Cytochrome P450 2D6 deficiency and its clinical relevance in a patient treated with risperidone.

In contrast to several authors who found hepatic cytochrome P 450 2D6 (CYP2D6) metabolising status to be clinically unimportant in treatment with the CYP2D6 substrate, risperidone, we report on a 17-year-old schizophrenic patient who suffered from severe extrapyramidal side effects (EPS) while being treated with risperidone at 4 mg per day. He was genotyped as a CYP2D6 poor metaboliser (PM). The active moiety of risperidone (sum of risperidone and 9-hydroxyrisperidone) was elevated and increased even further under co-medication with haloperidol and biperiden. We conclude that the PM phenotype for CYP2D6 of this patient had major clinical importance in treatment with risperidone. Most likely metabolic pathways other than CYP2D6 were also involved that are probably inhibited by haloperidol.

Influence of CYP2D6 genotype and medication on the sparteine metabolic ratio of psychiatric patients.

OBJECTIVE: To investigate the influence of CYP2D6 genotype and medication on the reliability of phenotyping in a naturalistic setting of psychiatric inpatients. METHODS: The phenotype of 160 psychiatric inpatients was estimated by taking the urinary metabolic ratio (MR) of the concentrations of sparteine to 2- and 5-dehydrosparteine. Genotyping identified CYP2D6*1, *3, *4, *5 and *6 alleles as well as duplication of the CYP2D6 gene. All subjects underwent detailed drug history including drug dose and therapeutic drug monitoring to control compliance and abuse of other psychotropic drugs. These data were compared with those of 195 unmedicated healthy Germans. RESULTS: The cumulative distribution of the MR in patients showed a significant shift to higher MR when compared with that of healthy subjects (P < or = 0.001). Patients medicated either with selective serotonin reuptake inhibitors (SSRIs, P < or = 0.001), antipsychotic drugs (P= 0.002) or other drugs known to be substrates or inhibitors of CYP2D6 (P < or = 0.001) showed a significantly higher mean MR than unmedicated patients. However, there was no significant effect of tricyclic antidepressants on the MR. Healthy subjects with CYP2D6 deficiency were separated by a MR of greater than 20 from those who expressed functional CYP2D6. Seven patients carrying at least one functional CYP2D6 allele revealed a MR of greater than 20, indicating the occurrence of phenocopying. CONCLUSION: The results of phenotyping may be falsified by drugs known to be substrates or inhibitors of CYP2D6; thus, this method is not sufficiently reliable. However, since we observed the phenomenon of phenocopying only in patients treated with a SSRI such as fluoxetine, fluvoxamine or paroxetine, we conclude that sparteine phenotyping of medicated patients detects CYP2D6 deficiency correctly, provided that patients treated with these SSRIs are excluded.

Allele and genotype frequencies of polymorphic cytochromes P4502D6, 2C19 and 2E1 in aborigines from western Australia.

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes. Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed. The wild-type allele *1 was the most frequent (85.81%) and the non-functional alleles (*4, * 5, * 16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5. For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype. For CYP2EI, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6* 10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively.

Telomeres in neonates: new insights in fetal hematopoiesis.

Progressive telomere shortening occurs in somatic cells, and with increasing donor age a significant decline in telomere length has been shown in various postnatal tissues. In contrast, little is known about changes in telomere length during human fetal development. Therefore, we measured telomere length in the leukocyte fraction of umbilical cord blood samples from 15 preterm (<37 wk of gestation) and 11 full-term (>37 wk of gestation) neonates using the telomere restriction fragment assay. Whereas no differences in mean (+/- SD) telomere restriction fragment between the groups of preterm neonates (8512 +/- 523 bp) and full-term newborns (8323 +/- 503 bp) could be found, significantly longer telomeres (p = 0.002) were found in very low birth weight preterm neonates when compared with low birth weight preterm neonates. In addition, a rapid and significant decline in mean telomere restriction fragment was observed between 27 and 32 wk of gestation (p = 0.02, r = 0.79) followed by a period of no significant loss of telomere repeats between 33 and 42 wk of gestation. These results are consistent with the known almost maximal proliferation rate of hematopoietic progenitor cells before 32 wk of gestation. The initial decrease in telomere restriction fragment could be caused by ontogeny-related functional alterations of hematopoietic cells or differences in stem cell turnover or the rate of telomere loss per cell division.

Elucidation of the genetic basis of the common 'intermediate metabolizer' phenotype for drug oxidation by CYP2D6.

A subgroup of 10-15% of Caucasians are termed phenotypical 'intermediate metabolizers' of drug substrates of CYP2D6 because they have severely impaired yet residual in-vivo function of this cytochrome P450. Genotyping based on the currently known CYP2D6 alleles does not predict this phenotype satisfactorily. A systematic sequencing strategy through 1.6 kb of the CYP2D6 5'-flanking sequence revealed six mutations of which three were exclusively associated with the functional CYP2D6*2 allele (-1496 C to G; -652 C to T; and -590 G to A), two were associated with the nonfunctional *4 and with the functional *10-alleles (-1338 C to T and -912 G to A) and one (-1147 A to G) was seen in all *2, *4 and *10-alleles investigated. The -1496 C to G mutation was found to be polymorphic within CYP2D6*2 alleles. In a family study, the wild-type CYP2D6 *2[-1496 C] and the novel variant [-1496 G] allele co-segregated with lower and higher CYP2D6 in-vivo function, respectively, as shown by phenotyping using sparteine as probe drug. In a representative population sample selected for genotypes comprising one CYP2D6*2 and one non-functional allele, the median urinary metabolic ratio (MRs) for sparteine oxidation was 4.4-fold reduced in individuals with the variant allele (*2[-1496 G], MRs = 0.53, n = 27) compared with individuals lacking the mutation (*2[-1496 C], MRs = 2.33, n = 12; P < 0.0001). The mutation -1496 C to G has an estimated frequency of approximately 20% in the general population and allows establishment of a genotype for the identification of over 60% of intermediate metabolizers in Caucasian populations.

Pharmacokinetics of dihydrocodeine and its active metabolite after single and multiple oral dosing.

AIMS: The pharmacokinetics of dihydrocodeine (DHC) and its active metabolite dihydromorphine (DHM) were assessed after a single oral dose of DHC and after increasing doses of DHC at steady-state. Methods Twelve healthy male volunteers (18-45 years, CYP2D6 extensive metabolizers (EMs), MR<1 took a single oral dose (s.d.) of DHC 60 mg after breakfast. After 60 h DHC 60 mg was administered twice daily for 3 days, the dose was increased to 90 mg twice daily for 3 days, the final dose of 120 mg was administered twice daily for 3 days (multiple dose: m.d.). Blood sampling and urine collection: during 60 h after s.d. and during 12 h after m.d. Results No significant differences in the area under the curve (AUC) of both, DHC and DHM could be detected after a single oral dose of 60 mg DHC (AUC (0,infinity)) and during steady-state doses of 60 mg DHC (AUC(0,12 h)). During increasing steady-state doses of DHC, the data showed a dose linearity of AUC, maximal serum concentration (Cmax ) and minimal steady-state serum levels (Cssmin) of both, DHC and DHM (P<0.0001), point estimates of DHC dose corrected AUCs were well within the bioequivalence range (60 mg: 0.989; 90%CI 0.951-1. 028, 90 mg: 0.997; 90%CI 0.959-1.036, 120 mg: 0.977; 90%CI 0.940-1. 016). O-demethylation from DHC to DHM remained constant within the increasing steady-state doses of DHC in the 12 extensive metabolizers of CYP2D6. CONCLUSIONS: In the studied dose range (60-120 mg) the pharmacokinetics of DHC and its active metabolite DHM are linear in EMs of CYP2D6.

Comparative evolutionary pharmacogenetics of CYP2D6 in Ngawbe and Embera Amerindians of Panama and Colombia: role of selection versus drift in world populations.

The development of CYP2D6 has been attributed to the need of earth-dwelling animals to detoxify toxic xenobiotics (phytoalexins) present in plants. This hypothesis has been extrapolated to humans, but is yet unconfirmed. Therefore, we studied two Amerindian populations as the best available model to test the effect of selection through diet on human CYP2D6 evolution. The frequency of sparteine poor metabolizers in Ngawbe was 4.4% (n = 344), while the frequency in Embera was 2.2% (n = 153). Among Ngawbe and Embera, CYP2D6*4 (allelic frequencies for each tribe, respectively: 0.171; 0.14), CYP2D6*6 (0.005; 0.011) and CYP2D6*10 (0.175; 0.069) were detected, while CYP2D6*3, CYP2D6*5, CYP2D6*9 and CYP2D6*16 were absent. All poor metabolizers possessed either CYP2D6*4 or CYP2D6*6 and there were no disagreements between genotypic and phenotypic data. The total frequency of mutant alleles showed no difference among Amerindians or when compared to Caucasians. It was higher than in Chinese, since the frequency of CYP2D6*4 was higher in Amerindians. XbaI restriction fragment length polymorphisms haplotypes were very homogeneous in Amerindians, because the only fragment that hybridized with the CYP2D6 cDNA probe was the 29 kb (not 42/44 kb or 11.5/13 kb). This indicated no gene cluster recombinations that generate insertions or deletions. We propose that in earlier hominids and humans, CYP2D6 had increasingly become a vestigial characteristic unconstrained by dietary stressors, as a result of cultural survival strategies. Human CYP2D6 evolution was preferentially affected by random genetic drift, and not by adaptive or purifying selection.

Analysis of the CYP2D6 gene mutations and their consequences for enzyme function in a West African population.

The data on differences in the metabolic handling of the CYP2D6 probe drugs sparteine and debrisoquine, and the relationship between phenotype and genotype and gene frequencies for several mutant CYP2D6 alleles in African populations are limited and sometimes controversial. Therefore, in a West African population (Ghana), we investigated (i) the phenotype for sparteine debrisoquine by phenotyping 201 individuals with both drugs and (iii) the genotype for CYP2D6 (n = 326) and debrisoquine (n = 201) oxidation, (ii) the coregulatory control of sparteine and alleles *3 and *4 in 133 individuals and for the alleles *1, *2, *3, *4, *5, *6, *7, *8, *9, *10, *14, *16, *17, *2b, *2xN, *2bxN in 193 individuals. Of the 326 individuals phenotyped with sparteine, eight had a metabolic ratio (MR)sp > 20 corresponding to a poor metabolizer frequency of 2.5% [95% (confidence interval) CI = 1.06-4.77]. The prevalence of the poor metabolizer phenotype for debrisoquine oxidation was 3% (95% CI = 1.1-6.39) with six of the 201 individuals having a MR greater than 12.6. The distribution of the MR of sparteine was trimodal whereas MR of debrisoquine was unimodally distributed with a pronounced kurtosis. In individuals phenotyped with both drugs, there was a significant correlation between the MRs (r(s) = 0.63, P < 0.001). The CYP2D6 alleles *1, *2 and *17 were the most common functional alleles occurring with frequencies of 43.7, 10.6 and 27.7%, respectively. The three other observed functional alleles *2xN, *10 and *20 had much lower frequencies (1.6%, 3.1% and 0.3%, respectively). Of the eight non-functional alleles, only *4 (6.3%) and *5 (6.0%) could be found. The allele *5 occurred with the same frequency as in Caucasian populations (4.1%) but the *4 allele had a much lower frequency (Caucasians 19.5%). One individual with *1/*1 was a poor metabolizer for sparteine and debrisoquine indicating the existence of as yet unknown non-functional alleles in this West African population. Although the prevalence of poor metabolizers and the number of heterozygotes for non-functional alleles was much lower in Ghanaians, the median MRsp of 0.7 was significantly higher in this population compared with a median MRsp of 0.4 in Caucasians, indicating a lower metabolic clearance for CYP2D6 substrates in the West Africans. The lower metabolic activity in Ghanaians could not be explained solely by the high frequency of the *17 allele, which is associated with an impairment of CYP2D6 enzyme function. In addition, a higher median MRsp of 0.5 corresponding to metabolic clearance of 346 ml/min was observed among extensive metabolizers with the genotype *1/*1. Thus, compared with the median of MRsp = 0.28 (CLmet 573 ml/min) in Caucasians homozygous for *1, the metabolic clearance of sparteine was 40% lower on average in respective Ghanaians.

Interaction of modafinil and clomipramine as comedication in a narcoleptic patient.

Modafinil is a psychostimulant compound that is just now becoming available in many countries for treatment of narcoleptic and hypersomnic patients. Whereas sleep attacks and drowsiness can be effectively improved, the drug does not sufficiently reduce cataplectic seizures. It therefore is often used in combination with tricyclic antidepressant medication, although little is known about the possible interactions. This case report describes a narcoleptic patient chronically treated with clomipramine who started receiving modafinil. The blood concentrations of clomipramine and its metabolite showed a significant dose-dependent and reversible increase under modafinil. Since the patient was genotypically and phenotypically a cytochrome P450 2D6 poor metabolizer, the authors attribute the relevant pharmacokinetic interaction to another clomipramine-metabolizing enzyme.

Assessment of the predictive power of genotypes for the in-vivo catalytic function of CYP2D6 in a German population.

The polymorphic cytochrome P450 CYP2D6 catalyses the biotransformation of at least 40 drugs. The CYP2D6 genetic polymorphism is responsible for pronounced interindividual differences in plasma concentrations and, hence, in drug action and side-effects after administration of the same dose. Provided there is a close relationship between CYP2D6 genotypes and catalytic function, genotyping could be used in the clinical setting for individualization of drug dose. In the present study, we evaluated the relationship between the in-vivo enzyme activity and 35 different genotypes in order to determine whether genotyping can be used to predict a person's metabolic capacity for CYP2D6-catalysed drug oxidation using sparteine as a probe drug. One hundred and ninety-five Caucasian individuals were genotyped for seven nonfunctional (CYP2D6 x 3, x 4, x 5, x 6, x 7, x 8, x 16) and eight functional alleles (CYP2D6 x 1, x 2, x 2 x 2, x 2B, x 2B x 2, x 9, x 10, x 17). The metabolic ratio distribution for sparteine showed trimodality, with 15 poor metabolizers, 21 intermediate metabolizers, and 1.59 extensive and ultrarapid metabolizers. All poor metabolizers were unambiguously identified as carriers of two nonfunctional alleles. In contrast, the most frequent functional genotypes extensively overlapped and, with few exceptions, genotype was not a useful predictor of function. Gene dose effects among homozygotes and heterozygotes of the major functional alleles were not significant and could not explain the wide variations. Only a minor fraction of phenotypical ultrarapid metabolizers, arbitrarily defined as individuals with a metabolic ratio < 0.2, could be identified as carriers of three functional gene copies, including duplicated CYP2D6 x 2 x 2 alleles. Similarly, only a minor fraction of the intermediate metabolizers had predictive genotypes involving alleles coding for enzyme with impaired function. Thus, genotyping correctly identifies poor metabolizers, but quantitative prediction of drug metabolism capacity among extensive metabolizers is not possible.

Analysis of CYP2D6 expression in human lung: implications for the association between CYP2D6 activity and susceptibility to lung cancer.

We have studied whether CYP2D6 is expressed in human lung tissue, using a specific and sensitive reverse transcriptase-polymerase chain reaction method and immunohistochemistry. Seven out of the eight patients were extensive metabolizers as shown by genotyping for the CYP2D6 (debrisoquine-sparteine) polymorphism. To investigate whether expression of CYP2D6 in lung tumours is different from that in normal lung tissue, tumour tissue samples were also obtained from the same eight patients. Correctly spliced CYP2D6 mRNA was detected by RT-PCR analysis in human liver and duodenum but not in any of the lung samples. In accordance with these negative results, immunoreactivity for CYP2D6 protein, using specific monoclonal and polyclonal antibodies, was very low or absent. No specific cell type of lung tissue showed strong immunoreactivity for CYP2D6, although expression of CYP3A could be clearly demonstrated in the same tissue samples. Moreover, a Western blot analysis revealed no signal in lung microsomes from two additional extensive metabolizers. Taken together, these results indicate that expression of CYP2D6 in human lung is absent or very low. These findings thus argue against a significant local metabolic activation of procarcinogenic agents by CYP2D6 in the lung.

Rapid detection of CYP2D6 null alleles by long distance- and multiplex-polymerase chain reaction.

The CYP2D6 gene on human chromosome 22 encodes a cytochrome P450 responsible for oxidative metabolism of over 30 clinically used drugs. The CYP2D6 gene is highly polymorphic with more than 20 alleles described to date. Some of these harbour loss-of-function mutations which lead to the poor metabolizer phenotype in 5-10% of Caucasians. These individuals are at increased risk of suffering from adverse side effects or to experience therapeutic failure following drug treatment. Phenotype determination requires ingestion of a probe drug and has other inherent problems. Due to the increasing number of alleles known, comprehensive CYP2D6 genotyping using the conventional assays has become cumbersome and time consuming. We have therefore developed a streamlined and more rapid CYP2D6 genotyping procedure. Use of long distance PCR allowed the amplification of a 4666 bp fragment which contains the entire CYP2D6 gene. The 4.7 kb fragment serves as a template for a multiplex allele-specific PCR assay to simultaneously identify the five PM-associated alleles, CYP2D6*3 (A), *4 (B), *6 (T), *7 (E), and *8 (G). Together with the CYP2D6 deletion allele CYP2D6*5 (D), which can be detected in a separate PCR assay, these alleles are responsible for the PM phenotype in approximately 99% of Caucasian individuals. We tested the reliability of the procedure by analysing DNA from more than 80 individuals with known CYP2D6 genotypes. Twelve different genotypes were present among these samples and all of them were correctly identified.

Expression of CYP3A4, CYP3A5 and CYP3A7 in human duodenal tissue.

The essential role of cytochrome P450 3A4 (CYP3A4) in human small intestine is well established, and CYP3A5 seems also to be present in most subjects. However, the role of CYP3A7 in the small intestine remains poorly characterized. We have therefore studied the expression of these CYP3A enzymes in the duodenal tissue from 19 patients, using a specific RT-PCR (reverse transcriptase-polymerase chain reaction) method. CYP3A4 and CYP3A5 were present at the mRNA level in the duodenum of 18 and 19 of the 19 patients studied, respectively. In contrast, mRNA for CYP3A7 was not found in the duodenum in any of the patients. These findings strongly suggest that, unlike CYP3A4 and CYP3A5, CYP3A7 is not expressed in human duodenum.

Expression of cytochrome P 450 3A enzymes in human lung: a combined RT-PCR and immunohistochemical analysis of normal tissue and lung tumours.

We have previously demonstrated expression of cytochrome P 450 3A (CYP3A) protein in pulmonary carcinomas and surrounding normal tissue, using immunohistochemistry. These results suggested that different CYP3A enzymes may be expressed in normal and tumour tissue. Therefore, the aim of the present study was to identify specific CYP3A enzymes expressed in normal human lung and lung tumours. Both normal lung tissue and tumour tissue from eight patients was analyzed for CYP3A4, CYP3A5 and CYP3A7 mRNA using a specific RT-PCR (reverse transcriptase-polymerase chain reaction) method. Identical samples were subjected to immunohistochemical analysis of CYP3A protein. CYP3A5 was the major enzyme of the CYP3A subfamily present at the mRNA level in both normal human lung and lung tumours. CYP3A5 mRNA was detected in normal lung tissue in all eight cases and in tumour tissue in four cases. CYP3A7 mRNA was detected in five cases in normal tissue and in one tumour. Notably, no CYP3A4 mRNA was found in any of the samples. Immunohistochemical staining for CYP3A protein was found in normal lung tissue in each case. Interestingly, all pulmonary carcinomas showed immunostaining for CYP3A, while mRNA for CYP3A enzymes was found in only four cases. In summary, our study indicates a specific expression pattern of the members of the CYP3A subfamily in normal human lung and lung tumours. These findings have potential clinical significance, since it has been recently shown that CYP3A5 catalyzes the activation of the anticancer pro-drugs cyclophosphamide and ifosfamide. Thus, local activation of these agents may take place in pulmonary carcinomas and surrounding normal tissues.

Propafenone in a usual dose produces severe side-effects: the impact of genetically determined metabolic status on drug therapy.

We report the case of an elderly lady presenting with dizziness, a head injury resulting from a fall and bradycardia. Propafenone 150 mg t.i.d. had been prescribed for atrial fibrillation with tachyarrhythmia, induced by hyperthyroidism, 18 months earlier. A toxic concentration of parent propafenone, and no 5-hydroxy metabolite, was detected in a plasma sample. Symptoms disappeared after the discontinuation of propafenone. The poor metaboliser (PM) phenotype of sparteine/debrisoquine was assumed and subsequently confirmed by phenotyping (sparteine test) and genotyping (allele-specific polymerase chain reaction). The PM phenotype is common in European populations, with a prevalence of about 7%. If drugs with narrow therapeutic ranges undergo genetically polymorphic metabolism, toxicity may arise even with recommended doses. Individualization of doses is required to avoid adverse effects.

Dihydrocodeine: a new opioid substrate for the polymorphic CYP2D6 in humans.

BACKGROUND: The opioid dihydrocodeine (DHC) is frequently used as an analgesic and antitussive agent. However, until now there have been no detailed data on dihydrocodeine metabolism in humans. We therefore investigated pathways that contribute to elimination of dihydrocodeine, and we tested the hypothesis that dihydrocodeine O-demethylation to dihydromorphine (DHM) is catalyzed by the polymorphic CYP2D6. METHODS: A single oral dose of dihydrocodeine was administered to six extensive (metabolic ratio [MR] < or = 1), two intermediate (1 < MR < 20) and six poor metabolizers (MR > or = 20) of sparteine/debrisoquin. Serum concentrations of dihydrocodeine and dihydromorphine were measured up to 25 hours, and urinary excretion of conjugated and unconjugated dihydrocodeine, dihydromorphine, and nordihydrocodeine were determined. RESULTS: There were no differences in the pharmacokinetics of dihydrocodeine between extensive and poor metabolizers. However, the area under the serum concentration-time curve (AUC), partial metabolic clearance, and total urinary recovery of dihydromorphine were significantly lower in poor metabolizers (10.3 +/- 6.1 nmol.hr/L; 7.0 +/- 4.1 ml/min; 1.3% +/- 0.9% of dose) compared with extensive metabolizers (75.5 +/- 42.9 nmol.hr/L; 49.7 +/- 29.9 ml/min; 8.9% +/- 6.2%; p < 0.01). There was a strong correlation between the AUCDHC/AUCDHM ratio and the urinary metabolic ratio of sparteine (rS = 0.89, p = 0.001). No significant differences between extensive and poor metabolizers were detected in urine for conjugated dihydrocodeine (extensive metabolizers, 27.7% of dose; poor metabolizers, 31.5%), unconjugated dihydrocodeine (extensive metabolizers, 31.1%; poor metabolizers, 31.1%), conjugated nordihydrocodeine (extensive metabolizers, 6.3%; poor metabolizers, 5.4%), or unconjugated nordihydrocodeine (extensive metabolizers, 15.8%; poor metabolizers, 19.5%). CONCLUSIONS: Dihydrocodeine O-demethylation to dihydromorphine is impaired in poor metabolizers of sparteine. The main urinary metabolites after administration of dihydrocodeine are the parent compound and its conjugates in extensive and poor metabolizers.

A missense mutation in exon 6 of the CYP2D6 gene leading to a histidine 324 to proline exchange is associated with the poor metabolizer phenotype of sparteine.

The sparteine/debrisoquine polymorphism is a clinically important genetic deficiency of cytochrome P4502D6-catalyzed oxidative drug metabolism. 5-10% of Caucasians designated as poor metabolizers have a severely impaired capacity to metabolize more than 30 therapeutically used drugs. Genotyping of a random Caucasian population for the known cytochrome P4502D6 mutations A, B and D which are associated with the poor metabolizer phenotype has revealed a substantial number of misclassified poor metabolizers indicating the existence of one or more unknown mutations which cannot be identified with the currently available genotyping assays. Therefore we have cloned and sequenced one nonfunctional cytochrome P4502D6 allele of a misclassified poor metabolizer and could identify a single missense mutation designated E mutation at position 3023(A-C) in exon 6. Direct sequencing analysis, FokI restriction analysis and a newly developed allele-specific polymerase chain reaction assay were applied to analyze for this mutation in a population study. Three out of 97 randomly selected Caucasians were carriers of this mutation and thus the E allele has a frequency of 1.5% (confidence interval95% = 0.33 - 4.54%). Since only 2 out of 4 misclassified poor metabolizers carried the E mutation, additional unknown mutant alleles must exist. Computer modelling suggests that the E mutation, which results in a histidine to proline exchange in position 324 of the protein, may cause an alteration of the 3D structure of CYP2D6 in close vicinity to the active site thereby leading to total loss of enzyme function.