ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Endothelin-1 (ET-1) is known as a potent vasoconstrictor peptide and stimulator of cell proliferation. The human preproendothelin-1 mRNA contains a frequent adenine insertion polymorphism (allele frequency = 0.28) within the 5′-untranslated region (5′-UTR), 138 bp downstream of the transcription start site, which was assumed to be related to hypertension. This 5′-UTR variant could putatively influence the mRNA secondary structure and stability, efficacy of translation initiation, or binding of sequence-specific mRNA-binding proteins. By cloning the entire ET-1 gene 5′-UTR in a pGL3 vector and transfection of two cell lines, we studied the effects on luciferase expressionin vitro. Luciferase activity was significantly increased in the insertion variant (I) compared to the wild-type (D) variant for both COS1 (2.97 ± 0.12 versus 2.17 ± 0.10;P= 0.002) and HepG2 cells (5.42 ± 0.90 versus 3.68 ± 0.37;P= 0.002). Investigations performedex vivousing human umbilical vein endothelial cells were performed to examine the influence of genotypes on the formation of mRNA and protein. Preproendothelin-1-mRNA was quantified in relation to GAPDH by a realtime polymerase chain reaction. Homozygous I-carriers showed significant elevated mRNA levels compared to I/D and I/I-carriers (I/I 9.03 ± 1.86, I/D 2.07 ± 1.15, D/D 2.33 ± 0.99;P= 0.001). ET-1 protein expression, determined by enzyme-linked immunosorbent assay, was increased among I-carriers (I/I 814 ± 144, I/D 528 ± 103, D/D 556 ± 75 pg/ml;P= 0.001). The observed effects may be due to an enhanced mRNA stability because the half-life of mRNA consisting of the I-variant was prolonged (35.4 ± 7.9 versus 19.9 ± 4.5 min). We were able to show that the +138 I/D polymorphism is of functional importance for ET-1 expression, and this may have consequences for vessel tonus regulation.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

BackgroundSibutramine, a centrally acting noradrenaline and serotonin re-uptake inhibitor, enhances satiety and is frequently used to support weight loss. However, a significant variability exists among individuals concerning the response to sibutramine.MethodsWe genotyped 111 participants of a randomized placebo-controlled clinical trial for theGNB3C825T polymorphism and analysed associations of genotypes with treatment outcome. Patients undergoing a structured weight loss programme were treated with either placebo or 15 mg sibutramine daily for 54 weeks.ResultsIn the placebo group, the non-pharmacological programme alone resulted in a significantly greater weight loss in individuals with theGNB3TT/TC genotypes as compared to individuals with the CC genotype (−7.1 ± 1.2 vs. −2.7 ± 1.5 kg,P= 0.031). Administration of 15 mg sibutramine was more effective in individuals with the CC genotype than in the subjects with the TT/TC genotypes (weight loss: 7.2 ± 2.2 vs. 4.1 ± 2.1 kg,P= 0.0013, sibutramine vs. placebo). In the CC genotype carriers, the odds ratio (OR) for a weight loss greater than 5% (sibutramine vs. placebo) was 6.6 (95% CI 1.8–25.6;P= 0.004) and for a weight loss greater than 10% was 9.6 (95% CI 1.7–53.8;P= 0.010).ConclusionGenotyping for theGNB3C825T polymorphism is highly predictive for the identification of obese individuals who will benefit from sibutramine treatment.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

ObjectivesGenetic polymorphisms of cytochromes P450 (CYPs) are a principal reason for inter-individual variations in the metabolism of therapeutic drugs and environmental chemicals in humans. The present study identifies 34 single nucleotide polymorphisms (SNPs) ofCYP3A5including 27 previously unidentified SNPs by direct sequencing of the exons, intron–exon junctions and 5′-upstream region of CYP3A5 from 92 racially diverse individuals (24 Caucasians, 24 Africans, 24 Asians, and 20 individuals of unknown racial origin).ResultsFour newCYP3A5SNPs produced coding changes: R28C, L82R, A337T, and F446S.CYP3A5 R28Coccurred in African populations (allelic frequency of 4%).CYP3A5 A337Toccurred in Asians (2% allelic frequency),CYP3A5 L82R(occurred in the racially unidentified group) andCYP3A5 F446S(identified in Caucasians with a 2% allelic frequency) were on an allele containing the splice change g.6986A>G known asCYP3A5*3. The newly identified allelic proteins were constructed by site-directed mutagenesis, expressed inEscherichia coliand purified. CYP3A5 L82R was expressed only as denatured CYP420, suggesting it may be unstable. CYP3A5*1 exhibited the highest maximal clearance for testosterone followed by CYP3A5 A337T>CYP3A5 R28C ≫ CYP3A5 F446S. CYP3A5*1 exhibited a higherVmaxfor nifedipine oxidation than CYP3A5 A337T>CYP3A5 R28C ≫ CYP3A5 F446S. CYP3A5 A337T and CYP3A5 R28C exhibited a 42–64% lowerVmaxfor nifedipine oxidation than CYP3A5*1. CYP3A5 F446S exhibited a>95% decrease in the intrinsic clearance for both 6β-hydroxytestosterone and nifedipine oxidation.ConclusionThis study identifies four new potentially defective coding alleles.CYP3A5 F446Sis predicted to be more catalytically defective than the splice change alone.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

In-vitro data indicate major effects of the genetically polymorphic cytochrome P450 enzyme 2C9 (CYP2C9) on the pharmacokinetics of celecoxib, a nonsteroidal anti-inflammatory drug acting as selective cyclooxygenase-2 inhibitor. Human studies report decreased clearance in heterozygous carriers of the CYP2C9 variant Ile359Leu (*3), but results appeared controversial and only data on single subjects carrying the homozygousCYP2C9*3/*3genotype have been published. We measured single-dose kinetics of celecoxib and its main metabolites hydroxy- and carboxy-celecoxib in 21 healthy volunteers who were selected as hetero- (n= 4) and homozygous (n= 3) carriers ofCYP2C9variants Arg144Cys (*2) and Ile359Leu (*3). Blood concentrations of celecoxib and its metabolites hydroxy-celecoxib and carboxy-celecoxib were quantified by high-performance liquid chromatography. A more than two-fold reduced oral clearance in homozygous carriers ofCYP2C9*3was seen for celecoxib compared to carriers of the wild-type genotypeCYP2C9*1/*1and heterozygous carriers of one *3allele were in-between (P= 0.003 for trend), whereasCYP2C9*2had no significant influence on celecoxib pharmacokinetics. Decreased concentrations of carboxy- and hydroxy-celecoxib in heterozygous and homozygous carriers ofCYP2C9*3were detected which supported the influence ofCYP2C9polymorphisms on celecoxib pharmacokinetic variability. Approximately 0.5% of Caucasians carrying the genotypeCYP2C9*3/*3will have greatly increased internal exposure to celecoxib. It remains to be shown whether this is associated with greater efficacy or with an increased incidence and severity of adverse events.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

ObjectivesThere is increasing evidence that polymorphism of theABCB1(MDR1) gene contributes to interindividual variability in bioavailability and tissue distribution of P-glycoprotein substrates. The aim of the present study was to (1) identify and describe novel variants in theABCB1gene, (2) understand the extent of variation inABCB1at the population level, (3) analyze how variation inABCB1is structured in haplotypes, and (4) functionally characterize the effect of the most common amino acid change in P-glycoprotein.Methods and resultsForty-eight variant sites, including 30 novel variants and 13 coding for amino acid changes, were identified in a collection of 247 ethnically diverse DNA samples. These variants comprised 64 statistically inferred haplotypes, 33 of which accounted for 92% of chromosomes analyzed. The two most common haplotypes,ABCB1*1 andABCB1*13, differed at six sites (three intronic, two synonymous, and one non-synonymous) and were present in 36% of all chromosomes. Significant population substructure was detected at both the nucleotide and haplotype level. Linkage disequilibrium was significant across the entireABCB1gene, especially between the variant sites found inABCB1*13, and recombination was inferred. The Ala893Ser change found in the commonABCB1*13 haplotype did not affect P-glycoprotein function.ConclusionThis study represents a comprehensive analysis ofABCB1nucleotide diversity and haplotype structure in different populations and illustrates the importance of haplotype considerations in characterizing the functional consequences ofABCB1polymorphisms.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

The primary genetic form of trimethylaminuria (TMAU) is caused by inherited defects in the flavin-containing monooxygenase 3 (FMO3) gene. Defective FMO3 has a decreased ability to catalyze theN-oxygenation of the dietary-derived malodourous amine, trimethylamine. We report two novel deleterious mutations identified in two unrelated individuals affected by the disorder. Sequence analysis of theFMO3coding exons amplified from genomic DNA revealed that the mutation from individual 1 was heterozygous for a G>A missense mutation in exon 2 of theFMO3gene. The mutation changed a GAG encoding Glu at codon 32 to AAG encoding Lys. Wild-type and mutant E32KFMO3were expressed inEscherichia colias maltose binding-fusion proteins and assayed for their ability to catalyze oxygenation of various FMO3 substrates. The results showed that the E32K mutation abrogated the catalytic activity of the enzyme. Individual 2 was identified as heterozygous for the P153L mutation. In addition, individual 2 was also heterozygous for a novel single nucleotide deletion of A191 in exon 3 at codon 64. The deletion resulted in a frame shift and caused premature termination of theFMO3gene immediately after codon 65. Family pedigree analysis revealed that the P153L and the deletion mutation were carried on different alleles for this individual. Therefore, both alleles of theFMO3gene for individual 2 were affected by mutations abolishing the catalytic activity of the enzyme, explaining the severe TMAU condition. The two deleterious mutations reported herein were rare mutations with estimated allelic frequencies of less than 1%.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

ObjectivesParaffin-embedded tumor samples are valuable in the study of cancer for routine staging, tumor marker analysis, and in retrospective studies to test new prognostic and predictive biomarkers. Their utility in retrospective pharmacogenetic analysis of clinical trials has yet to be evaluated. We set out to establish genotyping methods for relevant genes from archival tumor samples and determine if fixation, processing or somatic changes in the tumor might affect our ability to identify germ-line polymorphisms.Methods and resultsTo establish the assays, paraffin blocks were made using pellets prepared from eight tumor cell lines. DNA was isolated from viable cells and from sections from these blocks, and genotyped for polymorphisms in CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A5 and MDR1 using conventional PCR-RFLP assays. This demonstrated that fixation and processing did not alter the genotypes obtained (100% concordance). Next, sections were obtained from paraffin-embedded archival breast samples from 10 patients for whom gDNA isolated from peripheral blood was available for comparison. Concordance was complete with the same genotype being obtained for 100% of the samples tested. Attempts to extend these methods for the study of hematoxylin/eosin or immunohistochemically stained sections were not successful since the staining inhibited the PCR reactions. Only 25 of 50 samples were successfully amplified and of those only 14 produced accurate genotypes.ConclusionsAccurate genetic testing for polymorphisms in several genes of pharmacogenetic importance can be obtained from archival paraffin-embedded tumor samples. Thus, pharmacogenetic analysis can be applied to existing cancer therapy trials to test associations between these polymorphisms and treatment response.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Genetic factors influence alcohol consumption and alcoholism. A number of groups have bred alcohol drinker and non drinker rat strains, but genetic determinants remain unknown. The University of Chile rat lines UChA (low drinkers) and UChB (high drinkers) display differences in the relativeKmfor NAD+of mitochondrial aldehyde dehydrogenase (ALDH2) but noVmaxdifferences. The relativeKmdifferences may be due to mitochondrial changes or to genetic differences coding for ALDH2. We investigated whether there are differences in the coding regions of ALDH2 cDNA in these lines and whether theAldh2genotype predicts the phenotype of alcohol consumption and theKmof ALDH2 for NAD+. Liver cDNA was prepared, and theAldh2transcript was amplified, cloned and sequenced. Genotyping was conducted by DNA amplification and restriction enzyme digestion. When compared toAldh21of Sprague-Dawley, 94% of the UChA (low drinker) rats (n= 61), presented a mutation that changes Gln67to Arg in the mature enzyme (allele referred to asAldh22). In UChB (high drinker) rats (n= 69), 58% presented theAldh21allele, while 42% presented the Gln67Arg change plus a second mutation that changed Glu479to Lys (alleleAldh23). TheAldh22allele was absent in high drinker rats. Rats of differentAldh2genotypes displayed marked phenotypic differences in both ethanol consumption (g/kg/day; means±SE): (Aldh21/Aldh21) = 5.7±0.2, (Aldh22/Aldh22) = 0.9±0.2 and (Aldh23/Aldh23) = 4.6±0.2; andKms for NAD+of 43±3 μm, 132±13 μm and 41±2 μm, respectively (Aldh22versusAldh21orAldh23;P<0.0001 for both phenotypes). Overall, the data show that alleles ofAldh2strongly segregate with the phenotype of ethanol consumption and the relativeKmfor NAD+of ALDH2. Bases mutated suggest that non drinkerAldh22is ancestral with regard to the coding changes in eitherAldh21orAldh23, variants which would allow ethanol consumption and may provide an evolutionary advantage by promoting calorie intake from fermented products along with carbohydrates.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

UDP-glucuronosyltransferases (UGTs) belong to a superfamily of microsomal enzymes responsible for glucuronidation of numerous endogenous and exogenous compounds including bilirubin, hormones, various drugs as well as environmental carcinogens. Glucuronidation predominantly serves as a pathway for elimination of the different glucuronidated compounds. Seventeen human UGT transcripts have been identified thus far, and the UGT proteins are differentially expressed in a wide-range of human tissues. Genetic variants have been identified in coding and non-coding sequences of severalUGTgenes, and similar observations should be anticipated for allUGTs. As glucuronidation plays a critical part in the inactivation or elimination of countless substrates, genetic variants in this enzyme family that lead to altered expression or activity of UGTs are likely to have some physiologic and pharmacological consequences. This article focuses on the potential impact of variousUGTs or their variants on cancer risk and cancer therapeutics.

ISSN:0960-314X    

出版商:OVID    

年代:2003

数据来源: OVID