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.     

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.     

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.     

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.     

Reappraisal of human CYP isoforms involved in imipramine N-demethylation and 2-hydroxylation: a study using microsomes obtained from putative extensive and poor metabolizers of S-mephenytoin and eleven recombinant human CYPs

Cytochrome P450 (CYP) involved in the two major pathways of imipramine (IMI) was reappraised using human liver microsomes phenotyped for S-mephenytoin 4'-hydroxylation in vitro and 11 recombinant human CYP isoforms. Individual Eadie-Hoffstee plots for IMI N-demethylation and 2-hydroxylation showed a monophasic profile in microsomes obtained from three putative S-mephenytoin poor metabolizer (PM) livers, whereas the plots gave a biphasic relationship (except for one case in 2-hydroxylation) in those from the three extensive metabolizer (EM) livers. Effects of CYP-selective inhibitor/substrate probes on the two metabolic reactions were examined at the two IMI concentrations (2 and 400 microM) with microsomes obtained from the two PM and three EM livers. S-mephenytoin inhibited IMI N-demethylation by 50% at the low concentration in microsomes from the EM livers with no discernible effect on this pathway in those from the PM livers. Furafylline inhibited the N-demethylation by about 60% at the low and high substrate concentrations in microsomes from both the EM and PM livers. Quinidine abolished the 2-hydroxylation at the low and high concentrations in microsomes from both the EM and the PM livers. Among the recombinant human CYPs, CYP2C19, 2C18, 2D6, 1A2, 3A4 and 2B6 in rank order catalyzed the N-demethylation, whereas CYP2D6, 2C19, 1A2, 2C18 and 3A4 catalyzed the 2-hydroxylation. The Km values obtained from recombinant CYP2C19 and 1A2 approximated those of the high- and low-affinity components from human liver microsomes for IMI N-demethylation, respectively. For IMI 2-hydroxylation, the respective Km values obtained from recombinant CYP2D6 and 2C19 were close to those of the high- and low-affinity components from human liver microsomes. Our human liver microsomal study using the near-therapeutic IMI concentration (2 microM) suggests that 1) CYP2C19 and 1A2 are involved in the N-demethylation and the 2-hydroxylation is mediated exclusively by CYP2D6 and partially by CYP2C19 in the EM livers, and 2) CYP1A2 and 2D6 play a major role in IMI N-demethylation and 2-hydroxylation, respectively, in the PM livers. Our recombinant human CYP isoform study, in general, supports this conclusion.     

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.     

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.     

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.     

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.     

Binding of the pili of Pseudomonas aeruginosa to a low-molecular-weight mucin and neutral cystatin of human submandibular-sublingual saliva

The HLA-B*27 group of alleles has been extensively studied due to the association of particular B*27 alleles with ankylosing spondylitis (AS). We describe here an HLA-B*27 allele (B*2712) encoding an antigen that lacks reactivity with B27 monoclonal antibodies (moabs) and alloantisera but reacts with some B40/B60 moabs and alloantisera and expresses the Bw6 public epitope. This allele was discovered by the segregation of an HLA-B allele undetectable by PCR-SSP within a Caucasian family from the British population referred for routine bone marrow transplant HLA typing and found in the haplotype A*29; B*2712; Cw*1203; DRB1*13; DQB1*0603. Serological typing showed a lack of reactivity with four B27 moabs and four alloantisera but positive reactivity with moabs and alloantisera specific for B40/B60 and Bw6 public epitopes. Subsequent sequencing showed the closest homology was with B*2708 with three mismatches in exon 2 at positions 204, 209 and 210. The intron 2 sequence was identical with other B*27 lineage alleles including a 2 base pair deletion at positions 95 and 96. The relationship between HLA-B*2712 and reported B60 associations with susceptibility to AS remains to be determined.     

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.     

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.     

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.     

Circulating endotoxin during initial antibiotic treatment of severe gram-negative bacteremic infections

The second most common cause of congenital adrenal hyperplasia is 11 beta-hydroxylase deficiency, an autosomal recessive disorder. We performed genetic analysis of CYP11B1, the gene encoding steroid 11 beta-hydroxylase, in three patients with classic 11 beta-hydroxylase deficiency. Herein we describe the first splice donor site mutation, a new nonsense mutation, and a new missense mutation in this disorder. An African-American patient was found to be a compound heterozygote for a codon 318 + 1G --> A substitution at the 5'-splice donor site of intron 5, in combination with Q356X, a nonsense mutation previously reported in an African-American patient. A Caucasian patient was found to be a compound heterozygote with a novel missense mutation, T318R, in combination with a previously reported 28-bp deletion in exon 2. A different mutation at codon 318 (T318M) has been described previously. A Caucasian patient was heterozygous for a novel nonsense mutation (Q19X) in exon 2. The second mutation was not identified in this patient. Multiple apparent polymorphisms were also observed. Two of these polymorphisms in CYP11B1 represent sequences from CYP11B2, suggesting that gene conversion may have occurred. In summary, we have identified three novel mutations and two previously reported mutations in CYP11B1 patients with 11 beta-hydroxylase deficiency. Our data suggest the presence of a mutational hot spot at codon 318 of CYP11B1, and the possibility of a founder effect in frequently identified mutations.     

ApoE and CYP2D6 polymorphism with and without parkinsonism-dementia complex in the people of Chamorro, guam

Parkinsonism-dementia complex (PDC), a neurodegenerative disorder in the Chamorro, Guam population, has been epidemiologically ascribed to the ingestion of the neurotoxin cycasin. This disease is characterized neuropathologically by the presence of abundant neurofibrillary tangles (NFTs). We analyzed a genetic risk factor of Alzheimer's disease (AD), apolipoprotein E, hypothesized to be linked to NFT formation, and a genetic risk factor of Parkinson's disease (PD), CYP2D6 mutation, linked to slower metabolism of exogenous toxins, in Chamorro, Guam individuals with and without PDC. The representation of the G-to-C mutation in exon 9 of the CYP2D6 gene was higher in Chamorro and Filipino than in Caucasian individuals, but this mutant allele had similar high frequencies in both PDC patients and healthy Chamorro individuals. We found no alleles of these genes associated with AD or PD to be overrepresented among those with PDC.     

Allele-dropout using PCR-based diagnosis for the splicing mutation in intron-2 of the CYP21B-gene: successful amplification with a Taq/Pwo-polymerase mixture

The splicing mutation in intron 2 (nucleotide 656) of the 21-hydroxylase gene (CYP21B) is the most common mutation causing congenital adrenal hyperplasia (CAH). Homozygosity for nt656G is associated with the classical phenotype of CAH. In several studies, a number of clinically asymptomatic relatives of CAH-patients were genotyped as nt656G homozygotes. We have proposed that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one allele (in most cases nt656C) during PCR amplification. Here, we report the successful amplification of all alleles at nt656 with a Taq/Pwo DNA polymerase mixture in the primary PCR reaction. The results were independent from the type of polymerase used for sequencing reactions as the second step in mutation analysis.     

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.     

Association of HLA class I and class II alleles with myositis in Japanese patients

OBJECTIVE: To investigate the correlation of HLA class I and class II antigens and alleles with various forms of myositis in Japanese patients. METHODS: Eighty-four Japanese patients with myositis [22 with polymyositis (PM), 46 with dermatomyositis (DM), 16 with myositis overlapping with other collagen vascular diseases] were typed serologically for HLA-A, B, C antigens. HLA-DRB1, DQA1, and DQB1 alleles were determined by polymerase chain reaction dependent DNA typing methods. Fifty-eight Japanese controls were typed serologically while HLA-DRB1, DQA1, and DQB1 allele typing was carried out in 175, 95, and 104 controls, respectively. RESULTS: HLA-B7 was higher in patients than controls [20.2 vs 6.9% in controls: p=0.02, odds ratio (OR)=3.4]. The increase of HLA-B7 was largely dependent on the increase in overlap patients (37.5%; p=0.005, OR=8.1). HLA-A24 and B52 were significantly decreased in PM as compared to DM, while CW3 was significantly increased in PM versus DM. DRB1*08 alleles were significantly increased in patients (36.9 vs 20.5% in controls; p=0.004, OR=2.3), especially in PM and DM. DQA1*0501 and DQB1*0301 were significantly decreased in patients [4.8 vs 13.7% in controls; p=0.04, OR=0.32, and 8.3 vs 20.2% in controls; p=0.02, OR=0.36, respectively]. CONCLUSION: HLA-class I and class II alleles associated with Japanese patients with myositis may be different from those associated with Caucasian patients.     

Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450

The effects of methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied in vitro on nine individual, cDNAexpressed cytochrome P-450 activities (phenacetin O-deethylase for CYP1A1 and CYP1A2, coumarin 7-hydroxylase for CYP2A6, testosterone 6beta-hydroxylase for CYP3A4, 7-ethoxy-4-trifluoromethylcoumarin deethylase for CYP2B6, paclitaxel 6alpha-hydroxylase for CYP2C8, diclofenac 4'-hydroxylase for CYP2C9, S-mephenytoin 4-hydroxylase for CYP2C19, and (+/-)-bufuralol 1'-hydroxylase for CYP2D6) in commercially available human lymphoblastoid microsomes. These data show that specific solvents have enzyme-selective effects on P-450 activities. Methanol did not substantially inhibit (相似文献