Allele and genotype frequencies of polymorphic cytochromes P450 (CYP2C9, CYP2C19, CYP2E1) and dihydropyrimidine dehydrogenase (DPYD) in the Egyptian population

AIMS: The goal of this study was to determine the frequencies of important allelic variants of CYP2C9, CYP2C19, CYP2E1 and DPYD in the Egyptian population and compare them with the frequencies in other ethnic populations. METHODS: Genotyping of CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), c2 variant of CYP2E1 and DPYD alleles (*2 A-*6 ) was carried out in a total of 247 unrelated Egyptian subjects. An allele-specific fluorogenic 5' nuclease chain reaction assay was applied for detection of CYP2C9 and CYP2C19 variants. Other variants of the CYP2E1 and DPYD genes were determined using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR based assays. RESULTS: CYP2C9 allele frequencies in 247 Egyptian subjects were 0.820 for CYP2C9*1, 0.120 for CYP2C9*2 and 0.060 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.888, 0.110 and 0.002, respectively. CYP2C19*3, which is considered an Asian mutation, was detected in one subject (0.40%) who was heterozygous (*1/*3). Two subjects (0.80%) were homozygous for *2/*2, while no compound heterozygotes (*2/*3) or homozygotes for *3 were detected. For CYP2E1, only four subjects (1.70%) had the rare c2 variant, expressed heterozygously, giving an allele frequency of 0.009. Five variants of DPYD were analysed, with no splice sites (*2 A) or DeltaC1897 (*3) found in this population. The frequencies of other variants were 0.028, 0.115 and 0.090 for *4, *5 and *6, respectively. CONCLUSIONS: Comparing our data with that obtained in several Caucasian, African-American and Asian populations, we found that Egyptians resemble Caucasians with regard to allelic frequencies of the tested variants of CYP2C9, CYP2C19, CYP2E1 and DPYD. Our results may help in better understanding the molecular basis underlying ethnic differences in drug response, and contribute to improved individualization of drug therapy in the Egyptian population.     

Genotype and allele frequencies of TPMT,NAT2, GST,SULT1A1 and MDR-1 in the Egyptian population

AIMS: The goal of this study was to determine the frequencies of important allelic variants in the TPMT, NAT2, GST, SULT1A1 and MDR-1 genes in the Egyptian population and compare them with the frequencies in other ethnic populations. METHODS: Genotyping was carried out in a total of 200 unrelated Egyptian subjects. TPMT*2 was detected using an allele-specific polymerase chain reaction (PCR) assay. TPMT*3C and NAT2 variants (*5,*6 and *7) were detected using an allele-specific real-time PCR assay. Detection of GSTM1 and GSTT1 null alleles was performed simultaneously using a multiplex PCR assay. Finally, a PCR-restriction fragment length polymorphism assay was applied for the determination of TPMT*3A (*3B), SULT1A1*2 and MDR-1 (3435T) variants. RESULTS: Genotyping of TPMT revealed frequencies of 0.003 and 0.013 for TPMT*3A and TPMT*3C, respectively. No TPMT*2 or *3B was detected in the analysed samples. The frequencies of specific NAT2 alleles were 0.215, 0.497, 0.260 and 0.028 for *4 (wild-type), *5 (341C), *6 (590A) and *7 (857A), respectively. GSTM1 and GSTT1 null alleles were detected in 55.5% and 29.5% of the subjects, respectively. SULT1A1*2 was detected at a frequency of 0.135. Finally, the frequencies of the wild-type allele (3435C) and the 3435T variant in the MDR-1 gene were found to be 0.6 and 0.4, respectively. CONCLUSIONS: We found that Egyptians resemble other Caucasians with regard to allelic frequencies of the tested variants of NAT2, GST and MDR-1. By contrast, this Egyptian population more closely resemble Africans with respect to the TPMT*3C allele, and shows a distinctly different frequency with regard to the SULT1A1*2 variant. The predominance of the slow acetylator genotype in the present study (60.50%) could not confirm a previously reported higher frequency of the slow acetylator phenotype in Egyptians (92.00%), indicating the possibility of the presence of other mutations not detectable as T341C, G590A and G857A. The purpose of our future studies is to investigate for new polymorphisms, which could be relatively unique to the Egyptian population.     

Identification of a novel splice-site mutation in the CYP1A2 gene

AIMS: To identify the molecular basis for a low CYP1A2 metabolic status, as determined by a caffeine phenotyping test, in a 71-year-old, nonsmoking, Caucasian woman who presented with very high clozapine concentrations despite being administered a standard dose of the drug. METHODS: The nucleotide sequence of the 7 exons, exon-intron boundaries and 5'-flanking region of the CYP1A2 gene was analysed by direct sequencing. RESULTS: Only one heterozygous point mutation was identified in the donor splice site of intron 6 (3534G > A) of CYP1A2. This mutation could cause abnormal RNA splicing and therefore lead to a truncated nonfunctional enzyme. No other carrier of this mutation was identified in a population of 100 unrelated healthy Caucasians. CONCLUSIONS: This is the first report of a splice-site mutation affecting the CYP1A2 gene. This polymorphism is a likely explanation for the low CYP1A2 activity associated with high clozapine concentrations in this patient.     

CYP1A2、CYP2D6和CYP2C19基因多态性与氯氮平及其代谢物血药浓度的相关性研究

目的：研究CYP1A2、CYP2D6和CYP2C19基因多态性与精神分裂症患者氯氮平（clozapine,CLZ）及其活性代谢物去甲氯氮平（N-desmethy clozapine,N-CLZ）血药浓度的相关性。方法：纳入156例经CLZ单药治疗1个月以上的精神分裂症患者,采集清晨服药前空腹血,采用液相色谱-串联质谱（LC-MS/MS）法测定CLZ及N-CLZ稳态谷浓度。通过Axiom基因芯片分析技术检测CYP1A2（*1C、*1F）、CYP2D6（*2、*10）、CYP2C19（*2、*3）等6个SNP位点的基因型,比较不同基因型患者CLZ及其代谢物的浓度剂量比（C/D）及代谢物与CLZ血药浓度比值（C_N-CLZ/C_CLZ）的差异。结果：CYP2D6*10基因多态性与N-CLZ C/D具有相关性（P<0.01）。CYP1A2*1C、CYP2D6（*2、*10）基因多态性与C_N-CLZ/C_CLZ具有相关性（P<0.05,P<0.01,P<0.01）。CYP1A2*1F、CYP2C19*2、CYP2C19*3基因多态性与C/D及C_N-CLZ/C_CLZ无相关性（P>0.05）。结论：CYP1A2*1C和CYP2D6（*2、*10）基因多态性对CLZ代谢存在影响,建议临床在使用CLZ治疗前检测患者CYP1A2*1C和CYP2D6（*2、*10）基因型,为CLZ个体化治疗提供参考。     

中国人群中CYP1A2基因G-2964A和C734A多态性分布

目的:研究中国人群中CYPlA2基因G-2964A和C734A多态性分布.方法:应用聚合酶链反应-限制片长多态性技术对163名启东人和78名长沙人进行基因型分析.结果:等位基因A-2964在启东和长沙人中的发生率分别是0.25和0.22.A734的发生率在启东人中为0.68,而在长沙人中为0.66.一个受试者的两条等位基因中至多含有两个底活性位点.结论:G-2964A和C734A基因多态性在中国人和日本人中的分布没有显著性差异,C734A基因多态性在中国人中的分布也类似于白种人.     

Genetic polymorphisms in promoter and intronic regions of CYP1A2 gene in Roma and Hungarian population samples

The purpose of this study was to determine the interethnic differences of four CYP1A2 drug metabolizing enzyme variants. A total of 404 Roma and 396 Hungarian healthy subjects were genotyped for −163C>A, −729C>T, −2467delT and −3860G>A variants of CYP1A2 by RT-PCR and PCR-RFLP technique. The −3860A and −729T allele were not detectable in Roma samples, while in Hungarian samples were present with 2.02% and 0.25% prevalence, respectively. There was a 1.5-fold difference in presence of homozygous −163AA genotype between Hungarian and Roma samples (49.5% vs. 31.9%, p < 0.001). The −163A allele frequency was 68.6% in Hungarians and 56.9% in Romas (p = 0.025). The −2467delT allele frequency was 6.81% in Roma group and 5.81% in Hungarians. The most frequent allelic constellation was −3860G/−2467T/−729C/−163A in both populations. In conclusion, Hungarians have markedly elevated chance for rapid metabolism of CYP1A2 substrates, intensified procarcinogen activation and increased risk for cancers.     

Impact of CYP2D6, CYP3A5, CYP2C9 and CYP2C19 polymorphisms on tamoxifen pharmacokinetics in Asian breast cancer patients

AIM

To investigate the impact of genetic polymorphisms in CYP2D6, CYP3A5, CYP2C9 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Asian breast cancer patients.

METHODS

A total of 165 Asian breast cancer patients receiving 20 mg tamoxifen daily and 228 healthy Asian subjects (Chinese, Malay and Indian; n = 76 each) were recruited. The steady-state plasma concentrations of tamoxifen and its metabolites were quantified using high-performance liquid chromatography. The CYP2D6 polymorphisms were genotyped using the INFINITI™ CYP450 2D6I assay, while the polymorphisms in CYP3A5, CYP2C9 and CYP2C19 were determined via direct sequencing.

RESULTS

The polymorphisms, CYP2D6*5 and *10, were significantly associated with lower endoxifen and higher N-desmethyltamoxifen (NDM) concentrations. Patients who were *1/*1 carriers exhibited 2.4- to 2.6-fold higher endoxifen concentrations and 1.9- to 2.1-fold lower NDM concentrations than either *10/*10 or *5/*10 carriers (P < 0.001). Similarly, the endoxifen concentrations were found to be 1.8- to 2.6-times higher in *1/*5 or *1/*10 carriers compared with *10/*10 and *5/*10 carriers (P≤ 0.001). Similar relationships were observed between the CYP2D6 polymorphisms and metabolic ratios of tamoxifen and its metabolites. No significant associations were observed with regards to the polymorphisms in CYP3A5, CYP2C9 and CYP2C19.

CONCLUSIONS

The present study in Asian breast cancer patients showed that CYP2D6*5/*10 and *10/*10 genotypes are associated with significantly lower concentrations of the active metabolite of tamoxifen, endoxifen. Identifying such patients before the start of treatment may be useful in optimizing therapy with tamoxifen. The role of CYP3A5, CYP2C9 and CYP2C19 seem to be minor.     

Inducibility of CYP1A2 by omeprazole in vivo related to the genetic polymorphism of CYP1A2

AIMS: To evaluate the effect of the CYP1A2*1C and CYP1A2*1F polymorphisms on the inducibility of CYP1A2 by omeprazole in healthy subjects. METHODS: Mutations of CYP2C19 and CYP1A2 were identified by PCR-RFLP. Omeprazole, 120 mg day-1, was given to 12 extensive metabolizers (EM) with respect to CYP2C19 (six CYP1A2*1F/CYP1A2*1F and six CYP1A2*1C/CYP1A2*1F of CYP1A2) for 7 days. CYP1A2 activity was determined on three occasions, namely on day 1, day 9 and day 16 using the caffeine plasma index (the ratio of the concentrations of paraxanthine to caffeine), 6 h after oral administration of 200 mg caffeine. RESULTS: There was a significant difference (P = 0.002) between the caffeine ratios for CYP1A2*1F/CYP1A2*1F and CYP1A2*1C/CYP1A2*1F genotypes on day 9, but not on day 1 or day 16 (P > 0.05). The changes in the ratios from day 9 to day 1 (48% +/- 20%vs 19% +/- 20%) and from day 9 to day 16 (50% +/- 31%vs 15% +/- 22%) were significantly different (P < 0.05) between the CYP1A2*1F/CYP1A2*1F and CYP1A2*1C/CYP1A2*1F genotypes. CONCLUSION: The CYP1A2*1C and CYP1A2*1F genetic polymorphisms influenced the induction of CYP1A2 activity in vivo by omeprazole.     

The effect of the CYP1A2 *1F mutation on CYP1A2 inducibility in pregnant women

AIMS: To investigate the influence of the CYP1A2*1F mutation on CYP1A2 activity in smoking and nonsmoking pregnant women. METHODS: Pregnant women (n = 904) who served as control subjects in a case-control study of early fetal loss were investigated. They were phenotyped for CYP1A2 using dietary caffeine and the urinary ratio AFMU + 1X + 1 U/1,7 U. An assay for CYP1A2*1F using 5'-nuclease assay (Taqman) was developed to genotype the population. RESULTS: The frequencies of *1 A and *1F alleles among Swedish women were 0.29 and 0.71, respectively. There was no statistically significant difference in CYP1A2 activity between the genotypes, although a trend towards enhanced activity was observed in *1F/*1F (log MRc 0.77) and *1F/*1 A (log MRc 0.82) genotypes compared with the *1 A/*1 A genotype (log MRc 0.71) (anovaP = 0.07). The mean difference between the *1 A homozygotes and the heterozygotes was 0.11 [95% confidence interval of the difference: (-0.21, -0.01)] and that between the *1 A and *1F homozygotes was 0.05 [95% confidence interval of the difference: (-0.13, 0.03)]. No significant effect (P = 0.22) of the *1F on CYP1A2 activity was observed in smokers, tested using an interaction term (smoking * genotype) in the anova model (*1F/*1F log MRc 0.79, *1F/*1 A log MRc 0.86, and *1 A/*1 A log MRc 0.73). In smokers, there was no difference in ratio between homozygotes for the *1 A and *1F alleles [mean difference -0.06; 95% confidence interval of the difference: -0.22, 0.11] or between *1 A/*1 A and *1 A/*1F genotypes [mean difference -0.13; 95% confidence interval of the difference: -0.29, 0.04]. CONCLUSIONS: The effect of the CYP1A2*1F mutation on CYP1A2 activity in smoking pregnant women could not be confirmed.     

CYP3A4,CYP3A5和MDR1基因多态性对环孢素处置的影响

环孢素是一个广泛用于器官移植患者的免疫抑制剂,具有治疗指数窄,不同个体间药代动力学差异较大的特点。它主要通过肝脏和小肠的CYP3A4和CYP3A5代谢;同时它又是药物转运体的底物。不同个体间药物代谢酶和转运体活性的差异可能是造成不同器官移植患者环孢素药代动力学差异的主要原因。而遗传因素即编码药物代谢酶和转运体基因序列的差异可能是其产生活性差异的分子机制。因此,从编码药物代谢酶和转运体的基因入手,可能会为器官移植患者提供最优的治疗方案。     

中药止咳橘红颗粒对CYP3A4和CYP1A2抑制作用的研究

目的：在人体内研究止咳橘红对CYP3A4和CYP1A2的抑制作用，以预测止咳橘红与常用临床药物的相互作用。方法：咪哒唑仑和咖啡因分别作为CYP3A4和A2的探针药物，采取交叉设计，10名受试者在服用3d止咳橘红的前后均服用7.5mg咪哒唑仑和100mg咖啡因，服药后采血测定两者及代谢产物的代谢动力学参数，探讨针药物及代谢物的浓度用HPLC-MS法测定，Cmax,tmax从药时曲线中直接读出，AUC用梯形法计算，Ke用3P87程序进行拟合计算，分析服药前后CYP3A4和CYP1A2被抑制的情况，结果 服用止咳橘红后，咪哒唑仑的代谢受到了轻微的抑制，它的血药浓度，达峰时间和药时曲线下面积都有了升高趋势，但无显著差异。而咖啡因的代谢未受到影响。结论 止咳橘红对CYP3A4的活性有较弱的抑制作用，能够导致CYP3A4底物咪哒唑仑代谢的轻微抑制，而对CYP1A2的活性没有影响。止咳橘红长期使用或超过治疗剂量使用时是否会对CYP3A4产生显著性影响。尚需进一步的研究证明。     

慢性间断性低氧对大鼠肝脏CYP3A_2和CYP2E_1的影响研究

目的研究慢性间断性低氧对大鼠肝脏CYP3A2和CYP2E1的影响。方法将大鼠随机分为对照组、低氧3d组、低氧7d组、低氧14d组、低氧28d组,低氧处理结束24h后,常规腹腔注射麻醉,摘取眼球血液2ml制备血清,并测定丙氨酸氨基转移酶(ALT)、天门冬酸氨基转移酶(AST)、红霉素N-脱甲基酶(ERD)、苯胺羟化酶(ANH)活性;取新鲜肝组织以制备微粒体和提取核糖核酸(RNA),并以RT-PCR进行基因片段扩增以检测大鼠肝脏细胞色素CYP3A2、CYP2E1的mRNA表达水平。结果慢性间断性低氧对血清ALT、AST活性无明显影响;低氧7d后,大鼠肝脏ERD、ANH活性明显升高,28d时诱导率分别为155.5%、42.2%;同时,CYP3A2、CYP2E1mRNA的表达水平也分别增加了220.5%、102.8%。结论慢性间断性低氧能显著增加大鼠肝脏ERD、ANH活性,其机制可能与其在转录水平上提高肝脏CYP3A2和CYP2E1的基因表达水平有关。     

细胞色素P450 2C19、3A5和多药耐药基因多态性对兰索拉唑代谢的影响

目的研究细胞色素P4503A5*3、多药耐药基因C3435T和细胞色素P450 2C19*2突变对兰索拉唑(抗胃酸药)药代动力学的影响。方法24名健康志愿者单次口服兰索拉唑30 mg后,用HPLC方法测定血浆中的兰索拉唑浓度,研究在中国汉族健康人中兰索拉唑的体内过程与基因型的相关性。结果细胞色素P4502C19*1/*1与*2/* 2比较,AUC_(0-∞)存在显著性差异(P=0.04),表达CYP2C19*2/*2的受试者体内兰索拉唑的暴露量是*1/*1者的1.75倍;MDR1 3435C与3435TT比较,t_(max)、AUC_(0-2)存在显著性差异(P=0.026,P=0.03),但总暴露量(AUC_(0-∞))2组间没有差异,表明CYP3A5*3各基因型间药代动力学参数不存在显著性差异。结论CYP2C19*2/*2突变使兰索拉唑体内暴露量增大;MDR1 3435TT基因型者兰索拉唑起始吸收速率较大,达峰快;但该基因型对其总吸收量没有影响,CYP3A5*3对兰索拉唑药代动力学没有影响。     

三氯乙烯对3种细胞色素P450酶基因表达的影响

目的 探讨三氯乙烯（Trichloroethylene,TCE)对人体淋巴细胞瘤细胞株（MCL-5）中3种细胞色素P450酶基因（CYP1A1、CYP2E1、CYP3A4）表达的影响,并研究剂量反应关系和时间反应关系,方法 用常规的细胞培养方法,0.5、1.0、1.5、2.0mmol/L TCE处理细胞12、24、48、72h。利用提纯RNA和cDNA的药盒,合成cDNA,然后逆转录聚合酶反应（RT-PCR）表达3种CYP450基因,以β-Actin作为内对照,分析不同处理剂量和时间时基因表达的强度。结果 在MCL-5细胞株中都有基本的表达,CYP1A1表达在用1.0、1.5、2.0mmol/LTCE处理48h后有被上调的趋势,而且上调趋势随处理时间延长耐加强;CYP2E1、CYP3A4表达不受TCE处理时间长短的影响。3种CYP450酶基因的表达受TCE剂量的影响。3随0.5mol/L,1.0、1.5、2.0mmol/L剂量的增加有上调的趋势,结论 TCE对CYP450酶系统中的CYP2E1、CYP1A1、CYP3A4基因有明显的诱导作用。这些基因被诱导后的结果。可能会导致相对应酶活性的增加,同时加强对TCE的代谢,使TCE的代谢产物增加。     

The involvement of CYP1A2 and CYP3A4 in the metabolism of clozapine

Aims Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. Methods Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used. Results K_m and V_max values determined in human liver microsomes were lower for the demethylation (61±21 μm, 159±42 pmol min⁻¹ mg protein⁻¹ mean±s.d.; n=4), than for the N-oxidation of CLZ (308±1.5 μm, 456±167pmol min⁻¹ mg protein⁻¹; n=3). Formation of DCLZ was inhibited by fluvoxamine (53±28% at 10 μm ), triacetyloleandomycin (33±15% at 10 μm ), and ketoconazole (51±28% at 2 μm ) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34±16% at 10 μm ) and ketoconazole (51±13% at 2 μm ), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. K_m and V_max values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. Conclusions It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.     

膀胱癌患者CYP1A1基因MSPI多态性的相关分析

目的探讨细胞色素CYP1A1基因MSPI多态性与膀胱癌遗传易感性的关系。方法应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)分析技术,检测44例膀胱癌患者(病例组)和85例同期住院非膀胱癌患者(对照组),检测CYP1A1基因MSPI多态性位点的3种基因型及等位基因的分布频率。结果在病例组CYP1A1基因MSPI位点基因型分布频率为:TT(54·5%)、TC(36·4%)、CC(9·1%),等位基因分布频率为T(72·7%)、C(27·3%);在对照组CYP1A1基因MSPI位点基因型的分布频率为TT(61·2%)、TC(31·2%)、CC(7·1%),等位基因分布频率为T(77·1%)、C(22·9%)。各个基因型在两组中所占的比例差异无统计学意义(P>0·05);T、C等位基因频率两组比较差异亦无统计学意义(P>0·05)。结论CYP1A1基因MSPI位点多态性的单独存在可能与本地区膀胱癌易感性无关。     

复方银杏叶胶囊对肝损伤大鼠CYP2E1、CYP3A4的影响

目的研究复方银杏叶胶囊(CGB)对酒精性肝损伤大鼠CYP2E1、CYP3A4活性的影响。方法正常组和酒精性肝损伤模型组均以CGB[(250 mg/(kg.d)]灌胃,分别在灌胃CGB前及灌胃1周后,灌胃探针药氯唑沙宗(50 mg/kg)及氨苯砜(20 mg/kg),于探针药灌后24 h内不同时间点采血,测定各探针药血药浓度。结果灌胃CGB前,模型组氯唑沙宗和氨苯砜的AUC0-24、Cmax均显著低于正常组(P<0.05或P<0.01)。灌胃CGB后,模型组氯唑沙宗和氨苯砜的AUC0-24、Cmax均较灌胃前显著升高(P<0.05或P<0.01);且氯唑沙宗的t1/2灌胃CGB后明显高于灌胃前(P<0.05)。结论 CGB能够明显抑制酒精性肝损伤大鼠CYP2E1、CYP3A4酶活性。