Genetic polymorphism of CYP2D6 and lung cancer risk in African-Americans and Caucasians in Los Angeles County

The well described genetic polymorphism of the CYP2D6 gene influences response to a wide variety of therapeutic agents metabolized by the CYP2D6 enzyme product. CYP2D6 also appears to play a role, along with other cytochrome P450 enzymes, in the metabolic activation of the tobacco specific nitrosamine, NNK, as well as metabolism of nicotine to cotinine. While impaired activity of CYP2D6 was strongly protective against lung cancer in some studies, primarily based on phenotyping, the literature is conflicting. The molecular basis of CYP2D6 deficiency is now well understood, enabling the use of genotyping to classify individuals. We therefore examined whether lung cancer risk is reduced by the presence of four CYP2D6 alleles associated with impaired activity due to an inactivating mutation--CYP2D6*4, CYP2D6*3, CYP2D6*5 and CYP2D6*16--among 341 incident cases of lung cancer and 710 population controls of Caucasian or African-American ethnicity in Los Angeles County, California. We did not confirm a strong association between the presence of these inactivating alleles and lung cancer risk [odds ratio (OR) = 0.90, 95% confidence interval (CI) 0.60-1.35 for Caucasians], although there was a small decreased risk among the African-Americans (OR = 0.66, 95% CI 0.38-1.14). Among smokers, when the data are stratified according to lifetime smoking history, there is a suggestion of an association limited to Caucasian smokers of <35 pack-years, the median for all smokers in these data (OR = 0.49, 95% CI 0.23-1.04). However, among African-American smokers, who smoke less than Caucasians, the association did not differ between smoking categories. We also examined the possible role of additional copies of the CYP2D6 gene, which lead to enhanced CYP2D6 activity, in increasing lung cancer risk. Among controls the prevalence of having more than two copies of the CYP2D6 gene and no inactivating alleles was 4.3% for Caucasians and 4.9% for African-Americans. Relative to subjects with an inactivating allele, those with an additional copy of the CYP2D6 gene and no inactivating alleles may be at increased risk of lung cancer, particularly for adenocarcinoma (OR = 3.61, 95% CI 1.08-11.7 for African-Americans and OR = 2.20, 95% CI 0.69-6.0 for Caucasians). Our data suggest that the CYP2D6 genetic polymorphism is not the strong risk factor for lung cancer suggested by some studies of phenotype, but may play a minor role.     

Detection of CYP2D6*3 and 2D6*4 allelic variants by PCR-restriction fragment length polymorphism

The mutant of CYP2D6*3 allele with A2637 deletion in exon 5 and the mutant of CYP2D6*4 allele G1934-->A, splice site defect are among the most common polymorphic alleles of CYP2D6 gene, resulting in a decreased or no activity of CYP isoenzyme. In this study, a reliable polymerase chain reaction-restriction fragment length polymorphism method for identification of CYP2D6*3 and CYP2D6*4 alleles was used to investigate the genotype and phenotype prevalence in the groups of normal controls, and of cirrhosis and cancer patients. The results showed none of 36 controls genotyped for 2D6*3 and 2D6*4 allele to have the 2D6*3 allele with frameshift mutation in exon 5, while 33% (n=12) were found to bear the 2D6*4 allele with G to A mutation at the intron 3-exon 4 junction. In breast cancer patients (n=35) genotyped for 2D6*3 and 2D6*4 alleles, none with 2D6*3 allele was found either, but 60% (n=18) were found to bear the 2D6*4 allele. In patients with head and neck squamous cell cancer, there was only one subject with 2D6*3 allele and he was heterozygous. Among them, as many as ten (40%) patients were found to bear 2D6*4 allele. In the cirrhosis group, none of the patients was found to have the 2D6*3 allele, while the CYP2D6*4 allele was found in 23% (n=6) patients. The phenotype predicted according to the genotype was as follows: in the control group, 3% of individuals were identified as poor metabolizers, 70% as extensive metabolizers, and 27% as heterozygote extensive metabolizers. In the group of breast cancer, 7% of the patients were identified as poor metabolizer, 57% as extensive metabolizer and 36% as phenotype. In squamous cell cancer and cirrhosis patients, the incidence of poor metabolizer was zero, and of heterozygotes extensive metabolizer 42% and 31%, respectively.     

Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of .005 (*1x2), .013 (*2x2), and .001 (*4x2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T1957C), *2B (additional C2558T), and *4E (additional C2938T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EM/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment.     

Significance of the plasminogen activator inhibitor of placental type (PAI-2) in pregnancy

OBJECTIVE: Genetic oxidation polymorphisms of debrisoquine (CYP2D6) and proguanil (CYP2C19) were studied in unrelated healthy South Pacific Polynesian volunteers recruited in the South Island of New Zealand. METHODS: Phenotyping for CYP2D6 and CYP2C19 activities was determined using debrisoquine and proguanil, respectively, as probe drugs by measuring the urinary metabolic ratio of parent drug and its metabolite. RESULTS: Of 100 Polynesian subjects phenotyped, the metabolic ratio of debrisoquine ranged from 0.01 to 9.94. Therefore, all South Pacific Polynesians were classified as extensive metabolizers of debrisoquine according to previously established criteria of the antimode. The prevalence of poor metabolizers of debrisoquine (CYP2D6) in this Polynesian population is 0% (95% confidence interval of 0-3.6%). Oxidation polymorphism of CYP2C19 using proguanil as a probe was also studied in 59 Polynesian volunteers. The frequency distribution of the proguanil/cycloguanil ratio was bimodal. The proguanil/cycloguanil ratios for these subjects ranged from 0.09 to 34.4. Using a recommended proguanil/cycloguanil ratio cut-off point of 10 established in Caucasian populations, eight Polynesian subjects were identified as poor metabolizers of proguanil (CYP2C19), which corresponds to a poor metabolizer phenotype frequency of 13.6% (a 95% confidence interval of 5.9-24.6%). CONCLUSION: The incidence of poor metabolizer phenotypes for debrisoquine (CYP2D6) in South Pacific Polynesians appears to lower than in Caucasian populations, while the prevalence of poor metabolizers for proguanil (CYP2C19) in this ethnic population is higher. The frequencies of the poor metabolizer phenotype for debrisoquine and also for proguanil in South Pacific Polynesians are similar to those reported in Asian populations.     

Oral ulceration as a presenting sign of unknown sarcoidosis mimicking a tumour: report of 2 cases

OBJECTIVE: To test whether some genotypes for CYP2D6 or CYP2C19 could contribute to longevity, we genotyped 241 Danish nonagenarians and centenarians for CYP2D6 and CYP2C19. METHODS: For CYP2D6 we identified the alleles CYP2D6*1, CYP2D6*3 and CYP2D6*4 with allele-specific polymerase chain reaction (PCR). The CYP2D6*5 alleles were identified with a long PCR method. For CYP2C19 we identified the alleles CYP2C19*1, CYP2C19*2 and CYP2C19*3 with an oligonucleotide ligation assay. RESULTS: The four alleles for CYP2D6 did not occur in Hardy-Weinberg proportions. The frequency of poor metabolism was slightly higher (10.2%) than expected [7.7%; odds ratio (OR) = 1.36 (0.75-2.40)]. The genotypes for CYP2C19 occur in Hardy-Weinberg proportions. The frequency of poor metabolism (3.8%) was not significantly different from a young control group [3.1%; OR = 1.21 (0.26-5.75)]. CONCLUSION: CYP2D6 could play a role in human longevity due to the lack of Hardy-Weinberg proportions. If CYP2D6 only plays a role in longevity by protecting the poor metabolizers from cancer, we should expect a rise in the frequency in these genotypes in Denmark from 7.7% among young adults to 10-11% among very old people. We found a frequency of poor metabolism of 10.2% in the very old group. CYP2C19 is - due to the occurrence of Hardy-Weinberg proportions and the expected number of poor metabolizers unlikely to contribute to human longevity.     

Polymorphism of the thiopurine S-methyltransferase gene in African-Americans

The molecular basis for the genetic polymorphism of thiopurine S -methyltransferase (TPMT) has been estab-lished for Caucasians, but it remains to be elucidated in African populations. In the current study, we determined TPMT genotypes in a population of 248 African-Americans and compared it with allele frequencies in 282 Caucasian Americans. TPMT genotype was determined in all individuals with TPMT activity indicative of a heterozygous genotype (10.2 U/ml pRBC, n = 23 African-Americans, n = 21 Caucasians). No mutant alleles were found in the high activity control groups. The overall mutant allele frequencies were similar in African-Americans and Caucasians (4.6 and 3.7% of alleles, respectively). However, while TPMT*3C was the most prevalent mutant allele in African-Americans (52.2% of mutant alleles), it represented only 4.8% of mutant alleles in Caucasians ( P < 0.001). In contrast, TPMT*3A and TPMT*2 were less common in African-Americans (17.4 and 8.7% of mutant alleles), whereas TPMT*3A was the most prevalent mutant allele in Caucasians (85.7% of mutant alleles). A novel allele ( TPMT*8 ), containing a single nucleotide transition (G644A), leading to an amino acid change at codon 215 (Arg-->His), was found in one African-American with intermediate activity. These data indicate that the same TPMT mutant alleles are found in American black and white populations, but that the predominant mutant alleles differ in these two ethnic groups.     

CYP2C19 genotype and phenotype determined by omeprazole in a Korean population

Omeprazole (20 mg orally) was given to 103 healthy Korean subjects and blood was taken 3 h after administration. The plasma concentration ratio of omeprazole and hydroxyomeprazole, used as an index of CYP2C19 activity, was bimodally distributed. Thirteen subjects (12.6%) were identified as poor metabolizers (PMs) with an omeprazole hydroxylation ratio of 6.95 or higher. Among the 206 CYP2C19 alleles, CYP2C19*2 and CYP2C19*3 were found in 43 alleles (21%) and 24 alleles (12%), respectively. Twelve subjects (12%) carried two defect alleles (*2/*2, *2/*3 or *3/*3), 43 subjects (42%) were heterozygous for a mutated (*2 or *3) and a wild type (*1) allele, and the remaining 48 subjects (47%) were homozygous for the wild type allele. The distributions of the metabolic ratio between these three genotype groups were significantly different (Kruskal-Wallis test: p < 0.0001). The genotypes of 19 additional Korean PMs has been identified in a previous mephenytoin study. From a total of 32 PMs, 31 were genotypically PMs by analysis of the CYP2C19*2 and *3 alleles and only one PM subject was found to be heterozygous for the *1 and *2 alleles. At present it cannot be judged whether this subject has a defective allele with a so-far unidentified mutation or a true wild type allele. We thus confirm a high incidence (12.6%) of PMs of omeprazole in Koreans and of the 32 Korean PMs 97% could be identified by the genotype analysis.     

Competition and quality in health care: the UK experience

Intraindividual variability and the effects of menstrual cycle phase on CYP2D6 activity were evaluated by dextromethorphan phenotyping in 20 Caucasian normal volunteers. Dextromethorphan 30 mg was administered to 10 men every 14 days for 3 months, and to 10 premenopausal women during the mid-follicular and mid-luteal phases of each menstrual cycle for three complete cycles. Urinary dextromethorphan/dextrorphan molar ratios were obtained after an overnight urine collection. Ten women and nine men were extensive metabolizer phenotypes, and one man was a poor metabolizer phenotype (confirmed by genotyping). There was no difference in dextromethorphan metabolic ratios between the mid-follicular (mean +/- SD: 0.00728+/-0.00717) and mid-luteal (0.00745+/-0.00815) phases of the menstrual cycle (P = 0.88). Also, no significant difference was found in the intraindividual variability of the metabolic ratios between the two phases (P = 0.80). No statistically significant sex difference in CYP2D6 activity was found between men (0.00537+/-0.00431) and women (0.00737+/-0.00983) extensive metabolizers (P = 0.84). For all individuals, intraindividual variability in dextromethorphan ratios ranged from 12.1-136.6% with a median of 36.7%. Because hormonal fluctuations within the mid-follicular and mid-luteal phases of the menstrual cycle do not appear to affect CYP2D6 activity, pharmacokinetic or clinical investigations of CYP2D6 substrate activity may not require menstrual cycle phase stratification. Because baseline metabolic ratios may fluctuate an average of 37%, repeat baseline and treatment phenotyping assessments should be obtained for accurate determination of a given drug's effect on CYP2D6 activity when measured by dextromethorphan.     

Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities

A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.     

An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis

To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene.     

Primary palmar hyperhidrosis presenting with unilateral symptoms: a report of two cases and review of the literature

To further investigate the association between Parkinson's disease (PD) and genetic polymorphism of the CYP2D6 gene, a mutant allele (CVP2D6J) frequently observed in the Japanese population and related to EM/PM polymorphism (phenotypically, individuals are either extensive metabolizers [EM] or poor metabolizers [PM] of debrisoquine) was investigated. The CYP2D6J gene with a nucleotide substitution from C to T at position 188 (the HphI site in exon 1), which reduces CYP2D6 enzyme activity, was analyzed by polymerase chain reaction (PCR) and by digestion with HphI. No significant relationship was observed between PD patients and controls for this mutation. This suggests that the EM/PM polymorphism of CYP2D6 contributes little to the pathogenesis of PD. To further study the molecular basis for the relationship between PD and CYP2D6, the heterogeneity of CYP2D6 was investigated by combined genotype analysis of the two mutant CYP2D6 genes (ie, CYP2D6J, the HphI site mutation in exon 1, and CYP2D6L, the HhaI site mutation in exon 6). Although some characteristic patterns of the combined genotypes were observed in both PD patients and controls, a strong association between the heterogeneity of the CYP2D6 gene and PD was not shown by combined genotype analysis.     

CYP2D6 allelic frequencies in young-onset Parkinson's disease

Parkinson's disease (PD) is thought to develop as a result of interactions between genetic susceptibility factors and environmental exposures. One candidate gene is CYP2D6, which codes for the debrisoquine 4-hydroxylase cytochrome P450. Impairment of debrisoquine 4-hydroxylase activity has been associated with an increased risk of PD in patients with younger age at disease onset. Genotyping studies in patients with an older age at onset have reported modest increases in risk associated with the CYP2D6 B and A alleles; however, the risk for young-onset PD has not been adequately evaluated. We designed a case-control study to investigate the role of nonfunctional CYP2D6 allelic risk factors for young-onset PD in a sizable patient population and compared the distributions of CYP2D6 genotypes between young-onset ( < or = 51 years) PD patients (n = 108) and controls (n = 236). In contrast with the results from genotyping studies conducted among patients with an older age at onset, there were no significant differences in CYP2D6 allelic frequencies between young-onset PD cases and controls. The frequency of the B allele was slightly lower in the young-onset PD cases than in the controls (0.14 versus 0.20) (X2 = 2.66, p = 0.10). The presence of one or more B alleles was not associated with an increased risk of young-onset PD (odds ratio 0.58; 95% CI 0.33 to 1.00), nor was the presence of one or more nonfunctional alleles (i.e., A, B, D, and D2) (odds ratio 0.68; 95% CI 0.41 to 1.13). This study suggests that the young-onset PD population may differ from the older-onset population with respect to risk factors.     

Ultrarapid drug metabolism: PCR-based detection of CYP2D6 gene duplication

The enzyme debrisoquine 4-hydroxylase (CYP2D6), which metabolizes many widely used drugs, is highly polymorphic. The activity of the enzyme ranges between subjects from ultrafast to a complete absence. Therefore, metabolic capacity varies, producing intersubject differences in therapeutic efficacy and side effects at standard recommended doses. Up to 7% of Caucasians may demonstrate ultrarapid drug metabolism (UM) because of inherited alleles with multiplicate functional CYP2D6 genes, causing an increased amount of enzyme to be expressed. Identification of UM subjects is of potential clinical importance for adjustment of doses in drug therapy, as well as to avoid misidentification of noncompliance. In our study, we tested recently designed PCR assays for the detection of the UM genotype. We found a 3.5% prevalence of UMs carrying duplicate active CYP2D6 genes in a population consisting of 202 psychiatric patients.     

Use of omeprazole as a probe drug for CYP2C19 phenotype in Swedish Caucasians: comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype

A single oral dose of omeprazole (20 mg) was given orally to 160 healthy Caucasian Swedish subjects and tested as a probe for CYP2C19. The study was nonrandomized and included seven subjects previously classified as poor metabolizers (PM) of S-mephenytoin. The ratio between the plasma concentrations of omeprazole and hydroxyomeprazole (metabolic ratio; MR) was determined by HPLC in a blood sample drawn 3 h after drug intake. In 17 subjects the test was repeated and the MRs of omeprazole on the two occasions were correlated (rs = 0.85; p < 0.0001). There was a significant correlation between the MR of omeprazole and the S/R mephenytoin ratio among 141 subjects, in whom both ratios were determined (rs = 0.63, p < 0.001). All seven PMs of S-mephenytoin had higher MRs of omeprazole (7.1-23.8) than extensive metabolizers (EM) (0.1-4.9). All 160 subjects and another 15 Caucasian Swedish PMs previously phenotyped with mephenytoin were analysed with respect to the presence of the CYP2C19m1 allele by PCR amplification of the intron 4/exon 5 junction followed by Sma I digestion. EMs heterozygous for the CYP2C19m1 gene had MRs of omeprazole and S/R ratios of mephenytoin that were higher than those of subjects who were homozygous for the wild-type allele (p = 0.0001). Nineteen of the 22 PMs were homozygous for the CYP2C19m1 gene. Three were heterozygous for this allele. Thus, 41 of the 44 alleles (93%) of PMs were defective CYP2C19m1. One of the remaining three PM alleles was subsequently found to contain the CYP2C19m2 mutation, which has earlier been shown to be associated with the PM phenotype in Oriental populations. In conclusion, the phenotype determined by omeprazole correlated with that of mephenytoin, and was in good agreement with the genotype.     

Debrisoquine hydroxylase (CYP2D6) and prostate cancer

The p450 hepatic microsomal enzyme system metabolizes exogenous drugs and carcinogens. Debrisoquine hydroxylase (CYP2D6), one member of the p450 hemoproteins, has polymorphic expression leading to poor metabolism of debrisoquine and similar compounds in approximately 7% of Caucasians. The genetic locus for this enzyme has been characterized, and the mutations responsible for the slowed metabolism have been identified. Epidemiological studies of the CYP2D6 phenotype suggest an association between the normal or rapid metabolism phenotype and increased risk of lung and bladder cancer. Preliminary data have also suggested an association with prostate cancer (CaP). We used a PCR-based assay to investigate possible associations between the CYP2D6 B allele, the most common genetic mutation responsible for the poor metabolism phenotype, and CaP. Using genomic DNA isolated from peripheral blood, we genetically typed 571 men with CaP and 767 matched controls, all participants in the Physician's Health Study. Relative to men homozygous for the wild-type allele, heterozygotes for the B allele have an odds ratio of 1.19 (95% confidence interval, 0.94-1.51) for CaP, and men homozygous for the B allele have an odds ratio of 1.37 (95% confidence interval, 0.86-2.20). When analyzed as a trend over zero, one, or two copies of the B allele, there emerges a possible association between the B allele and an increased risk of CaP of borderline statistical significance (P = 0.07).     

Imipramine metabolism in relation to the sparteine and mephenytoin oxidation polymorphisms--a population study

1. Sparteine and mephenytoin phenotyping tests were carried out in 327 healthy Danish subjects. Two weeks later each subject took 25 mg imipramine followed by urine collection for 24 h. The urinary content of imipramine, desipramine, 2-hydroxy-imipramine and 2-hydroxy-desipramine was assayed by h.p.l.c. 2. The medians of the hydroxylation ratios (i.e. 2-hydroxy-metabolite over parent compound) were 6 to 14 times higher in 300 extensive metabolizers of sparteine (EMs) as compared with 27 poor metabolizers (PMs), but none of the ratios separated the two phenotypes completely. 3. There were 324 EM of mephenytoin (EMM) and three PM (PMM) in the sample. The demethylation ratios between desipramine, 2-hydroxy-desipramine and their corresponding tertiary amines showed statistically significant correlations with the mephenytoin S/R isomer ratio (Spearman's rs: -0.20 and -0.27, P < 0.05). 4. The demethylation ratios were higher in 80 smokers than in 245 non-smokers. This indicates that CYP1A2, which is induced by cigarette smoking, also catalyzes the N-demethylation of imipramine. 5. CYP2D6 genotyping was carried out by PCR in 325 of the subjects, and the D6-wt allele was amplified in 298 EMs, meaning that they were genotyped correctly. One PMs was D6-wt/D6-B, another PMs had the genotype D6-wt/ and hence both were misclassified as EMs. The remaining 25 PMs were D6-A/D6-B (n = 5), D6-B/ (n = 18) or D6-D/D6-D (no PCR amplification, n = 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative analysis of histology, DNA content, p53 and Ki-ras mutations in colectomy specimens with long-standing ulcerative colitis

CYP2C19 (S-mephenytoin hydroxylase) is a polymorphically expressed enzyme. Currently, two defective alleles are known--CYP2C19*2 and CYP2C19*3. The authors have developed an oligonucleotide ligation assay to detect these two alleles. This assay combines the hybridization of one common, biotinylated capture probe and two allele-specific probes to the target DNA, with the ability of a DNA ligase to distinguish mismatched nucleotides. The probes are only ligated if they are base paired correctly to the target strand. The biotin is bound to streptavidin, and all DNA not covalently bound to the biotin-labeled capture probe, is removed in a washing procedure. The allele-specific probes are labeled with either europium or samarium, and their emission can be measured simultaneously. The ratio between the emission separates the genotypes. This method was applied on DNA from 19 whites and 21 Vietnamese living in Denmark. All genotypes determined by the assay were consistent with the results from restriction enzyme cleavage. There were 12 poor metabolizers; 10 homozygous CYP2C19*2/CYP2C19*2, one heterozygous CYP2C19*2/CYP2C19*3, and one heterozygous CYP2C19*1/CYP2C19*2. The authors conclude that this assay is well-suited for a high throughput of samples in a routine laboratory. The finding of an apparently heterozygous CYP2C19*1/CYP2C19*2 poor metabolizer, confirms that there are still unknown mutations in CYP2C19.     

Hypolactasia and metabolic changes in post-menopausal women

The polymorphic cytochrome P450 CYP2D6 is involved in the metabolism of various drugs of wide therapeutic use and is a presumed susceptibility factor for certain environmentally-induced diseases. Our aim was to define the mutations and alleles of the CYP2D6 gene and to evaluate their frequencies in the European population. Using polymerase chain reaction-single strand conformation polymorphism analysis, 672 unrelated subjects were screened for mutations in the 9 exons of the gene and their exon-intron boundaries. A total of 48 point mutations were identified, of which 29 were novel. Mutations 1749 G-->C, 2938 C-->T and 4268 G-->C represented 52.6%, 34.3% and 52.9% of the mutations in the total population, respectively. Of the eight detrimental mutations detected, the 1934 G-->A, the 1795 Tdel and the 2637 Adel accounted for 65.8%, 6.2% and 4.8% respectively, within the poor metabolizer subgroup. Fifty-three different alleles were characterized from the mutation pattern and by allele-specific sequencing. They are derived from three major alleles, namely the wild-type CYP2D6*1A, the functional CYP2D6*2 and the null CYP2D6*4A. Five allelic variants (CYP2D6*1A, *2, *2B, *4A and *5) account for about 87% of all alleles, while the remaining alleles occur with a frequency of 0.1%-2.7%. These data provide a solid basis for future epidemiological, clinical as well as interethnic studies of the CYP2D6 polymorphism and highlight that the described single strand conformation polymorphism method can be successfully used in designing such studies.     

Coexisting mental illness and alcohol and other drug dependencies in pregnant and parenting women

1. To determine whether dexfenfluramine is a substrate of cytochrome P450 2D6 (CYP2D6), its disposition has been studied in nine extensive (EM) and eight poor metabolizers (PM) of debrisoquine. 2. Following a 30 mg dose of dexfenfluramine hydrochloride, urine was collected in all subjects for 96 h post-dose and plasma samples were collected in 11 subjects (six EMs and five PMs). Dexfenfluramine and nordexfenfluramine were measured in urine by h.p.l.c. and in plasma by g.c. 3. Urinary recovery of dexfenfluramine was greater in PMs than EMs (4136 +/- 1509 micrograms vs 1986 +/- 792 micrograms; 95% CI of difference 926-3374; P < 0.05) whereas that of nordexfenfluramine was similar in both phenotypes (PM: 1753 +/- 411 micrograms vs 1626 +/- 444 micrograms). 4. Dexfenfluramine AUC was higher in PMs (677 +/- 348 micrograms l-1 h) than EMs 359 +/- 250 micrograms l-1 h). The apparent oral clearance of dexfenfluramine was greater in EMs than PMs (93.6 +/- 42.4 l h-1 vs 45.6 +/- 19.5 l h-1; 95% CI of difference 1.2-94.7; P < 0.05). The renal clearance was similar in both phenotypes (EMs: 5.88 +/- 2.83 l h-1; PMs 6.60 +/- 2.01 l h-1), indicating that the higher urinary recovery of dexfenfluramine in PMs reflects higher plasma concentrations, rather than phenotype differences in the renal handling, of dexfenfluramine. 5. The apparent nonrenal clearance of dexfenfluramine was substantially lower (P < 0.05; 95% CI of difference 3.0-94.1) in PMs (39.0 +/- 19.5 l h-1) than EMs (87.6 +/- 41.2 l h-1). 6. There was a significant inverse correlation (rs = 0.776 95% CI-0.31-0.94; n = 11; p = 0.005) between the debrisoquine metabolic ratio and the apparent nonrenal clearance of dexfenfluramine. 7. PMs had a higher incidence of adverse effects (nausea and vomiting) than EMs. 8. In conclusion, the metabolism of dexfenfluramine is impaired in PMs. Thus CYP2D6, the isoenzyme deficient in poor metabolizers of debrisoquine, must catalyse at least one pathway of dexfenfluramine biotransformation.