ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Genetic polymorphisms are well-recognized causes of interindividual differences in disease risk and treatment response in humans. For genes containing multiple single-nucleotide polymorphisms (SNPs), haplotype structure is often the principal determinant of phenotypic consequences, and haplotype distribution represents the best approach for assessing patterns of linkage disequilibrium. To permit more widespread molecular determination of haplotypes, we developed a simple yet robust method to determine haplotype structure for multiple SNPs located up to 30 kb apart in genomic DNA using long-range polymerase chain reaction (LR-PCR) and intramolecular ligation. Complete concordance was shown between the new method and conventional approaches, such as family pedigree analysis or cloning and sequencing. The availability of a simple method to directly determine haplotype structure using genomic DNA, without family pedigree analysis, cloning or complex instrumentation, provides an important new tool for elucidating the genetic determinants of drug disposition and effects, disease risk, and molecular evolution.

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Tolbutamide is known to be metabolized by cytochrome P450 2C9 (CYP2C9), and the effects of the CYP2C9 amino acid polymorphisms*2(Arg144Cys) and*3(Ile359Leu) could be important for drug treatment with tolbutamide and for use of tolbutamide as a CYP2C9 test drug.Tolbutamide pharmacokinetics and plasma insulin and glucose concentrations were studied in 23 healthy volunteers with all six combinations of theCYP2C9alleles*1,*2and*3, including two subjects with the combinedCYP2C9*1/*1andCYP2C19*2/*2genotype. Volunteers received a single oral dose of 500 mg tolbutamide, followed by 75 g oral glucose at 1, 4.5 and 8 h after tolbutamide administration. Pharmacokinetic analysis was performed using a computer program for regression analysis of nonlinear mixed effects models.The mean oral clearances of tolbutamide were 0.97 (95% confidence interval [CI] 0.89–1.05), 0.86 (95% CI 0.79–0.93), 0.75 (95% CI 0.69–0.81), 0.56 (95% CI 0.51–0.61), 0.45 (95% CI 0.41–0.49) and 0.15 (95% CI 0.14–0.16) l/h in carriers ofCYP2C9genotypes1/*1,*1/*2,*2/*2,*1/*3,*2/*3and*3/*3, respectively. Tolbutamide pharmacokinetics in carriers of the functionally deficientCYP2C19*2/*2genotype were not different from those in theCYP2C19highly active genotype. Elimination in the sixCYP2C9genotype groups could be expressed as the linear combination of three constants (0.05, 0.04, 0.01 h−1), which were specific to the respectiveCYP2C9alleles*1,*2and*3, thus indicating a co-dominant mode of inheritance. Insulin and glucose concentration–time curves did not change with differingCYP2C9genotypes.Tolbutamide was confirmed as a substrate of the genetically polymorphic enzyme CYP2C9. The pronounced differences in pharmacokinetics due to the amino acid variants did not significantly affect plasma insulin and glucose concentrations in healthy volunteers.

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Several recent in-vitro data have revealed that CYP2C19, in addition to CYP2C9, is also involved in the 4-methylhydroxylation of tolbutamide. We evaluated the relative contribution of CYP2C9 and CYP2C19 genetic polymorphisms on the disposition of blood glucose lowering response to tolbutamide in normal healthy Korean subjects in order to reappraise tolbutamide as a selective in-vivo probe substrate of CYP2C9 activity. A single oral dose of tolbutamide (500 mg) or placebo was administered to 18 subjects in a single-blind, randomized, crossover study with a 2-week washout period. Twelve subjects (of whom six wereCYP2C19extensive metabolizer (EM) and six wereCYP2C19poor metabolizer (PM) genotype) were of the homozygous wild-typeCYP2C9*1genotype; the other six subjects were of theCYP2C9*1/*3andCYP2C19EM genotype. Pharmacokinetic parameters were estimated from plasma and urine concentrations of tolbutamide and 4-hydroxytolbutamide. Serum glucose concentrations were measured before and after oral intake of 100 g dextrose. In subjects heterozygous for theCYP2C9*3allele,Cmaxand AUC of tolbutamide were significantly greater and the plasma half-life significantly longer than those in homozygousCYP2C9*1subjects. No pharmacokinetic differences were found betweenCYP2C19EM and PM genotype subjects. The estimated AUC of the increase in serum glucose after oral intake of 100 g dextrose was 2.7-fold higher in subjects with the wild-typeCYP2C9genotype than in those withCYP2C9*1/*3, but CYP2C19 genetic polymorphism did not alter the blood glucose lowering effect of tolbutamide. The plasma AUC of 4-hydroxytolbutamide and the ratio of 4-hydroxytolbutamide/tolbutamide did not differ significantly betweenCYP2C19PM and EM genotype subjects, while these parameters were about twice as high in subjects with the wild-typeCYP2C9genotype than in heterozygousCYP2C9*3subjects (P<0.05). Our results strongly suggest that the disposition and hypoglycemic effect of tolbutamide are affected mainly by CYP2C9 genetic polymorphism, but not by CYP2C19 polymorphism. The in-vivo contribution of CYP2C19 to tolbutamide 4-methylhydroxylation appears to be minor in humans. This suggests that, at leastin vivo, tolbutamide remains a selective probe for measuring CYP2C9 activity in humans.

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Marked interindividual variability in expression of CYP3A4 influences the disposition of many endo- and xenobiotics, including the metabolism of steroids, environmental toxins and therapeutically useful drugs. The present study was designed to determine the genetic basis of CYP3A4 variability. We analysed DNA from 82 individuals with known CYP3A4 phenotype including 53 Caucasians and 21 African-American liver donors, seven individuals who were outliers in CYP3A4 metabolism and five individuals in a family of a poor nifedipine metabolizer. In addition, we analysed DNA from the eight person DNA Polymorphism Discovery Resource subset (Coriell Institute) and 89 individuals representing nine ethnic groups. Five non-synonymous mutations in the coding region ofCYP3A4were observed.CYP3A4*14(T44C) in exon 1 resulted in an L15P change;CYP3A4*15(G14387A) in exon 6 resulted in a R162Q substitution;CYP3A4*10(G14422C) in exon 6 resulted in a D174H substitution;CYP3A4*16(C15721G) in exon 7 resulted in a T185S amino acid substitution; andCYP3A4*12(C22002T) in exon 11 resulted in a L373F change in the CYP3A4 protein. An additional six single nucleotide polymorphisms (SNPs) in the 5′-UTR, 13 SNPs in the introns and three SNPs in the 3′-UTR were observed. Extensive population differences were observed in the frequencies of variousCYP3A4alleles. None of the 28CYP3A4SNPs identified in CYP3A4 phenotyped persons (most individuals being heterozygous for any CYP3A4 variant) was associated with low hepatic CYP3A4 protein expression or low CYP3A4 activityin vivo.

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

A pharmacogenetic syndrome caused by molecular defects in the dihydropyrimidine dehydrogenase gene (DPYD) results in partial to complete loss of dihydropyrimidine dehydrogenase (DPD) enzyme activity with patients exhibiting life-threatening toxicity following administration of routine doses of 5-fluorouracil. To date, more than 19 reported mutations have been putatively associated with DPD deficiency with 16 occurring within the open reading frame of the cDNA. The purpose of this study was to examine the conservation of functional domains (including the uracil, flavine adenine dinucleotide and NADPH binding sites) across three phyla (Chordata, Arthropoda and Nematoda) and the conservation of regions corresponding to the previously reported mutations. Comparative analysis of the uracil and NADPH binding sites in mammals and invertebrates demonstrated 100% amino acid identity between mammals andDrosophila melanogaster. Caenorhabditis elegansdemonstrated 89% and 88% identity in these domains, respectively. The mammalian sequences demonstrated 100% identity in two iron sulphur motifs (amino acids 953–964 and 986–997) with significant conservation inD. melanogaster(92% and 92% identity, respectively) andC. elegans(100% and 92% identity, respectively). Comparative amino acid analysis revealed non-conservation in the loci of four DPYD mutations [DPYD*12(R21Q),DPYD*5(I543V),DPYD*6(V732I),DPYD*9A(C29R)]. Seven mutations occurred in highly conserved regions [M166V,DPYD*8(R235W),DPYD*11(V335l),DPYD*4(S534N),DPYD*9B(R886H),D949V,DPYD*10(V995F)]. In summary, this comparative analysis identified conserved regions which may be critical to enzyme structure and/or function. The conservation of loci whereDPYDmutations occur was also examined to evaluate their functional significance on DPD enzyme activity. These data should prove useful in the evaluation of newly discovered mutations in theDPYDgene.

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

High red meat intake has been linked with an increased risk of colorectal cancer and adenomas. During high temperature cooking of red meats, heterocyclic amines (HCAs) are generated; however, to be carcinogenic, they must be metabolized by enzymes including cytochrome P450 1A2 (CYP1A2) andN-acetyltransferase 1 (NAT1) and/orN-acetyltransferase 2 (NAT2). We have conducted a clinic-based case–control study of colorectal adenomas that focused on assessment of exposure to HCAs (estimated by use of a HCA database and meat cooking module) and modification of these exposures by genetic factors. We have previously reported that intake of MeIQx was associated with an increased risk of colorectal adenomas [overall association at 80th percentile,>27.00 ng/day: odds ratio (OR) = 2.68, 95% confidence interval (CI) 1.58–4.55]. Here, we report our evaluation of whether variation in CYP1A2, NAT1 and/or NAT2 modify the association between HCAs and colorectal adenoma formation in 146 cases and 228 frequency-matched controls. TheNAT1*10allele was associated with a nonsignificant increased risk of colorectal adenomas (OR = 1.43; 95% CI 0.86–2.36). Further, when we analysed 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) intake as a categorical variable, we observed a six-fold increase in adenoma risk among rapid NAT1 acetylators who consumed more than 27 ng a day (OR = 6.50; 95% CI 2.16–19.7), whereas among slow NAT1 acetylators, the increase in risk was two-fold (OR = 2.32; 95% CI 1.12–4.81). While suggestive, the results were not significantly different from each other on either an additive or multiplicative scale. In contrast,NAT2genotype and CYP1A2 and NAT2 hepatic activity measured by caffeine urinary metabolites were not associated with adenoma risk, although an increase in risk with rapid CYP1A2 activity could not be ruled out (OR = 1.46; 95% CI 0.76–2.81). Moreover, there was no evidence that the effect of MeIQx was enhanced among subjects in any subgroup defined by variation in these measures. These results are compatible with the hypothesis that high HCA exposure is associated with an increased risk of colorectal adenomas, particularly in genetically susceptible subgroups. Further study of larger populations is needed to confirm and extend these observations.

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

TheAhrlocus encodes for the aryl hydrocarbon receptor (AHR), which plays an important toxicological and developmental role. Sequence variation in this gene was studied in 13 different mouse lines that included eight laboratory strains, twoMus musculussubspecies and three additionalMusspecies. The data presented represent the largest study of sequence variation across multiple mouse lines in a single gene (≈15.9 kb/mouse line). Among all mice, the average frequency of all polymorphisms in the intronic regions was 20.3 variants/kb and the average exonic frequency was 14.1 variants/kb. For substitutions alone, the average frequencies in the intronic and exonic regions for all mice were 13.3 and 8.9 substitutions/kb, respectively. Between laboratory strains, the average intronic and exonic frequencies for all polymorphisms dropped to 5.4 and 2.9 variants/kb, respectively. There were 111 non-synonymous polymorphisms that resulted in 42 different amino acid changes, of which only 10 amino acid changes had been previously identified. Based on the nucleotide sequence, the phylogenetic history of the gene showed mice from theAhrb2andAhrdalleles in separate branches while mice from theAhrb1andAhrb3alleles exhibited a more complex history. Evolutionarily, the AHR protein as a whole appears to be under purifying selective pressure (Ka: Ksratio = 0.237). Despite significant functional constraint in the basic helix-loop-helix and PAS domains, ligand binding is not constrained to the high-affinity allele, which supports further the role of the AHR in development and its importance beyond the adaptive response to environmental toxicants.

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Recently, another reserach group has reported an almost complete loss of function of the human norepinephrine transporter (hNET) in patients who had orthostatic intolerance and who were heterozygous for a guanine to cytosine exchange, resulting in a hNET Ala457Pro variant. To explore the reason for the deficiency in NET function, we compared in detail the pharmacology of the Ala457Pro variant with that of the wild-type hNET in COS-7 cells transiently transfected with hNET or Ala457Pro cDNA. Compared to the wild-type hNET, the Ala457Pro variant exhibited a five-fold higher affinity for cocaine, but a two-fold lower affinity for the NET inhibitor nisoxetine, and an unchanged affinity for the antidepressant desipramine. Plasma membrane expression (measured asBmaxof [3H]nisoxetine binding) of the Ala457Pro variant was only 40% of that of the wild-type hNET. The Ala457Pro variant showed a six- to 10-fold decrease in affinity for the substrates dopamine and 1-methyl-4-phenylpyridinium (MPP+). Compared with the wild-type hNET, the maximum rate (Vmax) of norepinephrine uptake by the Ala457Pro variant was slightly reduced, whereas the turnover number (calculated fromVmax/Bmax) was approximately two-fold higher. However, the Ala457Pro variant exhibited a 50-fold higherKm(i.e. lower apparent affinity) for norepinephrine than the wild-type hNET. Thus, the previously reported loss of function of the Ala457Pro variant associated with orthostatic intolerance is only partly due to a reduction in plasma membrane expression of the transporter, and is mainly caused by the pronounced reduction in the apparent affinity of norepinephrine.

ISSN:0960-314X    

出版商:OVID    

年代:2002

数据来源: OVID