Identification and characterization of a functional TATA box polymorphism of the UDP glucuronosyltransferase 1A7 gene

UDP glucuronosyltransferases (UGT) detoxify bilirubin and therapeutic drugs, a process influenced by single nucleotide polymorphisms (SNPs) in their structural genes and promoter elements. UGT1A1*28 is a functional UGT promoter polymorphism associated with Gilbert's disease and severe irinotecan toxicity, which also occurs in the absence of UGT1A1*28. The aim of this study was to identify and characterize UGT promoter variants relevant for irinotecan detoxification. Recombinant UGT1A proteins were analyzed for irinotecan metabolite glucuronidation by UGT activity assays. In 427 healthy blood donors and 71 homozygous UGT1A1*28 carriers, the 5'-untranslated region of the UGT1A7 gene locus was studied. An SNP was detected by allelic discrimination and characterized by reporter gene experiments. A novel -57 T--> G SNP with a gene frequency of 0.39 in healthy blood donors was identified in the putative TATA box of the UGT1A7 gene, reducing promoter activity to 30%. It is in linkage dysequilibrium with a variant of the UGT1A7 first exon that is present in the reduced-activity UGT1A7*3 and UGT1A7*4 alleles. Homozygous UGT1A1*28 carriers simultaneously carried this variant in 97%. We identified a novel reduced-function TATA box SNP of the UGT1A7 gene that catalyzes irinotecan metabolite detoxification. Its association with variants of the UGT1A1 promoter and UGT1A7 gene may influence irinotecan metabolism. Our finding emphasizes the importance of combinations of structural and regulatory gene polymorphisms that may be useful as markers of drug toxicity.     

抗结核药导致结核病患者肝损害与GSTM1、GSTT1基因多态性的关系

目的研究抗结核药物导致肝损害与GSTM1、GSTT1基因多态性的关系。方法收集结核病患者血标本124例,其中肝损害患者99例、非肝损害者25例。从外周血的白细胞提取DNA,用聚合酶链反应分析GSTM1及GSTT1无效基因型分布频率。结果 GSTM1、GSTT1无效基因分布频率均与肝损害呈显著正相关(P<0.01)。结论 GSTM1、GSTT1基因多态性与抗结核药导致肝损害呈正相关。     

Association study of a novel functional polymorphism of the serotonin transporter gene in bipolar disorder and suicidal behaviour

A serotonin transporter gene linked polymorphic region (5-HTTLPR) has been investigated in several genetic association studies, including studies of bipolar disorder (BD) and suicidality. The current study was designed to examine whether the new long (A/G) variant polymorphism of the 5-HTT gene may be associated with the suicide attempts in 305 families with at least one member having BD. No association with history of suicide attempt was found either in the multiallelic HTTLPR (LRS=0.15, df=2, P=0.92), or with the intron 2 variable number tandem repeat (VNTR) polymorphism (LRS=0.87 df=2 P=0.64). When we performed a haplotype analysis, we found no association between suicide attempt and haplotype distribution (LRS=1.84 df=4 P=0.76). These findings suggest that this new polymorphism in the 5-HTT gene may not influence suicidal behaviour in patients with bipolar disorder.     

Molecular cloning and functional characterization of a novel gene encoding human prostaglandin carrier, hPrC

In the present study, we isolated and determined the pharmacological characteristics of a novel gene encoding the human prostaglandin carrier (hPrC). The isolated cDNA consisted of 1431 base pairs that encoded a 477-amino acid protein, and we found that isolated hPrC does not belong to any drug transporter families. RT-PCR analysis revealed that the hPrC mRNA is expressed in various human tissues ubiquitously. When expressed in Xenopus laevis oocytes, hPrC mediated the transport of [(3)H]prostaglandin E(2) (PGE(2)) in a sodium-independent manner. The uptake of [(3)H] PGE(2) was not trans-stimulated by PG analogous. Although there are several PG transporters such as multidrug resistance-associated protein 4 (MRP4), organic cation transporter 1 (OCT1) [solute carrier (SLC) 22A1], organic anion transporter 1-3 (OAT1-3) [SLC22A6-8], OAT4 [SLC11], OATP-1 (LST-1) [SLCO1B1], OATP2B1 [SLCO2B1], OATP2A1 (PGT) [SLCO2A1], OATP4A1 (OATP-E) [SLCO4A1] have been isolated and well characterized, our findings suggest that hPrC functions as a novel transport peptide responsible for PG uptake. Our results should provide insight into the novel mechanism of the PG transport in the human body.     

镉应答新基因TEF-1δ的生物学功能研究

目的：探讨镉应答新基因TEF－1δ在细胞转化和致癌过程中的重要作用。方法：应用细胞转染、WesterBlot以及细胞转化等技术与方法，对克隆化基因TEF－1δ的生物学功能进行研究。结果：TEF－1δcDNA以pcDNA3.1/V5-His-TOPO为表达载体所转染的CHO细胞和猴肾COS1细胞均可表达相对分子质量为31000的TEF－1δ编码蛋白质，而非转染和单纯载体转染的对照细胞则无此蛋白质表达。 进一步研究表明，TEF－1δcDNA转染NIH3T3细胞可导致TEF－1δ蛋白质超额表达，并与细胞转化密切相关。结论：镉的细胞转化和致癌作用至少部分因TEF－1δ基因的高表达所致。TEF－1δ可能是一个新发现的镉应答原癌基因。     

Effect of genetic polymorphism of GSTM1 and GSTT1 genotypes on cytogenetic biomarkers among coaltar workers

Chromosomal aberrations (CAs) in peripheral blood lymphocytes and micronuclei (MN) in exfoliated buccal cells have been used for decades as cytogenetic biomarkers to investigate genotoxicity among occupationally or environmentally exposed population. In our study, we investigated the association of increased cytogenetic damage with genetic polymorphism in glutathione-S transferase genotypes among occupationally exposed 115 coaltar workers and 105 unexposed controls. We found higher mean value of chromosome aberrations (chromatid type-2.01 ± 1.76; chromosomal type-2.22 ± 1.73) and buccal micronuclei (BMN-7.10 ± 1.56) in exposed subjects when compared to referents (chromatid type-0.82 ± .51; chromosomal type-0.87 ± .54; BMN-5.09 ± 2.88). We observed that individuals having null genotype of GSTM1 and GSTT1 have significantly higher frequency of CAs and MN. Despite of small sample size, our findings suggest a significant association between polymorphism of glutathione-S transferase genotypes and cytogenetic biomarkers which are considered as early effects of genotoxic carcinogens.     

GSTT1 null genotype frequency in a Turkish population

In humans, glutathione-S-transferases (GSTs) are involved in the detoxification of xenobiotics. A deletion polymorphism in the glutathione S-transferase Theta 1 gene (GSTT1 null genotype) is associated with an increased risk of certain forms of cancer. The distribution of this polymorphism in 240 healthy individuals has been examined, using a polymerase chain reaction (PCR)-based method. The GSTT1 null genotype frequency was 20% in a Turkish Population. Received: 18 March 1998 / Accepted: 8 April 1998     

Dependence of Papanicolaou gradings of exfoliated urothelial cells upon GSTM1 and GSTT1 polymorphism in benzidine-exposed workers of the Shanghai dye industry

The distribution of the polymorphic alleles of the genes coding for glutathione S-transferases (GSTs) M1 and T1 was compared with the results of cytological grading of exfoliated urothelial cells (Pap test) in a non-diseased high-risk group of workers formerly exposed to benzidine in the Shanghai dyestuff industry (n = 317). All subjects were genotyped for GSTT1 and M1 gene polymorphism by allele-specific PCR. Individuals were stratified according to their job and duration of exposure. A subgroup of 78 individuals with cytological gradings of grade III or higher in the Pap test showed a significant under-representation of the combination of GSTT1 0/0 and M1 0/0 genotypes compared with 238 subjects with a cytological classification lower than grade III (OR 0.55, 95% CI 0.31-0.98. P=0.04). These results suggest that neither the GSTM1 0/0 or GSTT1 0/ 0 genotype alone nor their combination had a clear association with cytopathological changes in exfoliated urothelial cells from individuals previously exposed to benzidine in Shanghai. This contradicts the results of studies indicating that the GSTM1 0/0 genotype is associated with an increased risk for bladder cancer in the general population, mostly outside China.     

Cloning and characterization of a novel chromosomal drug efflux gene in Staphylococcus aureus

A novel drug efflux gene (named sepA, staphylococcal efflux pump gene) is cloned from antiseptic-resistant mutants of Staphylococcus aureus into Escherichia coli. The sepA gene conferred the reduction of susceptibility to acriflavine and the acceleration of ethidium bromide efflux from the E. coli cells. The sepA (474 bp) encoded the protein that has four predicted transmembrane segments. These results indicate that sepA gene is a multidrug-resistant gene and encodes a drug efflux protein.     

A Bayesian analysis of the influence of GSTT1 polymorphism on the cancer risk estimate for dichloromethane.

The carcinogenicity of dichloromethane (DCM) is related to metabolic activation mediated by glutathione transferase theta 1 (GSTT1), whereas oxidation serves as a detoxification pathway. The aim of this study was to calculate the excess cancer risk from DCM, using Bayesian statistics. In a first step, a previously developed population physiologically based pharmacokinetic (PBPK) model for DCM was simultaneously fitted to extensive human toxicokinetic data from 27 male volunteers exposed to 250-1000 ppm DCM (Astrand et al. Scand. J. Work Environ. Health 1, 78-94, 1975; Engstr?m and Bjurstr?m, Scand. J. Work Environ. Health 7, 215-224, 1977) using Markov chain Monte Carlo simulation. Improved population estimates were obtained for the PBPK model parameters. In a second step, excess cancer risk was calculated for lifelong exposure to 1-1000 ppm DCM by Monte Carlo simulation. Data on GSTT1 gene frequencies in the Swedish population were used, including all three genotypes. Estimated mean and median excess risks were in general agreement with those previously derived (El-Masri et al. Toxicol. Appl. Pharmacol. 158, 221-230, 1999). However, we estimate higher excess risks at the upper confidence limits. Furthermore, our simulations suggest that 1% of the Swedish population is not covered by a factor 4.2-7.1 away from the mean target dose. The majority of the fraction of the population not covered was classified as GSTT1 homozygote. This indicates that a higher uncertainty factor than the commonly used 3.16 should be considered in noncancer risk assessment for substances with polymorphic bioactivation.     

金丝桃素合酶基因的快速克隆及功能鉴定

金丝桃素合酶能催化大黄素生成金丝桃素,根据已发表的金丝桃素合酶的基因序列,设计了6对引物,通过连续重叠PCR快速克隆得到了金丝桃素合酶基因hyp-1。构建含hyp-1基因的原核表达载体pET32ahyp,将该载体导入大肠杆菌进行诱导表达。SDS-PAGE结果表明,hyp-1基因在大肠杆菌中获得表达;Western blot结果表明,重组的Hyp-1蛋白具有特异的免疫活性,表明hyp-1基因在E.coli中得到了表达。酶促反应表明,Hyp-1确实能催化大黄素形成金丝桃素,上述结果表明,本研究克隆的hyp-1基因是金丝桃素合酶基因,具备催化大黄素形成金丝桃素的能力,从而为通过合成生物学技术制备金丝桃素奠定了物质基础。     

中国人GGCX G3261A基因多态性分析

目的 了解γ-谷氨酰羧化酶(γ-glutamylcarboxylase,GGCX)基因多态位点G3261A在中国人群中的分布情况.方法 标准酚-氯仿法提取273份健康人外周血基因组DNA,应用聚合酶链反应/变性高效液相色谱(PCR/DHPLC)技术检测GGCX G3261A位点基因型.结果 273例无关个体中,基因型GG、GA和AA分别有110例、138例和25例,各占40.29%、50.55%和9.16%,G和A等位基因的频率分别为65.57%和34.43%.结论 获得中国人GGCX G3261A等位基因和基因型分布,为GGCX G3261A机应多态性与中国人华法林用量个体差异相关性的研究打下基础.     

Carboxylesterase 1 gene polymorphism and methylphenidate response in ADHD

Methylphenidate (MPH) is the most frequently prescribed drug in the treatment of attention deficit hyperactivity disorder (ADHD). Several pharmacogenetic studies suggested that catecholamine candidate genes influence individual MPH-responses, but these results are mostly contradictory. Genetic analyses of MPH metabolizing carboxylesterase 1 enzyme (CES1) have not been carried out, whereas, meta-analysis of CYP2D6 genetic variants has been already indicated significant pharmacogenetic differences in atomoxetine treatment. Here we present an association analysis of the CES1 Gly143Glu functional polymorphism in a Hungarian ADHD group (n = 173). The genotype frequencies were similar to that of the general population (5.8% vs 4.1% of Gly/Glu heterozygote). Pharmacogenetic analysis was conducted among 122 ADHD children treated with MPH. Neither the categorical analysis comparing 90 responders vs 32 non-responders, nor the dimensional analysis of Inattention and Hyperactivity–Impulsivity score reduction showed a significant main genotype effect. However, analyzing the daily dose, we observed an association with the rare 143Glu-variant: 5 patients in the responder group carrying the Glu-allele required lower doses of MPH for symptom reduction (0.410 ± 0.127 vs 0.572 ± 0.153 mg/kg, t(1,88) = 2.33, p = 0.022). This result warrants for further investigations of the CES1 gene in larger ADHD samples.     

Discovery and functional characterization of a novel small molecule inhibitor of the intracellular phosphatase,SHIP2

Background and purpose:

The lipid phosphatase known as SH2 domain-containing inositol 5′-phosphatase 2 (SHIP2) plays an important role in the regulation of the intracellular insulin signalling pathway. Recent studies have suggested that inhibition of SHIP2 could produce significant benefits in treatment of type 2 diabetes. However, there were no small molecule SHIP2 inhibitors and we, therefore, aimed to identify this type of compound.

Experimental approach:

The phosphatase assay with malachite green was used for high-throughput screening. The pharmacological profiles of suitable compounds were further characterized in phosphatase assays, cellular assays and oral administration in normal and diabetic (db/db) mice.

Key results:

During high-throughput screening, AS1949490 was identified as a potent SHIP2 inhibitor (IC₅₀= 0.62 µM for SHIP2). This compound was also selective for SHIP2 relative to other intracellular phosphatases. In L6 myotubes, AS1949490 increased the phosphorylation of Akt, glucose consumption and glucose uptake. In FAO hepatocytes, AS1949490 suppressed gluconeogenesis. Acute administration of AS1949490 inhibited the expression of gluconeogenic genes in the livers of normal mice. Chronic treatment of diabetic db/db mice with AS1949490 significantly lowered the plasma glucose level and improved glucose intolerance. These in vivo effects were based in part on the activation of intracellular insulin signalling pathways in the liver.

Conclusions and implications:

This is the first report of a small molecule inhibitor of SHIP2. This compound will help to elucidate the physiological functions of SHIP2 and its involvement in various diseases, such as type 2 diabetes.     

宫颈癌中p53基因多态性的检测及其临床意义

目的探讨p53基因多态性在宫颈癌中的表达及其临床意义。方法采用等位基因特异性PCR（allele specific polymorphism chain reaction,ASP）的方法检测p53基因型在46例宫颈癌患者及84例对照者中的表达。采用SPSS14.0软件分析p53基因多态性与宫颈癌临床特征的关系。结果 p53基因多态性分析显示,Pro/pro基因型在宫颈癌和对照组中的表达差异有统计学意义。HPV状态分层分析,HPV阴性者中Pro/pro基因型在宫颈癌和对照组中的表达差异有统计学意义,HPV阳性者则无显著统计学差异。p53基因多态性与患者的年龄、宫颈癌的临床分期、组织类型、细胞分化程度、肿瘤大小、有无淋巴结转移均无显著相关。不同临床分期、组织类型、分化程度、淋巴结转移的宫颈癌中Pro/pro基因型表达差异与HPV状态无显著相关。结论 p53基因72密码子Pro/pro基因型可能是HPV阴性妇女患宫颈癌的高危因素。p53基因多态性与宫颈癌临床病理性特征无相关性。