Structure-function analyses of single nucleotide polymorphisms in human N-acetyltransferase 1

Human N-acetyltransferase 1 (NAT1) alleles are characterized by one or more single nucleotide polymorphisms (SNPs) associated with rapid and slow acetylation phenotypes. NAT1 both activates and deactivates arylamine drugs and carcinogens, and NAT1 polymorphisms are associated with increased frequencies of many cancers and birth defects. The recently resolved human NAT1 crystal structure was used to evaluate SNPs resulting in the protein substitutions R64W, V149I, R187Q, M205V, S214A, D251V, E261K, and I263V. The analysis enhances knowledge of NAT1 structure-function relationships, important for understanding associations of NAT1 SNPs with genetic predisposition to cancer, birth defects, and other diseases.     

Shares in single nucleotide polymorphisms

The announcement of a consortium between the public and private sectors to produce a public single nucleotide polymorphism (SNP) map of the human genome is a unique development which acknowledges the need for research tools to be widely available. The human genome project has been characterised by public-private tensions over information access and ownership. This new initiative demonstrates the scope for collaboration between the interests of the two sectors that are very much in the public interest.     

单核苷酸多态性与阿司匹林抵抗的相关性研究

目的：本研究选取4个基因研究其中7个单核苷酸多态性与阿司匹林抵抗是否存在相关性。方法：2010年10月-2012年3月在本院急诊内科和心内科病房住院患者中入选服用阿司匹林的心血管疾病患者330例,对其COX-1、COX-2及GPIIIa、P2Y1基因的多态性位点通过直接测序法进行基因型检测。结果：29％仰：95）的患者存在阿司匹林抵抗,P2RY,基因的893T〉C变异与阿司匹林抵抗发生率增加有关（OR3．16、95％CI1.36～7．16、P=0．05）,而其余的ITGB3、PTGS1、PTGS2基因与阿司匹林抵抗无相关性。且统计临床资料发现,年龄大、有糖尿病史、HDL水平低的患者阿司匹林抵抗发生率高。结论：P2RY1基因的893T〉C变异与阿司匹林抵抗有关。     

单核苷酸多态性与阿司匹林抵抗的相关性研究

目的:本研究选取4个基因研究其中7个单核苷酸多态性与阿司匹林抵抗是否存在相关性。方法:2010年10月-2012年3月在本院急诊内科和心内科病房住院患者中入选服用阿司匹林的心血管疾病患者330例,对其COX-1、COX-2及GPIIIa、P2Y1基因的多态性位点通过直接测序法进行基因型检测。结果:29%(n=95)的患者存在阿司匹林抵抗,P2RY1基因的893T>C变异与阿司匹林抵抗发生率增加有关(OR3.16、95%CI1.36~7.16、P=0.05),而其余的ITGB3、PTGS1、PTGS2基因与阿司匹林抵抗无相关性。且统计临床资料发现,年龄大、有糖尿病史、HDL水平低的患者阿司匹林抵抗发生率高。结论:P2RY1基因的893T>C变异与阿司匹林抵抗有关。     

Structure/function evaluations of single nucleotide polymorphisms in human N-acetyltransferase 2

Arylamine N-acetyltransferase 2 (NAT2) modifies drug efficacy/toxicity and cancer risk due to its role in bioactivation and detoxification of arylamine and hydrazine drugs and carcinogens. Human NAT2 alleles possess a combination of single nucleotide polymorphisms (SNPs) associated with slow acetylation phenotypes. Clinical and molecular epidemiology studies investigating associations of NAT2 genotype with drug efficacy/toxicity and/or cancer risk are compromised by incomplete and sometimes conflicting information regarding genotype/phenotype relationships. Studies in our laboratory and others have characterized the functional effects of SNPs alone, and in combinations present in alleles or haplotypes. We extrapolate this data generated following recombinant expression in yeast and COS-1 cells to assist in the interpretation of NAT2 structure. Whereas previous structural studies used homology models based on templates of N-acetyltransferase enzyme crystal structures from various prokaryotic species, alignment scores between bacterial and mammalian N-acetyltransferase protein sequences are low (approximately 30%) with important differences between the bacterial and mammalian protein structures. Recently, the crystal structure of human NAT2 was released from the Protein Data Bank under accession number 2PFR. We utilized the NAT2 crystal structure to evaluate the functional effects of SNPs resulting in the protein substitutions R64Q (G191A), R64W (C190T), I114T (T341C), D122N (G364A), L137F (A411T), Q145P (A434C), E167K (G499A), R197Q (C590A), K268R (A803G), K282T (A845C), and G286E (G857A) of NAT2. This analysis advances understanding of NAT2 structure-function relationships, important for interpreting the role of NAT2 genetic polymorphisms in bioactivation and detoxification of arylamine and hydrazine drugs and carcinogens.     

Single nucleotide polymorphisms in human P-glycoprotein: its impact on drug delivery and disposition

Drug efflux pumps belong to a large family of ATP-binding cassette transporter proteins. These pumps bind their substrate and export it through the membrane using energy derived from ATP hydrolysis. P-glycoprotein, the main efflux pump in this family, is expressed not only in tumour cells but also in normal tissues with excretory function (liver, kidney and the intestine). It has a broad specificity of substrates and plays an important role in drug delivery and disposition. Recently, genetic screening of P-glycoprotein has yielded multiple single nucleotide polymorphisms, which seem to alter transporter function and expression. This review discusses the various polymorphisms of this gene and its impact on drug disposition and diseases.     

Role of single nucleotide polymorphisms in pharmacogenomics and their association with human diseases

Global statistical data shed light on an alarming trend that every year thousands of people die due to adverse drug reactions as each individual responds in a different way to the same drug. Pharmacogenomics has come up as a promising field in drug development and clinical medication in the past few decades. It has emerged as a ray of hope in preventing patients from developing potentially fatal complications due to adverse drug reactions. Pharmacogenomics also minimizes the exposure to drugs that are less/non-effective and sometimes even found toxic for patients. It is well reported that drugs elicit different responses in different individuals due to variations in the nucleotide sequences of genes encoding for biologically important molecules (drug-metabolizing enzymes, drug targets and drug transporters). Single nucleotide polymorphisms (SNPs), the most common type of polymorphism found in the human genome is believed to be the main reason behind 90% of all types of genetic variations among the individuals. Therefore, pharmacogenomics may be helpful in answering the question as to how inherited differences in a single gene have a profound effect on the mobilization and biological action of a drug. In the present review, we have discussed clinically relevant examples of SNP in associated diseases that can be utilized as markers for “better management of complex diseases” and attempted to correlate the drug response with genetic variations. Attention is also given towards the therapeutic consequences of inherited differences at the chromosomal level and how associated drug disposition and/or drug targets differ in various diseases as well as among the individuals.     

Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms

N-Acetyltransferase 2 (NAT2) catalyses the activation and/or deactivation of a variety of aromatic amine drugs and carcinogens. Polymorphisms in the N-acetyltransferase 2 (NAT2) gene have been associated with a variety of drug-induced toxicities, as well as cancer in various tissues. Eleven single nucleotide polymorphisms (SNPs) have been identified in the NAT2 coding region, but the specific effects of each of these SNPs on expression of NAT2 protein and N-acetyltransferase enzymatic activity are poorly understood. To investigate the functional consequences of SNPs in the NAT2 coding region, reference NAT2*4 and NAT2 variant alleles possessing one of the 11 SNPs in the NAT2 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Reductions in catalytic activity for the N-acetylation of a sulfonamide drug (sulfamethazine) and an aromatic amine carcinogen (2-aminofluorene) were observed for NAT2 variants possessing G191A (R64Q), T341C (I114T), A434C (E145P), G590A (R197Q), A845C (K282T) or G857A (G286T). Reductions in expression of NAT2 immunoreactive protein were observed for NAT2 variants possessing T341C, A434C or G590A. Reductions in protein stability were noted for NAT2 variants possessing G191A, A845C, G857A or, to some extent, G590A. No significant differences in mRNA expression or transformation efficiency were observed among any of the NAT2 alleles. These results suggest two mechanisms for slow acetylator phenotype(s) and more clearly define the effects of individual SNPs on human NAT2 expression, stability and catalytic activity.     

Prediction and functional analysis of single nucleotide polymorphisms

With the high speed DNA sequencing of genome, databases of genome data continue to grow, and the understanding of genetic variation between individuals grows as well. Single nucleotide polymorphisms (SNPs), the most common type of genetic variation, are an increasingly important resource for understanding the structure and function of the human genome and become a valuable resource for investigating the genetic basis of disease. During the past years, in addition to experimental approaches to characterize specific variants, intense bioinformatics techniques were applied to understand the effects of these genetic changes. In the genetics studies, one intends to understand the molecular basis of disease, and computational methods are becoming increasingly important for SNPs selection, prediction and understanding the downstream effects of genetic variation. The review provides systematic information on the available resources and methods for SNPs discovery and analysis. We also report some new results on DNA sequence-based prediction of SNPs in human cytochrome P450, which serves as an example of computational methods to predict and discover SNPs. Additionally, annotation and prediction of functional SNPs, as well as a comprehensive list of existing tools and online recourses, are reviewed and described.     

Whole-genome genotyping of haplotype tag single nucleotide polymorphisms

The International HapMap Consortium recently completed genotyping over 3.8 million single nucleotide polymorphisms (SNPs) in three major populations, and the results of studying patterns of linkage disequilibrium indicate that characterization of 300,000-500,000 tag SNPs is sufficient to provide good genomic coverage for linkage-disequilibrium-based association studies in many populations. These whole-genome association studies will be used to dissect the genetics of complex diseases and pharmacogenomic drug responses. As such, the development of a cost-effective genotyping platform that can assay hundred of thousands of SNPs across thousands of samples is essential. In this review, we describe the development of a whole-genome genotyping (WGG) assay that enables unconstrained SNP selection and effectively unlimited multiplexing from a single sample preparation. The development of WGG in concert with high-density BeadChips has enabled the creation of three different high-density SNP genotyping BeadChips: the Sentrix Human-1 Genotyping BeadChip containing over 109,000 exon-centric SNPs; the HumanHap300 BeadChip containing over 317,000 tag SNPs, and the HumanHap550 Beadchip containing over 550,000 tag SNPs.     

Three novel single nucleotide polymorphisms of the human thiopurine S-methyltransferase gene in Japanese individuals

In this study, the entire coding sequence and the exon-intron junctions of the thiopurine S-methyltransferase (TPMT) gene from 200 Japanese individuals were screened for mutation. Three novel single nucleotide polymorphisms (SNPs) were identified-106G>A in exon 3 (Gly36Ser, *20 allele), 967A>G in 3'-untranslated region, and -87C>T in intron 8. The allele frequencies were 0.003 for 106G>A, 0.003 for 967A>G, and 0.010 for IVS8 -87C>T. In addition, the three known SNPs, 474T>C (Ile158Ile), 719A>G (Tyr240Cys, *3C allele), and IVS4 +35C>T were detected at frequencies of 0.299, 0.010, and 0.421, respectively.     

apelin-APJ系统基因多态性与大动脉僵硬度相关性研究

目的 探讨福建沿海地区高血压人群apelin及其受体APJ基因的单核苷酸多态性(SNPs)与臂踝脉搏波传导速度(baPWV)反应的大动脉僵硬度相关性.方法 以2011年福建沿海地区的横断面调查为基础,纳入556例高血压患者,其中男性173例,女性383例,年龄(58±11)岁,均进行体格检查、实验室检查,及动脉硬化检测仪测baPWV及采用Taqman@MGB探针法获得apelin基因rs56204867、rs3761581、rs3115757和APJ基因rs9943582、rs7119375共5个SNPs的基因分型.结果 556例高血压患者中,动脉硬化组423例的baPWV值为(1764±296)mm/s,正常组133例的baPWV值为(1266±111) mm/s,认为baPWV≥1400 mm/s为动脉硬化;无论男女,动脉硬化组与正常组间5个SNPs相应的等位基因分布差异无统计学意义(P均＞0.05);调整年龄、协方差分析提示男性apelin基因rs3115757-C突变等位基因携带者baPWV明显快于rs3115757-G野生等位基因携带者[(1536±375) mm/s vs(1 383±304) mm/s,P=0.01)].二元logistic回归分析示,女性高血压人群中携带apelin基因rs56204867-C的动脉硬化风险增加(OR=2.087,95％CI:1.018～4,276,P=0.04).结论 女性高血压人群apelin基因rs56204867与其大动脉弹性下降有关,提示调查地区人群apelin-APJ系统基因多态性与高血压血管损害有关.     

The use of single nucleotide polymorphisms in the isolation of common disease genes

The numerous successes using positional cloning to identify genes mutated in monogenic disorders has galvanised geneticists to start using similar techniques to tackle common complex diseases such as asthma, osteoarthritis, depression and early onset heart disease. The technology is currently at an intermediate stage in which linkage in family studies is being supplemented with locus-specific association studies in populations, enabling accurate localisation of the disease causing or susceptibility gene. These studies are often labour and time intensive unless focus is placed on biological candidate genes. In general, most candidate gene studies for common diseases have been unrewarding. However, single nucleotide polymorphisin (SNP) mapping has accelerated complex disease gene localisation, providing a tool to narrow the linkage region by the detection of multiple SNPs associated with the disease in a relatively small linkage disequilibriuln (LD) region. Identification of susceptibility genes will enable a better understanding of the mechanisms of the disease processes and will facilitate the discovery of new and more efficacious medicines. Whole genome SNP maps will also allow abbreviated SNP profiles to be developed for pharmacogenetic applications, enabling physicians to tailor therapeutic regimens (i.e., identify patients likely to receive therapeutic benefit and not suffer adverse reactions).     

Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes

Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O?-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min?1 · kg?1, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H?1?O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (～5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O?-benzylguanine was within ～80% of the observed total clearance in humans after intravenous administration (15 ml · min?1 · kg?1), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.     

Interethnic and intraethnic variability of NAT2 single nucleotide polymorphisms

Genetic polymorphisms of human arylamine N-acetyltransferase 2 (NAT2) are responsible for interindividual variation in the acetylation of numerous drugs and in the transformation of aromatic and heterocyclic amines into carcinogenic intermediates. Although large interethnic variability in the frequency for NAT2 variant alleles has been reported, comparison of allele frequencies is hampered by differences in the criteria for the assignment of allelic variants. To avoid such sources of bias, in this review we analyze the occurrence of both interethnic and intraethnic variability for the seven commonest single nucleotide polymorphisms (SNP) in the NAT2 gene by using raw SNP data instead of inferred haplotypes. Besides the large interethnic variability observed for all SNPs except C282T, intraethnic variability for NAT2 SNPs was identified for the SNPs G191A among Caucasians (p<0.0001), T341C among Oriental (p<0.001) or African individuals (p<0.012), C481T among Oriental (p<0.001) or African individuals (p<0.001), and G590A among Oriental individuals (p<0.001). In contrast, no major intraethnic differences were identified for the SNPs C282T, A806G or G857A. Intraethnic variability may have relevant clinical implications. For instance, case-control NAT2 studies should not be extrapolated from one Oriental population to another. Nonsynonymous SNPs occur in 32% of alleles in Japanese individuals and in 47% of alleles in Chinese individuals, therefore the frequency of adverse effects and cancer related to slow acetylation is expected to be higher in individuals with Chinese descent than in those with Japanese descent. Intraethnic variability reinforces the need for proper selection of control subjects and points against the use of surrogate control groups for studies involving association of NAT2 alleles with adverse drug effects or spontaneous diseases.     

Potent inhibition of human liver aldehyde oxidase by raloxifene.

The selective estrogen receptor modulator, raloxifene, has been demonstrated as a potent uncompetitive inhibitor of human liver aldehyde oxidase-catalyzed oxidation of phthalazine, vanillin, and nicotine-Delta1'(5')-iminium ion, with K(i) values of 0.87 to 1.4 nM. Inhibition was not time-dependent. Raloxifene has also been shown to be a noncompetitive inhibitor of an aldehyde oxidase-catalyzed reduction reaction of a hydroxamic acid-containing compound, with a K(i) of 51 nM. However, raloxifene had only small effects on xanthine oxidase, an enzyme related to aldehyde oxidase. In addition, several other compounds of the same therapeutic class as raloxifene were examined for their potential to inhibit aldehyde oxidase. However, none were as potent as raloxifene, since IC(50) values were orders of magnitude higher and ranged from 0.29 to 57 micro M. In an examination of analogs of raloxifene, it was shown that the bisphenol structure with a hydrophobic group on the 3-position of the benzthiophene ring system was the most important element that imparts inhibitory potency. The relevance of these data to the mechanistic understanding of aldehyde oxidase catalysis, as well as to the potential for raloxifene to cause drug interactions with agents for which aldehyde oxidase-mediated metabolism is important, such as zaleplon or famciclovir, is discussed.     

单核苷酸多态性与胃癌易感性的相关性研究

目的研究单核苷酸多态性与胃癌易感性的相关性。方法选取2009年5月～2012年5月笔者所在医院收治的242例胃癌患者血样(实验组)与344例来自健康人群的正常血样(对照组)。对两组血样EZH2rs734004、rs734005、rs2072407、rs6464926、rs12670401多态性测序,运用组织芯片技术与免疫组化检测240例同一来源胃癌组织与30例正常胃黏膜EZH2蛋白表达。结果检测发现EZH2中rs12670401基因型CC与rs6464926基因型TT在胃癌患者中所占的比例较高,EZH2与rs2072404比例增加会严重影响到胃癌发生率与风险(P=0.007,OR=1.785,95%CI=1.171～2.724;P=0.012,OR=1.720,95%CI=1.121～2.623)。胃癌患者中的EZH2包含的杂合基因型rs2072407TC、rs734004TC、rs734004CG所占比例低于正常血液对照组,差异有统计学意义(P<0.05)。所占比例减少可降低患者胃癌发生的几率与风险(P=0.032,OR=0.712,95%CI=0.520～0.975;P=0.033,OR=0.708,95%CI=0.517～0.971;P=0.035,OR=0.712,95%CI=0.517～0.977)。应用连锁不平衡分析LD进一步对上述观察进行证实。胃癌不同组织学类型与不同临床参数间的EZH2免疫组化染色强度差异无统计学意义(P>0.05)。结论单核甘酸多态性与胃癌易感性相关。