RESUMO
Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High-titre autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in approximately 7% of APS1 patients, with immunoreactivity to pituitary tissue frequently described. Using APS1 patient serum to immunoscreen a pituitary cDNA expression library, testis specific, 10 (TSGA10) was isolated. Immunoreactivity against TSGA10 was detected in 5/99 (5.05%) patients with APS1, but also in 5/135 (3.70%) systemic lupus erythematosus (SLE) patients and 1/188 (0.53%) healthy controls. TSGA10 autoantibodies were not detected in the serum from patients with any other autoimmune disease. Autoantibodies against TSGA10 were detectable from a young age in 4/5 positive APS1 patients with autoantibody titres remaining relatively constant over time. Furthermore, real-time PCR confirmed TSGA10 mRNA to be most abundantly expressed in the testis and also showed moderate and low expression levels throughout the entire body. TSGA10 should be considered as an autoantigen in a subset of APS1 patients and also in a minority of SLE patients. No recognizable clinical phenotype could be found to correlate with positive autoantibody reactivity.
Assuntos
Autoanticorpos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas/imunologia , Proteínas do Citoesqueleto , Feminino , Regulação da Expressão Gênica , Humanos , Lúpus Eritematoso Sistêmico/genética , Masculino , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/imunologia , Proteínas/genética , RNA Mensageiro/genéticaRESUMO
The development of a testis requires the proper spatiotemporal expression of the SRY gene and other genes that act in a dosage-sensitive manner. Mutations in the SRY gene account for only 10-15% of patients with 46,XY gonadal disorder of sex development (DSD). To enable the diagnostics of deletions and duplications of genes known to be involved in different forms of DSD, we developed a synthetic probe set for multiplex ligation-dependent probe amplification (MLPA) analysis. Here, we report the results from the analysis of 22 patients with 46,XY gonadal DSD. The analysis with the DSD probe set has led to the identification of two copy number variations, an 800-kb NR0B1 (DAX1) locus duplication on Xp21 in a patient with isolated partial gonadal dysgenesis and a duplication of the SRD5A2 gene that represents a rare normal variant. The described MLPA kit represents an optimal complement to DNA sequence analysis in patients with DSD, enabling screening for deletions and duplications of several genes simultaneously. Furthermore, the second identification of an NR0B1 locus duplication in a patient with isolated gonadal dysgenesis, without dysmorphic features and/or mental retardation, highlights the importance of evaluating NR0B1 duplication in patients with gonadal dysgenesis.
Assuntos
Proteínas de Ligação a DNA/análise , Dosagem de Genes , Disgenesia Gonadal 46 XY/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Receptores do Ácido Retinoico/análise , Proteínas Repressoras/análise , Receptor Nuclear Órfão DAX-1 , Proteínas de Ligação a DNA/genética , Feminino , Amplificação de Genes , Duplicação Gênica , Humanos , Masculino , Sondas de Oligonucleotídeos , Receptores do Ácido Retinoico/genética , Proteínas Repressoras/genéticaRESUMO
Testatin has been implicated in fetal testis development due to its restricted expression in pre-Sertoli cells immediately after the onset of Sry gene expression. However, testatin knockout mice showed normal testis development and fertility. We investigated the spatial and temporal expression pattern of the Cres/testatin subgroup of genes, including the novel gene Cstl1/Cres4, in fetal mouse gonads and in adult testis, epididymis and ovary. The genes are related to the family 2 cystatins of protease inhibitors. Using real-time PCR and in situ hybridization we could show that 4 subgroup genes, testatin, CstSC, CstTE-1/Cres3 and Cres are expressed in fetal testis. We also confirmed the expression of testatin, CstE2, CstSC, CstTE-1/Cres3, Cres, CstT and Cstl1/Cres4 in adult testis and CstE2, CstTE-1/Cres3, Cres and CstE1/Cres2 in adult epididymis. In testatin knockout animals, the expression of CstE2 was heavily downregulated in adult testis, but not in adult epididymis, compared to wildtype controls. In conclusion, an explanation for the lack of phenotype in testatin knockout mice could be functional redundancy with another member of the Cres/testatin subgroup. The most likely candidate/s would be CstSC, CstTE-1/Cres3 or Cres as they are expressed in the fetal testicular tubules in early testis differentiation together with testatin.
Assuntos
Cistatinas/genética , Expressão Gênica , Reprodução/genética , Testículo/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Cistatinas/química , Cistatinas/deficiência , Epididimo/química , Feminino , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Ovário/química , Ovário/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Testículo/química , Testículo/embriologiaRESUMO
In view of conflicting data in the literature regarding the enzyme(s) responsible for metabolism of selegiline, a drug used in the treatment of Parkinson's disease, investigations were carried out in vitro using the human cytochrome P450 enzymes CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 recombinantly expressed in yeast to elucidate the enzyme specificity in selegiline metabolism. In the yeast microsomes used, desmethylselegiline and levomethamphetamine were formed from selegiline at significant rates. The highest contribution to the hepatic clearance of selegiline was calculated to be exerted by CYP2B6 (124 l/h) CYP2C19 (82 l/h), whereas CYP3A4 (27 l/h) and CYP1A2 (21 l/h) were of less importance. Antibodies against CYP2B6 inhibited metabolism of selegiline in microsomes containing CYP2B6 but not in microsomes without significant amounts of the enzyme. In contrast to previous reports, we could not find any role for CYP2D6 in the metabolism of selegiline. The data strongly indicate that the high extent of interindividual variation seen in vivo for selegiline clearance is caused by the metabolism of the compound by the highly polymorphic CYP2B6 and CYP2C19.
Assuntos
Antiparkinsonianos/metabolismo , Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Oxigenases de Função Mista/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Proteínas Recombinantes/metabolismo , Selegilina/metabolismo , Antiparkinsonianos/química , Antiparkinsonianos/uso terapêutico , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C19 , Humanos , Microssomos Hepáticos/enzimologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Saccharomyces cerevisiae/enzimologia , Selegilina/química , Selegilina/uso terapêuticoRESUMO
Losartan, an angiotensin II receptor antagonist, is oxidized by hepatic cytochromes P450 to an active carboxylic acid metabolite, E-3174. The aim of the present investigation was to study the contribution of CYP2C9 and CYP3A4 in losartan oxidation in vitro and to evaluate the role of CYP2C9 polymorphism. Kinetic properties of different genetic CYP2C9 variants were compared both in a yeast expression system and in 25 different samples of human liver microsomes where all known genotypes of CYP2C9 were represented. Microsomes were incubated with losartan (0.05-50 microM), and the formation of E-3174 was analyzed by high-performance liquid chromatography to estimate V(max), K(m), and intrinsic clearance for all individual samples. Sulfaphenazole, a CYP2C9 inhibitor, blocked the formation of E-3174 at low losartan concentrations (<1 microM), whereas the inhibitory effect of triacetyloleandomycin, a CYP3A4 inhibitor, was significant only at high concentrations of losartan (>25 microM). In comparison to the CYP2C9.1 variant, oxidation of losartan was significantly reduced in yeast expressing the rare CYP2C9.2 or CYP2C9.3 variants. Moreover, the rate of losartan oxidation was lower in liver microsomes from individuals hetero- or homozygous for the CYP2C9*3 allele, or homozygous for the CYP2C9*2 allele. The difference between the common and rare CYP2C9 variants was mainly explained by a lower V(max), both in yeast and human liver microsomes. In summary, these in vitro results indicate that CYP2C9 is the major human P450 isoenzyme responsible for losartan oxidation and that the CYP2C9 genotype contributes to interindividual differences in losartan oxidation and activation.
Assuntos
Antagonistas de Receptores de Angiotensina , Anti-Hipertensivos/farmacocinética , Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Losartan/farmacocinética , Polimorfismo Genético , Esteroide 16-alfa-Hidroxilase , Esteroide Hidroxilases/genética , Sequência de Bases , Citocromo P-450 CYP2C9 , Sistema Enzimático do Citocromo P-450/metabolismo , Primers do DNA , Diclofenaco/farmacocinética , Humanos , Oxirredução , Esteroide Hidroxilases/metabolismoRESUMO
Human cytochrome P450 2A6 (CYP2A6) constitutes the major nicotine oxidase, and large interindividual differences are seen in the levels of this enzyme, to a great extent caused by the distribution of several different polymorphic gene variants mainly located in the open reading frame (ORF). In the present study, we report a common polymorphism located in the 5' flanking region of CYP2A6 affecting its expression. DHPLC analysis and complete sequence of the open reading frame of the gene from a Turkish individual revealed a -48T > G substitution disrupting the TATA box. Using dynamic allele-specific hybridization (DASH), genotyping of this novel variant (named CYP2A6*9) was carried out in 116 Swedish, 132 Turkish, and 102 Chinese subjects, and the allele frequencies were found to be 5.2, 7.2, and 15.7%, respectively. The significance of the polymorphism was investigated by the construction of luciferase reporter plasmids containing 135 or 500 bp of the 5'-upstream region of the gene transfected into human hepatoma B16A2 cells. The constructs carrying the -48T > G mutation were only expressed at about 50% of the wild-type alleles. It is concluded that the CYP2A6*9 allele might be one of the most common CYP2A6 variants in Caucasians that alters the levels of enzyme expression.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Oxigenases de Função Mista/genética , Oxirredutases/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/fisiologia , TATA Box/genética , Alelos , Povo Asiático/genética , Linhagem Celular , China/etnologia , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2A6 , Análise Mutacional de DNA , Expressão Gênica/fisiologia , Genes Reporter , Testes Genéticos , Variação Genética , Genótipo , Humanos , Desnaturação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/análise , Ácidos Nucleicos Heteroduplexes/genética , Hibridização de Ácido Nucleico/métodos , Suécia/etnologia , Transfecção , Turquia/etnologia , População Branca/genéticaRESUMO
On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Oxirredutases N-Desmetilantes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular , Clonagem Molecular , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/biossíntese , Etiquetas de Sequências Expressas , Humanos , Fígado/metabolismo , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Oxirredutases N-Desmetilantes/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transfecção , Células Tumorais CultivadasAssuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Mutação/fisiologia , Esteroide 16-alfa-Hidroxilase , Esteroide Hidroxilases/genética , Varfarina/administração & dosagem , Citocromo P-450 CYP2C9 , Relação Dose-Resposta a Droga , Humanos , Varfarina/uso terapêuticoRESUMO
Cytochrome P450 2A6 (CYP2A6) is involved in the C-oxidation of nicotine and in the metabolic activation of tobacco nitrosamines. Recent data have suggested that CYP2A6 genetic polymorphisms might play a role in tobacco dependence and consumption as well as in lung cancer risk. However, the previously published studies were based on a genotyping method that overestimated the frequencies of deficient alleles, leading to misclassification for the CYP2A6 genotype. In this study, we genotyped DNA from 244 lung cancer patients and from 250 control subjects for CYP2A6 (wild-type allele CYP2A6*1, and two deficient alleles: CYP2A6*2, and CYP2A6*4, the latter corresponding to a deletion of the gene) using a more specific procedure. In this Caucasian population, we found neither a relation between genetically impaired nicotine metabolism and cigarette consumption, nor any modification of lung cancer risk related to the presence of defective CYP2A6 alleles (odds ratio = 1.1, 95% confidence interval = 0.7-1.9).
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Neoplasias Pulmonares/genética , Oxigenases de Função Mista/genética , Polimorfismo Genético/genética , Estudos de Casos e Controles , Citocromo P-450 CYP2A6 , DNA Ligases/genética , França/epidemiologia , Predisposição Genética para Doença , Genótipo , Humanos , Isoenzimas/genética , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/epidemiologia , Masculino , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Fumar/genéticaRESUMO
With the aid of the htgs and dbEST databases, a novel cytochrome P450 cDNA was found by homology searches, and the corresponding gene was identified on chromosome 19. Nested PCR was used to amplify a full-length sequence of 1515 bp. The predicted 504 amino acid sequence displays 38--49% identity with CYP2 family members and the protein was designated CYP2S1. mRNA dot blot analysis demonstrated high expression levels in trachea, lung, stomach, small intestine, and spleen. The expression pattern was confirmed by Northern blot, which also revealed a single transcript of approximately 2.4 kb. Western blot analysis, using an antiserum directed against the C-terminus of the enzyme, detected a protein in human lung with the same mobility as recombinant CYP2S1. Subcellular fractionation and immunostaining revealed that CYP2S1 was localized in the endoplasmic reticulum. We conclude that CYP2S1 represents a novel abundantly expressed human P450 with potential importance for extrahepatic xenobiotic metabolism.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Oxigenases/genética , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular Transformada , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/imunologia , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/química , DNA Complementar/genética , DNA Recombinante , Feminino , Humanos , Soros Imunes/imunologia , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Pulmão/metabolismo , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Oxigenases/imunologia , Oxigenases/metabolismo , Filogenia , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Distribuição Tecidual , TransfecçãoRESUMO
During the last couple of years, cytochrome P450 2A6 (CYP2A6; coumarin 7-hydroxylase) has received a lot of attention because it has been shown that it is the principle human nicotine C-oxidase. This enzyme also activates a number of structurally unrelated precarcinogens including many nitrosamines and aflatoxin B1, and metabolizes certain clinically used drugs. There is a pronounced interindividual and interethnic variability in CYP2A6 levels and activity, and much of this can be attributed to polymorphisms in the CYP2A6 gene, where a few inactivating mutations as well as gene deletions have been described. The frequency of the inactive alleles is low in European populations and very few poor metabolizers for the probe drug coumarin have been described in these populations. In contrast, a relatively high allele frequency (15-20%) of the CYP2A6 gene deletion has been found in Asians, resulting in a generally reduced activity in these populations. Because of the importance of CYP2A6 in nicotine metabolism, it has been suggested that the CYP2A6 genotype influences the interindividual differences in smoking behavior as well as lung cancer susceptibility. Several case-control studies have been conducted in this area, but these have yielded conflicting results. The recent progress in the field of CYP2A6 genetics and the development of more specific genotyping methods will facilitate molecular epidemiological studies aimed at clarifying these important issues.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Nicotina/metabolismo , Polimorfismo Genético , Citocromo P-450 CYP2A6 , Humanos , IndividualidadeRESUMO
Cytochrome P450 1B1 (CYP1B1) is a human extrahepatic P450 that activates procarinogens, metabolizes 17beta-estradiol, and may well have a role in the pathogenesis of some forms of cancer. Besides rare deleterious mutations reported for the CYP1B1 gene, six single-nucleotide polymorphisms have been reported, of which four cause amino acid exchanges. We have expressed two of the common CYP1B1 alleles in yeast cells and mammalian COS-1 cells in order to functionally characterize the alleles with respect to kinetic properties and protein stability. The CYP1B1.2 variant contains the two linked amino acid substitutions R48G and A119S compared to CYP1B1.1. The kinetic parameters of two structurally unrelated CYP1B1 substrates for the two variants were examined. No kinetic differences were seen of 17beta-estradiol hydroxylation activities between the two CYP1B1 variants and an only minor increase in the apparent Km for ethoxyresorufin was observed for CYP1B1.2. It therefore appears that they have very similar catalytic properties and the substitutions do not appear to alter CYP1B1 catalytic function. The two CYP1B1 variants were similarly stable when expressed in mammalian COS-1 cells, indicating that the substitutions have no effect on protein folding or stability. The combined results indicate that these two CYP1B1 variants show very similar properties with respect to catalytic activities and protein stability and do not alter CYP1B1 function.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Células COS , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/química , Primers do DNA/genética , Estabilidade Enzimática , Variação Genética , Humanos , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C-oxidase. A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed. Two defective alleles have hitherto been identified, one of which is very common in Asian populations. Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption. Here, we have re-evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3' flanking region of 30-40% of CYP2A6*1 alleles results in genotype misclassification. In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects. In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles. One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D). The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events. We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Nicotina/metabolismo , Esteroide Hidroxilases/genética , Apoproteínas/metabolismo , Sequência de Bases , Southern Blotting , China , Citocromo P-450 CYP2A6 , Sistema Enzimático do Citocromo P-450/metabolismo , Genótipo , Humanos , Masculino , Oxigenases de Função Mista/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Polimorfismo Genético , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido Nucleico , Fumar/genética , Espanha , TransfecçãoRESUMO
Cytochrome P450 2E1 (CYP2E1) is a toxicologically very important enzyme with a high extent of interindividual variability in expression. We sequenced and characterized the 5'-flanking region of the human and rat CYP2E1 genes. The identity between the human and rat sequences (-3.8 kb to +1 kb) was generally between 35 and 60%, and the most similar regions were found in the proximal part of the sequence. Two more distant regions at -1.6 to -2.0 kb and -2.5 to -2. 8 kb in the human sequence were also found to have high identity to the rat sequence. A polymorphic repeat sequence in the human gene was found between -2178 to -1945 bp. The common allele (CYP2E1*1C) contained 6 repeats (each 42-60 bp long) and the rare allele (CYP2E1*1D) had 8 repeats with an allele frequency of 1% among Caucasians and 23% among Chinese. The CYP2E1 5'-flanking regions of the human (-3712 bp to +10 bp) and rat (-3685 bp to +28 bp) genes were ligated in front of a luciferase reporter gene and transfected into rat hepatoma Fao and human hepatoma B16A2 cells. Important species specificity was noted in the control of gene expression and regions of negative and positive cis-acting elements were localized. No difference was seen in the constitutive expression between the two polymorphic forms. The importance of this repeat polymorphism for high and low inducible CYP2E1 phenotypes is discussed.
Assuntos
Citocromo P-450 CYP2E1/genética , Polimorfismo Genético , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Alelos , Animais , Povo Asiático/genética , Clonagem Molecular , Citocromo P-450 CYP2E1/biossíntese , Indução Enzimática , Frequência do Gene , Genes Reporter , Humanos , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/genética , Neoplasias/etiologia , Neoplasias/genética , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Análise de Sequência de DNA , População Branca/genéticaRESUMO
Approximately 40% of human P450-dependent drug metabolism is carried out by polymorphic enzymes, which can cause abolished, quantitatively or qualitatively altered or enhanced drug metabolism. The latter situation is due to stable duplication, multiduplication or amplification of active genes, most likely in response to dietary components that have resulted in a selection of alleles with multiple non-inducible genes. Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy due to ultrarapid metabolism or, alternatively, adverse effects from the drug treatment due to the presence of defective alleles. Knowledge in this field has grown rapidly and can now be applied to both drug development and clinical practice. This is facilitated by the recent development of high-throughput methods for mutation detection and oligonucleotide chips array technology for the identification of a multitude of mutations in the genes encoding drug-metabolizing enzymes. The outcome will allow for safer and more efficient drug therapies.
Assuntos
Alelos , Anti-Inflamatórios não Esteroides/metabolismo , Citocromo P-450 CYP2D6/genética , Inibidores Seletivos de Recaptação de Serotonina/metabolismo , Citocromo P-450 CYP2D6/sangue , Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular , Humanos , Polimorfismo GenéticoRESUMO
Cytochrome P450 2A6 is an important human hepatic P450 which activates pre-carcinogens, oxidises some drugs and constitutes the major nicotine C-oxidase. In fact, results have been presented in the literature which suggested a relationship between the distribution of defective CYP2A6 alleles and smoking behaviour as well as cigarette consumption. In the present report, we describe the structure of a novel CYP2A locus where the whole CYP2A6 gene has been deleted, resulting in an abolished cytochrome P450 2A6-dependent metabolism. The origin of this locus is apparently due to an unequal crossover event between the 3'-flanking region of the CYP2A6 and CYP2A7 genes. A rapid PCR-based method for the detection of the CYP2A6del allele was developed and the allele frequency was 15.1% among 96 Chinese subjects, but only 1.0% in Finns (n=100) and 0.5% in Spaniards (n=100). In the Chinese population, we did not detect any CYP2A6*2 alleles using an improved genotyping procedure, in contrast to the 11-20% previously reported. It is concluded that genotyping for the CYP2A6del allele is of great importance in studies correlating, for example, smoking behaviour, pre-carcinogen activation or drug metabolism to the CYP2A6 genotype, in particular when oriental populations are investigated.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Povo Asiático/genética , Sistema Enzimático do Citocromo P-450/genética , Deleção de Genes , Oxigenases de Função Mista/genética , Reação em Cadeia da Polimerase/métodos , Alelos , Sequência de Bases , Citocromo P-450 CYP2A6 , DNA Complementar , Finlândia , Frequência do Gene , Genótipo , Humanos , Dados de Sequência Molecular , FenótipoRESUMO
Cytochrome P450 2A6 (CYP2A6) is a polymorphic enzyme responsible for the oxidation of certain precarcinogens and drugs and is the major nicotine C-oxidase. The role of CYP2A6 for nicotine elimination was emphasised recently by the finding that smokers carrying defective CYP2A6 alleles consumed fewer cigarettes [Pianezza et al. (1998) Nature 393, 750]. The method used for CYP2A6 genotyping has, however, been found to give erroneous results with respect to the coumarin hydroxylase phenotype, a probe reaction for the CYP2A6 enzyme. The present study describes an allele-specific PCR genotyping method that identifies the major defective CYP2A6 allele and accurately predicts the phenotype. An allele frequency of 1-3% was observed in Finnish, Spanish, and Swedish populations, much lower than described previously.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Oxigenases de Função Mista/genética , Oxirredutases/genética , Fumar/genética , População Branca/genética , Citocromo P-450 CYP2A6 , Sistema Enzimático do Citocromo P-450/metabolismo , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Etnicidade , Finlândia , Genótipo , Humanos , Oxigenases de Função Mista/metabolismo , Oxirredutases/metabolismo , Fenótipo , Reação em Cadeia da Polimerase , Espanha , Suécia , Tabagismo/enzimologia , Tabagismo/genéticaRESUMO
The polymorphic cytochrome P450 2D6 (CYP2D6) causing poor, extensive or ultrarapid metabolism of several clinically important drugs exhibits pronounced interethnic variation. Ultrarapid metabolism is caused by multiple copies of active CYP2D6 genes and recently 29% of an Ethiopian population has been shown to carry duplicated or multiduplicated CYP2D6 genes, whereas the corresponding frequency in other black, Oriental and European populations investigated is 1-2%. In order to characterize the distribution of alleles with multiple CYP2D6 copies in a neighbouring population and to characterize the CYP2D locus in general among Saudi Arabians, the CYP2D6 genotype of a Saudi Arabian population was examined using restriction fragment length polymorphism (RFLP) analysis and allele-specific polymerase chain reaction (PCR) amplification. Of 101 Saudi Arabians studied, 21 subjects had an EcoRI fragment indicative of CYP2D6 gene duplication. In contrast, only two individuals were heterozygous for a deletion of the whole gene (CYP2D6*5). The allele frequency of CYP2D6*4, the most common defective allele among Caucasians, was only 3.5% in the Saudi population. Two other alleles, CYP2D6*10 and *17, common in certain populations and which cause diminished enzyme activity, were found only at low allele frequencies of 3.0% each. These findings are in agreement with earlier Saudi Arabian phenotyping studies which reported a low frequency (1-2%) of poor metabolizers for CYP2D6 probe drugs. In conclusion, the Saudi Arabian population studied exhibited very few defective alleles and a large number of subjects carried duplicated CYP2D6 genes, implying a high conservation on functional CYP2D6 genes possibly due to dietary reasons and reveal the Saudi Arabians as an unique population in comparison with others examined.