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.     

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.     

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.     

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.     

The effects of genetic polymorphisms of CYP2C9 and CYP2C19 on phenytoin metabolism in Japanese adult patients with epilepsy: studies in stereoselective hydroxylation and population pharmacokinetics

PURPOSE: The aim of this study was to clarify the effects of genetic polymorphisms of cytochrome P450 (CYP) 2C9 and 2C19 on the metabolism of phenytoin (PHT). In addition, a population pharmacokinetic analysis was performed. METHODS: The genotype of CYP2C9 (Arg144/Cys, Ile359/Leu) and CYP2C19(*1, *2 or *3) in 134 Japanese adult patients with epilepsy treated with PHT were determined, and their serum concentrations of 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) enantiomers, being major metabolites of PHT, were measured. A population pharmacokinetic analysis (NONMEM analysis) was performed to evaluate whether genetic polymorphism of CYP2C9/19 affects the clinical use of PHT by using the 336 dose-serum concentration data. RESULTS: The mean maximal elimination rate (Vmax) was 42% lower in the heterozygote for Leu359 allele in CYP2C9, and the mean Michaelis-Menten constants (Km) in the heterozygous extensive metabolizers and the poor metabolizers of CYP2C19 were 22 and 54%, respectively, higher than those without the mutations in CYP2C9/19 genes. (R)- and (S)-p-HPPH/PHT ratios were lower in patients with mutations in CYP2C9 or CYP2C19 gene than those in patients without mutations. CONCLUSIONS: Although the hydroxylation capacity of PHT was impaired with mutations of CYP2C9/19, the impairment was greater for CYP2C9. In view of the clinical use of PHT, two important conclusions were derived from this population study. First, the serum PHT concentration in patients with the Leu359 allele in CYP2C9 would increase dramatically even at lower daily doses. Second, the patients with CYP2C19 mutations should be treated carefully at higher daily doses of PHT.     

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.     

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.     

CYP2D6 phenotype-genotype relationships in African-Americans and Caucasians in Los Angeles

CYP2D6 genotyping (CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*13, CYP2D6*16 alleles and gene duplications) was previously performed on 1053 Caucasian and African-American lung cancer cases and control individuals and no significant difference in allele frequencies between cases and control individuals detected. We have carried out additional genotyping (CYP2D6*6, CYP2D6*7, CYP2D6*8, CYP2D6*9, CYP2D6*10, CYP2D6*17 alleles) and debrisoquine phenotyping on subgroups from this study to assess phenotype-genotype relationships. African-Americans showed significant differences from Caucasians with respect to frequency of defective CYP2D6 alleles, particularly CYP2D6*4 and CYP2D6*5. The CYP2D6*17 allele occurred at a frequency of 0.26 among 87 African-Americans and appeared to explain higher average metabolic ratios among African-Americans compared with Caucasians. CYP2D6*6, CYP2D6*8, CYP2D6*9 and CYP2D6*10 were rare in both ethnic groups but explained approximately 40% of higher than expected metabolic ratios among extensive metabolizers. Among individuals phenotyped with debrisoquine, 32 out of 359 were in the poor metabolizer range with 24 of these (75%) also showing two defective CYP2D6 alleles. Additional single strand conformational polymorphism analysis screening of samples showing large phenotype-genotype discrepancies resulted in the detection of three novel polymorphisms. If subjects taking potentially interfering drugs were excluded, this additional screening enabled the positive identification of 88% of phenotypic poor metabolizers by genotyping. This sensitivity was comparable with that of phenotyping, which identified 90% of those with two defective alleles as poor metabolizers.     

Updated sequence information for TEM beta-lactamase genes

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercapto-purine, 6-thioguanine and azathioprine. TPMT activity is inherited as an autosomal co-dominant trait, and several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. Although ethnic differences in TPMT activity have been described, population frequency analysis of TPMT alleles has not been well defined in different ethnic groups. The frequency of four allelic variants of the TPMT gene, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C were compared in British Caucasian (n = 199) and Ghanaian (n = 217) populations using PCR-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 14.8% of Ghanaians (31 heterozygotes, one homozygote). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the Ghanaian samples analysed. In contrast, 10.1% of British subjects had variant alleles, consisting of TPMT*2 (n = 2), TPMT*3A (n = 17) and TPMT*3C (n = 1) alleles. The frequencies of mutant alleles in this study were 5.3 and 7.6% in British Caucasians and Ghanaians, respectively. Among Ghanaian tribes, Ewe subjects had a lower frequency of mutant alleles (5.9%) than Ga (13.2%) or Fanti (11.6%), although this did not reach statistical significance. This study provides the first analysis of TPMT mutant allele frequency in an African population and indicates that, unlike Caucasians, TPMT*3C is the most common allele in African subjects.     

Association between CYP2C19 genotype and proguanil oxidative polymorphism

AIMS: To examine the relationship between proguanil metabolism and the number of mutations in CYP2C19 by comparing the CYP2C19 genotype and proguanil phenotype of 10 subjects. METHODS: Partial clearance and urinary metabolic ratio data were obtained from a previous study of 10 subjects [5]. Analysis of CYP2C19 genotypes was performed using PCR amplification followed by restriction endonuclease digestion of genomic DNA from a blood sample. RESULTS: The intrinsic partial clearance of PG to CG ranged from 0.41-10.11 h-1, and was related to the number of functional CYP2C19 alleles present. Genotypic PMs had metabolic ratios > 13, while genotypic heterozygote EMs had metabolic ratios < 9. CONCLUSIONS: Proguanil may be a suitable phenotyping probe for the CYP2C19 genetic polymorphism, however the exact antimode of the urinary metabolic ratio chosen to separate poor and extensive metabolisers needs further investigation.     

The effect of carbohydrates on affect

Data on both the incidence of slow acetylator phenotype of probe drugs isoniazid, sulfadimidine or sulfamethazine, caffeine and dapsone in mainland or overseas Chinese, and the distribution of NAT2 genotypes and the frequency of NAT2 alleles in the Chinese populations were summarized and reanalysed using a meta-analysis method. Frequency of the slow acetylator phenotype in 3516 healthy Han Chinese gave an overall mean of approximately 19.9 +/- 4.0%, with the range of the combined data being between 15.8% and 25.5%. In addition, frequencies of the slow acetylator phenotype differ between the different minorities in Chinese populations and the range was between 3.2% and 50.6%, with a mean value of 20.6 +/- 12.9% in a total of 1842 individuals from 17 Chinese minorities. In addition, there was no significant heterogeneity in overseas Chinese between the probe drugs isoniazid and sulfadimidine or sulfamethazine (chi 2 = 5.97, df = 4; p > 0.05), and the mean value of slow acetylator phenotype incidence was 24.5% (119/485; 95% CI: 20.7-28.3%), consistent with that of the native Chinese. As expected, frequency of the slow acetylator genotypes in Chinese populations was 25.4% (112/441; 95% CI: 21.3-29.5%), which was in accordance with that of the slow acetylator phenotype in native or overseas Chinese. For all genotypes, *4/*4 (29.9%, 132/441), *4/*6A (27.4%, 121/441), *4/*7A (12%, 53/441) and *6A/*6A (11.3%, 50/441) occupied 80.6%, but *5A/*7A (0.2%, 1/441), *5A/*5A (1.1%, 5/441) and *7A/*7A (1.8%, 8/441) were not frequently found. From this report, the genotype frequencies of homozygous rapid acetylator, heterozygous rapid acetylator, and homozygous slow acetylator were found to be 0.299 (132/441), 0.447 (197/441) and 0.254 (112/441), respectively. Furthermore, both *4 (52.3%; 95% CI: 49-56%) and *6A (30.5%; 95% CI: 28-34%) were major NAT2 alleles, while *7A (11.2%; 95% CI: 9-13%) and *5A (6%; 95% CI: 4-8%) were uncommonly present. Frequency of the mutant alleles was observed at 0.477 (421/882 alleles). The *7A constituted 23.5% t(99/421) of slow acetylator alleles in Chinese populations, showing that this point mutation exists not only in Oriental or Asiatic, but also in Chinese populations. According to the Hardy-Weinberg equilibrium, in the phenotyped Chinese populations, the mean estimate of predicted allelic frequencies of the genotypes RR, Rr, and rr was 0.294, 0.496, and 0.210 for the Chinese, and the expected frequency of the deficient gene r was 0.458. By comparison, the predicted values are in complete agreement with the observed ones. In conclusion, this meta-analysis determined the accurate population frequencies of phenotype and genotype of the NAT2 genetic deficiency in healthy Chinese subjects.     

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.     

A method for a selective local application of radioactive precursors of nucleic acids to the cow ovary

OBJECTIVE: To determine whether genetic polymorphism of cytochrome P450 (CYP) 2C9 affects the in vivo metabolism of warfarin enantiomers. METHODS: Eighty-six Japanese patients heart disease who were given warfarin participated in the study. Plasma unbound concentrations of warfarin enantiomers and urinary (S)-7-hydroxywarfarin concentrations were measured by means of a chiral HPLC and ultrafiltration technique to calculate the unbound oral clearance (CLpo,u) for the enantiomers and the formation clearance (CLm) for (S)-warfarin 7-hydroxylation. Genotyping for CYP2C9 (the wild type [wt], Arg144/Cys, and I1e359/Leu) and for CYP2C19 (wt, ml, and m2) was performed with a polymerase chain reaction method. RESULTS: Three patients were heterozygous for the CYP2C9 Leu359 mutation but none were homozygous for the mutation (the allele frequency of 0.017). None had a CYP2C9 Cys144 allele. The medians for (S)-warfarin CLpo,u and its 7-hydroxylation CLm obtained from heterozygotes of CYP2C9 Leu359 were significantly less than those obtained from homozygotes of the wt allele, as follows: 234 ml/min (range, 156 to 269 ml/min) versus 632 ml/min (range, 180 to 2070 ml/min) (p < 0.001) and 0.20 ml/min (range, 0.05 to 0.77 ml/min) versus 0.80 ml/min (range, 0.05 to 14.9 ml/min) (p < 0.05), respectively. In contrast, no difference was observed in (R)-warfarin CLpo,u between the groups. The allele frequencies for CYP2C19 m1 and CYP2C19 m2 were 0.26 and 0.14, respectively, indicating 15% of patients were genotypically poor metabolizers of CYP2C19. No difference in CLpo,u for warfarin enantiomers was observed between the assumed CYP2C19 phenotypes. CONCLUSION: Heterozygotes for CYP2C9 I1e359/Leu allele have reduced in vivo metabolism of (S)-warfarin but not (R)-warfarin. Because (S)-warfarin has a greater anticoagulant potency than its (R)-congener, the genetic polymorphism of CYP2C9 may partly account for the large interpatient variability in therapeutic dosages of warfarin.     

Elimination of interference component in Wigner-Ville distribution for the signal with 1/f spectral characteristic

Human populations are thought to metabolize coumarin almost exclusively by 7-hydroxylation. We have identified an individual who is homozygous for a single amino acid substitution (Leu160His) in the cytochrome P450 CYP2A6 arising from the variant CYP2A6*2 allele. On administration of coumarin (2 mg orally) no detectable 7-hydroxycoumarin was excreted in the 0-8-hr urine, rather, approximately 50% of the dose was eliminated as 2-hydroxyphenylacetic acid, the end-product of coumarin 3-hydroxylation. His immediate family members, who were heterozygous for the CYP2A6*2 allele, excreted little 2-hydroxyphenylacetic acid and mainly 7-hydroxycoumarin, when similarly tested. These findings raise a question regarding human risk evaluations for environmental coumarin exposures, since 7-hydroxylation is regarded as a detoxication pathway, but 3-hydroxylation as the process required to lead to macromolecular covalent binding of coumarin. Persons homozygous for the CYP2A6*2 allele may constitute 1-25% of various populations.     

Redox potential affects the measured heat resistance of Escherichia coli O157:H7 independently of oxygen concentration

A South Korean population from Kongju (n = 350) was screened by isoelectric focusing and immunoblotting procedures to determine the distribution of genetic variations in 3 apolipoprotein genes including APOA-IV, APOE and APOH. Although the known APOA-IV protein polymorphism was not observed, sporadic examples of 2 putative new variants were identified. The frequencies of the APOE*2, APOE*3 and APOE*4 alleles were 0.069, 0.823 and 0.107, respectively. At the APOH structural locus 3 common alleles, APOH*1 (0.010), APOH*2 (0.913) and APOH*3 (0.073) were observed. In addition, a unique APOH allele designated APOH*3 Kongju was identified in this Korean population.     

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.     

Genetic variability of apolipoprotein E in a Polish population

Apolipoprotein E (apoE, protein; APOE, gene) is a component of very low density lipoprotein and high-density lipoprotein and plays an important role in lipoprotein metabolism. There are three common alleles of APOE (*2, *3, and *4), which encode the E2, E3, and E4 isoforms of the protein. Distribution of apoE isoforms shows marked variation among various ethnic groups. Direct phenotyping of human APOE in plasma was used to estimate APOE allele frequencies in 137 unrelated blood donors from 3 regions of Poland. The relative frequencies observed for the APOE*2, APOE*3, and APOE*4 alleles were 0.055, 0.839, and 0.106, respectively. The data have been compared with data found in other population groups. The frequency of the APOE*2 allele in Poles was among the lowest in Europe.     

Tumour necrosis factor beta alleles and hyperinsulinaemia in coronary artery disease

BACKGROUND: Hyperinsulinaemia and dyslipoproteinaemia are markers and risk factors for coronary artery disease (CAD) and non-insulin-dependent diabetes mellitus (NIDDM). We investigated the influence of a tumour necrosis factor beta (TNF-beta) gene polymorphism on serum parameters related to these metabolic disorders in patients with CAD. METHODS: A total of 199 patients with CAD and 81 control subjects with angiographically normal coronary arteries were studied. A digestion of amplified DNA with NcoI revealed three fragment patterns: homozygosity for TNF-beta *1 or TNF-beta *2 and heterozygosity (TNF-beta *1/*2). RESULTS: Patients with CAD who had increased serum insulin or C-peptide (fasting and after glucose load) were predominantly heterozygous for TNF-beta (72% vs. 47%) and less frequently homozygous for TNF-beta *2 (22% vs. 43%, P = 0 x 0.03). CONCLUSION: This study demonstrates an association of TNF-beta alleles with the risk factor hyperinsulinaemia in CAD. Genomic variants of TNF-beta may therefore contribute to the complex susceptibility for the metabolic syndrome in patients with CAD.     

Evaluation of omeprazole and lansoprazole as inhibitors of cytochrome P450 isoforms

The human clearance of omeprazole and lansoprazole is conducted primarily by the hepatic cytochrome P450 (CYP) system. Efficacy data indicate few differences between these two drugs, but they may exhibit discrete drug interaction profiles. To compare the potency and specificity of these drugs as inhibitors of CYP isoforms, we performed in vitro studies with human liver microsomal preparations. Both drugs were potent, competitive inhibitors of CYP2C19, as measured by the conversion of S-mephenytoin to 4-hydroxymephenytoin (k(i) = 3.1 +/- 2.2 microM for omeprazole, K(i) = 3.2 +/- 1.3 microM for lansoprazole). For omeprazole, the highest concentration at which >70% inhibition of CYP2C19 was observed with no significant inhibitory effect on other isoforms was at least 20 times greater than K(i). Both drugs were competitive inhibitors of CYP2C9-catalyzed conversion of tolbutamide to 4-hydroxytolbutamide (K(i) = 40.1 +/- 14.8 microM for omeprazole, K(i) = 52.1 +/- 1.4 microM for lansoprazole) and were noncompetitive inhibitors of CYP3A-catalyzed conversion of dextromethorphan to 3-methoxymorphinan (K(i) = 84.4 +/- 4.0 microM for omeprazole, K(i) = 170.4 +/- 7.1 microM for lansoprazole). Lansoprazole was at least 5 times more potent (K(i) = 44.7 +/- 22.0 microM) than omeprazole (k(i) = 240.7 +/- 102.0 microM) as an inhibitor of CYP2D6-mediated conversion of dextromethorphan to dextrorphan. No inhibition of CYP1A2, assessed by measuring the conversion of phenacetin to acetaminophen, was noted. Our data suggest that whereas the inhibitory profiles of these two drugs are similar, lansoprazole may be the more important in vitro inhibitor of CYP2D6. Since its inhibition is very potent and has a broad "window of selectivity," omeprazole seems to be a useful, selective inhibitor of CYP2C19.     

Validation of methods for CYP2C9 genotyping: frequencies of mutant alleles in a Swedish population

Cytochrome P450 2C9 (CYP2C9) catalysis the metabolism of important drugs such as phenytoin, S-warfarin, tolbutamide, losartan, torasemide, and nonsteroidal anti-inflammatory drugs. A functional polymorphism of the CYP2C9 gene has been described. The variant alleles include CYP2C9*2 having a point mutation in exon 3 causing an Arg144Cys exchange, and CYP2C9*3 with a point mutation in exon 7 resulting in an Ile359Leu exchange. Genotyping of these variant forms was carried out in 430 Swedish healthy volunteers and three different methods were compared. Sequence analysis of the different PCR products revealed that other genes in the CYP2C locus were co-amplified in one of the methods applied, whereas the other two methods were specific for CYP2C9. The frequencies of the CYP2C9*1, CYP2C9*2 and CYP2C9*3 alleles in the population examined were found to be 0.819, 0.107, and 0.074, respectively. The need for careful evaluation of the genotyping procedure by sequence analysis of PCR products is emphasised.