers is presented.

CONTENTS 1. Introduction 2. Clinical Relevance 3. Molecular Mechanism 3.1. Principal considerations 3.2. Identification of P-450IID1 (P-450dbl, P-450bufI) as the deficient enzyme 3.3. Immunoquantitation of P-4501ID1 in human liver 3.4. Autoantibodies against P-450IIDI in chronic active hepatitis 3.5. Cloning and characterization of a human P-450IID1 (dbl) cDNA and of genes related to P-450IIDI 3.6. Aberrant splicing patterns of P-450IIDI pre-mRNA in livers of poor metabolizers 3.7. Detection of mutant P-4501IDI genes by restriction fragment length polymorphisms (RFLPs) 4. Future Perspectives Acknowledgements References

303 303 303 306 306 306

1986). A dramatic event in a pharmacokinetic study prompted the initial search for a specific metabolic defect: the investigator who was participating in a study on debrisoquine, a sympatholytic antihypertensive drug, had a much more pronounced hypotensive response than his colleagues, collapsing from a subtherapeutic dose. This was found to be due to impaired 4-hydroxylation of debrisoquine (Smith, 1986; Mahgoub et al., 1977). A group of physicians in Bonn at the same time independently observed increased side effects associated with decreased oxidative metabolism of sparteine, an oxytocic and antiarrhythmic alkaloid (Eichelbaum et al., 1979; Smith, 1986). These initial observations were followed both for debrisoquine and sparteine by population studies with determination of the urinary metabolic ratio (MR). Poor metabolizers of debrisoquine and sparteine were defined (by statistical analysis of the

1. I N T R O D U C T I O N Genetic polymorphisms of drug metabolizing enzymes give rise to distinct subgroups in the population which differ in their ability to perform a certain drug biotransformation reaction. Individuals with deficient metabolism of a certain drug are called 'poor metabolizers' or 'PM-phenotypes', as compared to the normal ~extensive metabolizer' or 'EM-phenotype'. Most genetic polymorphisms of drug metabolism were discovered by the observation of unusual or exaggerated drug reactions after normal doses of drugs in a few patients or volunteers, followed by studies in larger populations and in families of poor metabolizers. Between 1975 and 1977 two groups independently discovered a genetic deficiency of debrisoquine and sparteine metabolism (Eichelbaum et al., 1979; Smith, J.PT.~/2--J

297 298 298 298 300 301 301

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FIG. 1. Molecular characterization of the debrisoquine/ sparteine polymorphism. The scheme should give an idea how different splicing defects may cause mutant mRNAs which yield no detectable P-4501ID1 (P-450dbl) protein and result in the debrisoquine poor metabolizer phenotype. Mutated alleles of the P-45011D1 (P-450dbl) gone can be detected in genomic DNA of poor metabolizers. antimode in the bimodal distribution) as individuals with a metabolic ratio of greater than 12.6 and 20, respectively, for the ratio of debrisoquine to 4-OHdebrisoquine and of sparteine to 2- and 5-dehydrosparteine (Eichelbaum et al., 1986; Evans et al., 1980). Family studies established that in both cases poor metabolizers are homozygous for a recessive gene whereas extensive metabolizers are either homozygous or heterozygous for the dominant gene (Evans et al., 1980; Steiner et al., 1985) (Fig. 1). It soon became evident that the two deficient reactions must be under identical or linked genetic control; in most populations studied a poor metabolizer of debrisoquine is also a poor metabolizer of sparteine and vice versa. However, there are apparent exceptions to this rule. For instance, in a recent study in Ghana the ability of Ghanaians to oxidize sparteine was independent of their capacity for debrisoquine oxidation (Eichelbaum and Woolhouse, 1985). In the following years it was discovered that poor metabolizers also have an impaired capacity for the oxidative metabolism of now over 20 other drugs including fl-adrenergic blocking agents, antiarrhythmics, antidepressants, opioids and many other clinically used drugs (Fig. 2; Eichelbaum and Gross, 1989). New substrates are still being discovered. Of course a large number of drugs whose metabolism is not controlled by the debrisoquine/sparteine-type polymorphism have also been identified. Common to the polymorphically metabolized substrates is that they have a basic nitrogen and are oxidized at a site with a distance of 0.54).7 nm from the basic nitrogen (Meyer et al., 1986). But a prediction of which drug will or will not be subject to polymorphic oxidation on the basis of structural considerations alone is not

Clinical studies have demonstrated that poor metabolizers of debrisoquine, sparteine and other drugs represent a high risk group in the population with a propensity to develop adverse drug effects. Moreover, recent studies also have indicated that a link might exist between the debrisoquine phenotype and some forms of cancer (Ayesh et al., 1984; Ayesh and Idle, 1985; Kaisary et al., 1987; Roots e t a l . , 1988), with early onset Parkinson's disease (Barbeau et al., 1985; Fonne-Pfister et al., 1987; Pottier et al., 1987), with systemic lupus erythematosus (Baer et al., 1986) and Balkan nephropathy (Ritchie et al., 1983). The reasons for these associations are unexplained. The clinical consequences of the debrisoquine/sparteine polymorphism are discussed by Eichelbaum and Gross (1989). Genetic polymorphisms typically occur with variable frequency in relation to different ethnic (genetic) backgrounds (Kalow e t a l . , 1986). Thus, the poor metabolizer phenotype of the debrisoquine/sparteine polymorphism is most frequently observed among Caucasians (5 10%) and is rarer in other populations, probably about 2% in Oriental and 1% in Arabic populations (Kalow, 1982).

3. MOLECULAR MECHANISM The debrisoquine/sparteine polymorphism probably represents the most extensively studied example at the protein and gene level of a genetic variation of a drug-metabolizing enzyme. In this review studies in our and other laboratories on the molecular mechanisms of this variation are summarized. Some of these data have previously been reviewed (Meyer et al., 1986, 1988; Meyer, 1987). 3.1. PRINCIPALCONSIDERATIONS The principal mechanisms causing quantitative or functional deficiencies of drug-metabolizing enzymes in human liver are summarized in Table 1. Thus, a mutation in the regulatory or structural sequence of the gene for a drug-metabolizing enzyme may cause decreased intracellular concentration or absence of the enzyme protein or lead to a structural alteration with consequent changes in enzyme function. It is evident from these considerations that the elucidation of the molecular basis of these polymorphisms requires access to human tissue (liver, leucocyte DNA, etc.) from subjects or families phenotyped in rive to establish the causal relationship between in vitro and in rive findings. Moreover, sensitive assays for the involved metabolic reactions are necessary to monitor the purification of the enzymes with affinity for the substrates in question. The various types of oxidative reactions affected by the debrisoquine/sparteine polymorphism (e.g. alicyclic, aliphatic and aromatic hydroxylations, oxidative dealkylation, N-oxidation; Fig. 2) suggested a

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FIG. 2. Chemical structures of drugs with deficient metabolism in poor metabolizers of debrisoquine.

cytochrome P-450 enzyme ('P-450') as the possible target of the defect. But it was not clear which of the multiple P-450 enzymes of the cytochrome P-450 superfamily may be the target of the debrisoquine/sparteine polymorphism. The liver contains multiple P-450 enzymes, probably between 20 and 100, and these have variable and often overlapping reactivities toward various drug substrates (Gonzalez, 1989, 1990; Meyer, 1984; Nebert and Gonzalez,

1987; Ortiz de Montellano, 1986). Recent data on primary amino acid sequence and chromosomal localization for a large number of human and animal P-450s have revealed that the different enzymes are produced from a gene superfamily, i.e. each P-450 enzyme is derived from a different gene. To date 9 gene families have been described in mammals and a nomenclature based on sequence has been proposed for these multiple enzymes (Gonzalez, 1989, 1990;

300

U.A. MEYERet al. TABLE 1. Possible Mechanisms Causing a Genetic Deficiency of a Drug-Metaboli-ing Enzyme

At DNA/RNA level (a) Deletion, insertion or rearrangement mutation of gene (b) Defect in transcription, RNA processing or RNA stability At enzyme protein level (a) Decreased intracellular concentration or absence of enzyme protein --Diminished rate or lack of synthesis --Accelerated degradation of labile enzyme variant (b) Normal intracellular concentration of mutant enzyme protein At level of enzyme function (a) Decreased affinity for substrates (increased K,n) (b) Decreased maximal velocity (decreased Vm~X) (c) Combination of (a) and (b) (d) Change in the stereoselectivity of the reaction

Nebert et al., 1987), Accordingly, the human P-450 enzyme catalyzing debrisoquine and sparteine oxidation is called P-450IID1, although in a recent update of the proposed nomenclature (Nebert et al., 1989) its designation has been changed to IID6. The reasons for this change was the discovery of 5 rat genes related to P-450IID1 (Matsunaga et al., 1989). However, to conform with previous reviews and with most of the cited literature in this manuscript we will maintain the designation of P-450IID1. It also should be realized that P-450s are difficult to purify and to separate from each other because of their similar physical properties, their labile prosthetic heine group and their integration into the membranes of the endoplasmic reticulum. Another complication in their study is overlapping substrate specificity, i.e. one P-450 enzyme can metabolize multiple substrates and the same substrate can often be metabolized by different P-450s. A further problem is that antibodies against purified P-450s frequently recognize the structurally related P-450s of the same family or subfamily. All these complications had to be overcome with P-450IID1 and initially delayed progress in the elucidation of the debrisoquine/sparteine polymorphism. 3.2. IDENTIFICATIONOF P-450IID1 (P-450dbl, P-450bufI) AS THE DEFICIENTENZYME Initial studies by Davies et al. (1981) revealed no detectable debrisoquine 4-hydroxylation in microsomes prepared from the liver biopsy of one poor metabolizer and provided evidence for NADPHrequirement and inhibition by carbon monoxide of debrisoquine 4-hydroxylation in livers of extensive metabolizers, consistent with the involvement of a P-450-mediated monooxygenase reaction. More detailed studies in our laboratory in biopsies of extensive and poor metabolizers excluded the presence of specific inhibitors or a generally decreased microsoreal content in total spectral P-450, of abnormalities in typical other P-450 activities or of electron transferring proteins (P-450 reductase and cytochrome bs) as cause of the metabolic defect of poor metabolizers (Meier et al., 1983; Minder et al., 1984). Of particular significance for the identification and functional characterization of the involved P-450 isozyme was the introduction of the beta-blocking agent bufuralol (Fig. 2) as a prototype substrate (Dayer et al., 1982; Minder et al., 1984). The fluor-

escent properties of bufuralol provided the basis for a sensitive HPLC-assay of the metabolism to the major metabolite l'-OH-bufuralol (Kronbach et al., 1987), allowing the study of small (5 mg wet weight) liver samples. Moreover, bufuralol is a chiral compound which displays stereoselective metabolism primarily of the (+)-enantiomer to l'-OH-bufuralol in vivo and in human liver microsomes. Interestingly, this stereoselectivity was virtually absent as determined in serum or in microsomes from liver biopsies of poor metabolizers (Dayer et al., 1984, 1986, 1987; Gut et al., 1984). Considerable residual nonselective bufuralol l'-hydroxylase activity was detectable in poor metabolizers in t,ivo and in vitro. This suggested to us that more than one enzyme may be involved in microsomal bufuralol l'-hydroxylation. Kinetic analysis of microsomal bufuralol l'-hydroxylation (Dayer et al., 1987; Kronbach et al., 1987) and debrisoquine 4-bydroxylation (Boobis et al., 1985; Kronbach et al., 1987) indeed revealed the contribution of high- and low-affinity P-450 enzymes to the total metabolism of the various substrates. Finally, these observations in microsomes were confirmed by partial purification from human liver of two functionally distinct P-450 isozymes, both able to catalyze bufuralol l'-hydroxylation, namely P-450IIDI (previously designated P450bufl (Gut et al., 1986) or P450dbl (Gonzalez et al., 1988b; Zanger et al., 1988a) with high affinity for bufuralol and high stereoselectivity for the (+)-enantiomer, and the low affinity/nonstereoselective P450buflI. Functionally, the two isozymes could be further differentiated by their ability to produce characteristic patterns of additional bufuralol metabolites, by their different specificity for other substrates, and their sensitivity to quinidine, a potent inhibitor of polymorphic drug oxidation reactions of the debrisoquine/sparteine type in ritro (Gut et al., 1986; Otton et al., 1984) and in vivo (Brinn et al., 1986', Brosen et al., 1987; Leemann et al., 1986). Detailed kinetic studies in liver microsomes of poor metabolizers (Dayer et al., 1984, 1987; Gut et al., 1986) strongly suggested the specific deficiency of only the high affinity/highly stereoselective enzyme (P-4501ID1) in poor metabolizers. But in the usual microsomal assays the different enzymatic activities contributing to bufuralol l'-hydroxylation are always measured together. We since have discovered that P-4501ID1 and the corresponding microsomal activity can be assessed alone by using the unique

Debrisoquine/sparteine metabolism peroxygenase function of this isozyme in a cumene hydroperoxide (CuOOH)-supported reaction (Zanger et al., 1988b). By kinetic analysis of microsomes from liver biopsies of in vivo phenotyped extensive metabolizer and poor metabolizer individuals, we could demonstrate an increased selectivity of the CuOOHmediated activity of P-450IIDI for the in t, itro identification of the poor metabolizer condition manifested by (a) a drastically reduced Vm,x for l'-OH-bufuralol formation, (b) an increased Km (for + bufuralol), and (c) the loss of stereoselectivity in poor metabolizer microsomes (Zanger et al., 1988b). The more selective assay for P-450IID1 proved to be highly valuable during further purification because it measures a relatively detergent-insensitive P-450 function. This advantage and the application of monoclonal antibodies to remove copurified proteins such as epoxide hydrolase enabled us to improve decisively the purification of P-450IID1 from human liver. The obtained preparation had the highest specific activity reported so far and allowed sequencing of the N-terminal 22 amino acids (Gonzalez et al., 1988c; F. Vilbois, U. M. Zanger, F. Lottspeich and U. A. Meyer, unpublished data). Furthermore, the results obtained with this assay and with purified P-450IIDI demonstrated convincingly the specificity of the polyclonal antibody previously prepared against rat P-450IIDI (P450dbl; Gonzalez et al., 1987) for a single human P-450 isozyme, P-450IIDI (Zanger et al., 1988b). This antibody was used for the cloning of the rat P - 4 5 0 I I D I and the human P - 4 5 0 H D I gene (see below). Human cytochromes P-450 with an increased catalytic activity for debrisoquine and other substrates of the debrisoquine/sparteine polymorphism have also been purified by Distlerath et al. (1985) and Birgersson et al. (1986). It is assumed that these enzymes are identical to P-450IIDI, although no sequence information is available. 3.3. IMMUNOQUANTITATIONOF P-450IID1 IN HUMAN LIVER

Different types of antibodies all specifically recognizing P-4501ID1 subsequently were developed or discovered in our laboratory: they include rabbit polyclonal antibodies, mouse monoclonal antibodies and human autoantibodies. All these antibody preparations were used to test the alternative hypotheses that (1) quantitative absence of P450IID1, or (2) presence of a functionally deficient P-450IIDI protein, may explain deficient drug metabolism in poor metabolizers (Table 1). The amount of P-450IID1 immunoquantitated on Western blots with these antibodies closely correlated with the Vm~Xof CuOOH-mediated bufuralol-l'-hydroxylation (Gonzalez et al., 1988b; Zanger et al., 1988b; Fig. 3). No P-4501IDI protein could be demonstrated in the liver biopsies of poor metabolizers (Zanger et al., 1988b). Thus, our immunological data provide convincing evidence for a specific absence of P-450IIDI rather than a functional alteration as cause of the debrisoquine polymorphism (Fig. 1).

301

3.4. AUTOANTIBOD1ESAGAINSTP-450IID1 IN CHRONIC ACTIVE HEPATITIS Chronic active hepatitis is the collective term for an etiologically heterogeneous group of progressive inflammatory liver diseases. Most cases are either associated with hepatitis B virus markers, are drug induced or are of the autoimmune-type (Odi6vre et al., 1983). Autoimmune hepatitis has been further differentiated into two classes depending on the mutually exclusive occurrence of antiactin antibodies in autoimmune hepatitis I (lupoid hepatitis) or of anti-liver kidney microsome antibodies type 1 (antiLKM1) in autoimmune hepatitis II (Homberg et al., 1987). Anti-LKMl-defined chronic active hepatitis occurs predominantly in girls between 2 and 14 years of age and is frequently associated with a number of other autoimmune diseases but not with common viral infections or drug-induced hepatitis (Homberg et al., 1987; Maggiore et al., 1986). Anti-LKMI autoantibodies were discovered in indirect immunofluorescence studies by their characteristic reaction with cytoplasm of hepatocytes and proximal renal tubules of rat (Rizzetto et al., 1973). Further studies revealed binding of the antibody to rough and smooth endoplasmic reticulum and, in rat liver microsomes, to a 50 kDa integral membrane protein (De Lemos-Chiarandini et al., 1987). Recently, a second type of anti-LKM antibody (anti-LKM2) has been described; it can be distinguished from antiLKM 1 by a different immunofluorescence pattern in kidney (Homberg et al., 1984). Anti-LKM2 exclusively occurs in patients with hepatitis induced by tienilic acid (ticrynafen) and the corresponding antigen has been identified as a human cytochrome P-450 isozyme which metabolizes the drug (Beaune et al., 1987) and is identical or closely related to a P-450 isozyme metabolizing mephenytoin (Meier and Meyer, 1987). We have discovered that IgGs prepared from sera of several patients with autoimmune hepatitis II specifically inhibit and immunoprecipitate a microsomal protein with activity for the hydroxylation of bufuralol and debrisoquine and have identified the LKM-1 antigen as P-450IID1 (Zanger et al., 1988a). This was evidenced by complete inhibition and immunoprecipitation of CuOOH-mediated microsomal bufuralol-l'-hydroxylation by anti-LKM1 sera. We also used anti-LKMl IgG covalently linked to Protein A-Sepharose to isolate the microsomal antiLKM1 antigen. The eluted protein was identical to P-450IIDI in its M r of ~ 5 0 k D a and its N-terminal amino acid sequence (Zanger et al., 1988a). By the same procedure the anti-LKM1 antigen was isolated from solubilized microsomes of human livers including liver biopsies from in vivo phenotyped extensive metabolizer and poor metabolizer individuals. The amount of P-450IIDI immunoisolated in this way closely correlated with the V,,,~ of bufuralol-l'hydroxylation. No-protein could be isolated from poor metabolizer livers. Thus human anti-LKM1 autoantibodies indeed are specifically directed against cytochrome P-450IID1. The important question of course is whether the occurrence of anti-P-450IID1 autoantibodies is causally related to the function of this enyzme, to the

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Fro. 3. Relationship of immunodetectable P-4501lDI and Vm,Xof (+)bufuralol l'-hydroxylation in 14 human livers. Eight of the samples were liver biopsies from patients phenotyped in vivo with debrisoquine or sparteine (from Zanger et al., 1988b). Reprinted with the permission of the copyright holder, American Chemical Society, Washington, D.C. genetic polymorphism of debrisoquine metabolism and ultimately to the development of chronic active hepatitis. In patients with hepatitis induced by the drug tienilic acid (ticrynafen) and anti-LKM2 autoantibodies a mechanism was proposed involving covalent modification of this enzyme by reactive metabolites of tienilic acid followed by immunological response to the altered protein structure (Beaune et al., 1987). If a similar mechanism applies to the generation of anti-LKM 1 antibodies, a substrate with high specificity for P-450IIDI and possibly leading to modification of the protein structure has to be postulated. As already mentioned, a large number of drugs are metabolized by P-450IID1 (Fig. 2). They include

known hepatotoxins such as perhexiline (Shah et al., 1982) or N-propylajmaline (Zekorn et al., 1985). There is no evidence, however, that the patients with anti-LKMl positive chronic active hepatitis were exposed to such drugs, or to any other potential substrates of P-450IID1. Most recent data suggest that anti-LKM 1 positive patients are of the extensive metabolizer phenotype for sparteine (Manns et al., 1989) and thus may produce more of a toxic metabolite. We observed normal enzyme activity and normal behavior on electrophoresis of the P-450IID1 protein in the diseased liver of a anti-LKM1 positive patient (U. M. Zanger and U. A. Meyer, unpublished data). This interesting liver tissue became available when the

Debrisoquine/sparteine metabolism patient with L K M I positive hepatitis received a liver transplant. The predominance of female patients with antiLKM 1 autoantibodies and the association with other autoimmune diseases in 30-50% of these patients may point to a deleterious combination of sex-specific and genetic factors in the generation of hepatotoxicity. No endogenous substrates (e.g. steroids) of P450IID1 have been described so far. The high activity of P-450IID1 with organic hydroperoxides may offer yet another possibility whereby intermediate products of lipid peroxidation could alter this specific protein. It may even be speculated that these patients develop antiidiotypic antibodies against a circulating drug- or metabolite-protein complex, This hypothesis would explain the amazing inhibitory capacity of anti-LKM 1 antibodies for bufuralol l'-hydroxylation because they would be directed against the drug-binding or catalytically active site. All the proposed mechanism must account for the generation of antibodies against one discrete P-450 isozyme among the numerous related proteins and for the fact that these antibodies are strongly inhibitory. As patients with autoirnmune hepatitis type II have a poor prognosis, the disease progressing rapidly to cirrhosis and hepatic failure, it is important that these hypotheses are tested in the near future. 3.5. CLONINGAND CHARACTERIZATION OF A HUMAN P-450IID1 (dbl) cDNA AND OF GENES RELATEDTO P-450IID1 The polyclonal antibody against the rat P-450IIDI protein described above (Gonzalez et al., 1987; Zanger et al., 1988b) was used to screen a human liver ,;,gtll library for IID1 cDNA clones. A cDNA containing the full protein coding sequence was isolated and characterized in a collaborative effort between the laboratory of F. J. Gonzalez (National Cancer Institute, NIH, Bethesda, U.S.A.) and our laboratory. The cDNA-deduced NH2-terminal sequence was in complete agreement with that of our purified P-450IID1 protein (Gonzalez et al., 1988c). Moreover, the cDNA was functionally expressed in the SV40-based COS cell system and the expressed enzyme had the same molecular weight and functional characteristics (Kin, stereoselectivity for ( + ) bufuralol, inhibition by anti-LKM1 antibodies, inhibition by quinidine, etc.) as the human microsoreal activity and the purified P-450IID1 enzyme (Gonzalez et al., 1988b; U. M. Zanger, T. Catin, F. J. Gonzalez and U. A. Meyer, unpublished observations). By use of human-rodent somatic cell hybrids the P - 4 5 0 H D ! gene was localized to the long arm of human chromosome 22 ( C Y P 2 D locus; Gonzalez et al., 1988c), in accordance with linkage studies with genetic markers in poor metabolizer families by Eichelbaum et al. (1987). More recently, the human C Y P 2 D locus has been further characterized by analyzing genomic clones derived from human lymphocyte DNA of a positively identified homozygous extensive metabolizer of debrisoquine (Kimura et al., 1989). The human C Y P 2 D locus contains three genes, designated l i D ! (111)6 according to the recent 'update' of the nomenclature; Nebert et al., 1989), l l D 7 a n d lIDS, located on a contiguous

303

region of about 45 kbp on chromosome 22. The l i d genes have 9 exons and 8 introns as other genes of the P-450II gene family. The sequence of the l i D 7 and I I D 8 genes suggest that they probably represent mutants or pseudogenes. Expression of l i D 7 and I I D 8 has not been detected suggesting that only l i d 1 is expressed in normal human liver. 3.6. ABERRANTSPLICINGPATTERNSOF P-450IIDI

PRE-mRNA IN LIVERSOF POOR METABOLIZERS To study the molecular basis of the debrisoquine polymorphism, Northern blot analysis with m R N A initially isolated from 7 organ transplant donor livers and hybridization to the IIDI cDNA was performed. Three of the livers had markedly decreased bufuralol l'-hydroxylase activity, indistinguishable from livers of poor metabolizers of debrisoquine, although the donors for obvious reasons could not be phenotyped in t, it, o. Variant mRNAs of larger ( ,~ 2.1 kb, variants a and b) or smaller ( ~ 1.0 kb, variant b') size were observed in three low activity livers as compared to the normal 1.Bkb m R N A species predominantly represented in livers of extensive metabolizers. To determine the nature of the variant mRNAs, cDNA libraries were constructed from them and screened with the IID1 cDNA. When the isolated cDNAs were sequenced we discovered that the variant a from two of the low activity livers had retained the complete intron 5, another variant (b) had retained intron 6. A third variant, (b') probably representing the smaller size m R N A was truncated and was missing part of the 6th exon which was removed by incorrect splicing (Gonzalez et al., 1988b; Fig. 4). Variant b' could represent alternative splicing of the same allele as variant b. These data suggest that at least 2 different mutated alleles can cause aberrant splicing of pre-mRNAS in the low activity livers. It is likely that these abnormal mRNAs lead to prematurely terminated, truncated and unstable proteins. This would explain the absence of the P-450IID1 protein in the poor metabolizer livers (Zanger et al., 1988b). However, further studies are necessary to determine which changes in the D N A sequence are responsible for incorrect splicing of IIDI pre-mRNA in poor metabolizers. An important aspect of our findings is that apparently different mutations cause deficient P-450IID1. We do not know how many additional mutant alleles are responsible for the poor metabolizer phenotype. This can only be estimated when a larger number of poor metabolizers have been studied. 3.7. DETECTIONOF MUTANT P - 4 5 0 I I D I GENES BY RESTRICTION FRAGMENTLENGTH POLYMORPHISMS (RFLPs) Some mutations in exons, introns or flanking regions of a gene, even at considerable distance from the affected gene, are likely to create or eliminate restriction sites where endonucleases recognize specific sequences and cleave DNA. When the sizes of restriction fragments are changed in this way, the alterations are referred to as restriction fragment length polymorphisms, or RFL15s. A RFLI i may be directly generated by the primary mutational event

304

U. A. MEYERet al. HUMAN P450dM GENE ON CHROMOSOME 22 1 2 3 4 5 6

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Loss3'half exon 6, truncation

FIG. 4. Scheme of the exons and approximate sizes of the 8 introns of the P-4501IDl gene. The normal splicing pattern of the wild-type (wt) pre-mRNA, and aberrant splicing of three variant pre-mRNAs from livers with markedly decreased activity of bufuralol-l'-hydroxylase and absent P-450IID1 protein. causing the genetic deficiency or can be 'linked' to it because changes in nonfunctional flanking DNA occurr and identify the mutated or the normal allele. Leucocyte DNA from in vivo phenotyped extensive and poor metabolizer individuals was digested with a large number of different restriction endonucleases and the separated DNA fragments hybridizing with the P-450IIDI cDNA were analyzed (Southern analysis). RFLPs generated by several endonucleases were found to be associated with the poor metabolizer phenob, pe when DNA of 24 unrelated poor metabolizers and of 29 unrelated extensive metabolizers was compared (Skoda et al., 1988). The segregation of these RFLPs was studied in 6 families of poor metabolizers in which obligate heterozygote carriers of the recessive gene could be identified by pedigree analysis (Skoda et al., 1988). The results are best illustrated with the XbaI endonuclease with the 6 families shown in Fig. 5. At least one 29 kb fragment was present in all extensive metabolizers and represents the normal allele. A polymorphic 44 kb fragment was found in 58% of poor metabolizers but only in 3.4% of extensive metabolizers, and a polymorphic 11.5 kb fragment was present in 33% of poor metabolizers but in none of the extensive metabolizers tested in this study. Only one individual was homozygous for the 44 kb fragment and recently one for the 11.5 kb allele was discovered, both were as expected of poor metabolizer phenotype by urinary metabolic ratio determination. Thus, 75% of poor metabolizers had either the 44kb or the 11.5 kb polymorphic DNA fragment, which in the family

studies were allelic with the 29 kb fragment present in all extensive metabolizers. The remaining 25% of poor metabolizers have a 29 kb/29 kb genotype as have the majority of the extensive metabolizers; their mutated alleles cannot be identified by RFLPs with the enzymes tested to date. Our data on this small population of phenotyped individuals thus indicate that at least three different independent mutant alleles of the P - 4 5 0 I I D I gene locus are associated with the poor metabolizer phenotype for debrisoquine hydroxylation and occur singly or in combination (Table 2), In analogy to other genetic polymorphisms and diseases, we suspect that within the group of poor metabolizers having one or two 'uninformative' 29 kb fragments many additional mutations will be found. Genomic DNA of these individuals is presently being sequenced to identify these mutational events. The elucidation of the mechanisms accounting for the observed RFLPs also will require sequence analysis of the normal and mutant P - 4 5 0 I I D I genes. The fact that several other restriction enzymes demonstrate a similar increase in fragment size in individuals with the 44 kb XbaI fragment, whereas a decrease in fragment size is again seen with several other restriction enzymes (which are in linkage disequilibrium with XbaI) in individuals with the 11.5 kb XbaI variant, suggests an insertion or deletion mechanism, respectively (Skoda et al,, 1988). However, other mechanisms such as gene conversion, unequal crossovers, etc., also are possible. It also cannot be decided yet if the same mutation causes the observed RFLPs and the deficiency of the P-450IID1 enzyme or if different but linked mutations are involved. In

TABLE2. Genotyping using Xbal R F L P s with a P - 4 5 0 I I D I cDNA Probe Phenotype ~25% of PMs ~50% of PMs ~25% of PMs

Genotype* 44/44 or 44/11.5 kb or 11.5/11.5 kb fragments 44/29 or 11.5/29 kb fragments 29/29 kb fragments

Interpretation RFLPs predict PM phenotype RFLPs predict heterozygous EM or PM with only one identified mutated allele Xbal RFLPs not informative

EM: extensivemetabolizer ofdebrisoquine; PM: poor metabolizer ofdebrisoquine. From Skoda et al., 1988.

Debrisoquine/sparteine metabolism

11 [2

Go

[1 [2

111 112 113

We

305

11 12

111 11'2 113

44.0

29.0

Me

111 112113114

44.0 29.0 --11.5

44.0 29.0

--4.5 - - 4.0

4.5

--

--

11.5

4.0

4.5 4.0

I1 12

MI1 12

MI1 12 lI5

II 1 I12 1"[3

111 112 11"3 m

1TIr

44.0 29.0

44.0 29.0

44.0 29.0

--

4.5

~.5

a.5

--

4.0

40

4.0

- -

- -

FIG. 5. Detection of mutant P - 4 5 0 I I D I genes by RFLP analysis of genomic DNA in 6 families of poor metabolizers of debrisoquine (from Skoda et al., 1988).

306

U.A. MEYERet aL

any case, the C Y P 2 D gene locus is highly polymorphic. Of over 20 restriction enzymes tested, only 7 displayed no RFLPs, and with the remaining 13 enzymes, 14 allelic forms of the P - 4 5 0 I I D I gene can be described. However, only 2 of these alleles were linked to the PM phenotype.

et al., 1986) and Balkan nephropathy (Ritchie et al.,

1983) remains a puzzle. Acknowledgements--The work from our department cited

in this review was supported by Grants 3.806.84 and 3.817.87 from the Swiss National Science Foundation.

4. F U T U R E PERSPECTIVES

REFERENCES

Because of its clinical importance the polymorphic P-4501ID1 enzyme and its gene have become one of the most extensively studied human P-450s to date. These investigations also represent the initial steps toward a direct determination of the poor metabolizer genotype as opposed to the less reliable phenotyping procedure which requires drug administration and urine collection and is hampered by potential drug~trug interaction and analytical problems (Brosen and Gram, 1989). In the future, additional mutations affecting the I I D I gene which cause deficient P-450IIDI function will be identified and ultimately, the majority of poor metabolizer will probably be identifiable with a combination of gene probes recognizing the different mutations. Moreover, new drug substrates will be identified. One approach in this direction is to use the extremely sensitive CuOOH-mediated bufuralol-l'-hydroxylation assay in human liver microsomes (Zanger et al., 1988b) and to screen potential endogenous and exogenous substrates for competitive inhibition (Fonnd-Pfister and Meyer, 1988). Of considerable interest for instance was the observation that MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a synthetic chemical inducing a syndrome resembling Parkinson's disease in man and in animals, is a competitive inhibitor of P-450IID1 (FonnG-Pfister et al., 1987). Further evidence for a possible cerebral metabolism of this substance by P-450IID1 has been provided by the detection in brain tissue of P450IID1 with described monoclonal antibodies (Zanger et aL, 1988a). Whether P-450IIDI deficiency in liver and/or brain predisposes to early onset of Parkinson's disease, as has been suggested (Barbeau et al., 1985) however, remains speculative. Another approach with a high potential for the future is the study of new substrates of the human P-450IID1 enzyme or other P-450 enzymes in the absence of interfering other enzymes by cDNA expression, as described above (Gonzalez et al., 1988a,b). With this test system, potential substrates can simply be added to the culture medium and the metabolites measured in the medium several hours later. Finally, we would like to understand why extensive metabolizers of debrisoquine have an apparently higher incidence of cancer of the lung, bladder and liver/gastrointestinal tract (Ayesh and Idle, 1985; Kaisary et al., 1987), or in other terms why poor metabolizers appear protected. Whether the association reflects activation by P-4501IDI of an unknown procarcinogen or promoter or simply reflects genetic linkage with a protooncogene is unknown. Similarly, the role of the affected pathway in predisposing to other diseases such as lupus erythematosus (Baer

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