Pharmacogenomics

Systems literature analysis (SLA) is the literature-driven version of systems biology. It treats collections of scientific literature as a system of millions of interconnections between research parameters, such as genes, diseases, tissues, cell events, model organisms, experiment types, and reagents. SLA aims to replace the traditional keyword-based querying of literature databases, which return sorted lists of papers, with a systems-based approach that returns integrated networks of relationships. Major applications include literature-based discovery of novel targets that link, for example, previously unlinked diseases and phenotypes to common genes and cellular events, and experiment design.

Response to: Chan LF, Zai C, Monda M et al. Role of ethnicity in antipsychotic-induced weight gain and tardive dyskinesia: genes or environment? Pharmacogenomics 14(11), 1273-1281 (2013).

Aim: Allopurinol is widely used as an effective urate-lowering drug and is one of the most frequent causes of cutaneous adverse drug reactions (cADRs). Recently, a strong association of HLA-B*58:01 with allopurinol-induced severe cADRs was identified. This study investigated the predisposition to different types of allopurinol-cADRs conferred by HLA-B*5801 in a Han population from mainland China. Patients & methods: HLA-B genotyping was performed on 38 Chinese patients with different types of allopurinol-cADRs from 2008 to 2011. Results: All the allopurinol-cADR patients carried HLA-B*58:01, in contrast with only 11.11% (7/63) in the allopurinol-tolerant patients (odds ratio [OR] = 580.07; p < 0.0001) and 13.99% (80/572) in a Han Chinese population from the human MHC database (dbMHC; OR: 471.09; p < 0.0001) carried the genotype. Each type of allopurinol cADRs revealed a statistically significant association with HLA-B*58:01. In particular, the risk of allopurinol-induced maculopapular eruption was significantly higher in patients with HLA-B*58:01 (OR: 339.00; p < 0.0001). Conclusion: The strong association of both the mild and severe types of allopurinol cADRs with the HLA-B*58:01 allele were observed. The results indicated that the prospective use of a genetic test of HLA-B*58:01 might reduce the prevalence of allopurinol-induced cADRs. Original submitted 7 March 2012; Revision submitted 21 May 2012.

A letter in response to: Sanchez-Giron F, Villegas-Torres B, Jaramillo-Villafuerte K et al. Association of the genetic marker for abacavir hypersensitivity HLA-B*5701 with HCP5 rs2395029 in Mexican Mestizos. Pharmacogenomics 12(6), 809-814 (2011).

International HIV treatment guidelines recommend HLA-B*57:01 typing before abacavir administration, in order to reduce the incidence of abacavir hypersensitivity reactions, the major cause of early therapy discontinuation. A fast, sensitive and specific test for HLA-B*57:01 detection has been developed in the present study. Two sets of sequence-specific primers were designed, and amplification rapidly detected by real-time PCR. A total of 108 samples were analyzed in a single-blind fashion, and 41 samples were identified as positive. Complete agreement, with κ = 1 (standard error = 0.0962, p < 0.0001), was found, with a validated methodology used in the EPI109367 clinical trial funded by GlaxoSmithKline, and consisting of low-resolution sequence-specific oligonucleotide PCR, followed by high-resolution sequence-specific oligonucleotide PCR carried out on the HLA-B*57-positive samples. We provided a detailed characterization of a novel HLA-B*57:01 screening test, which can be easily implemented by those laboratories already involved in the detection of viral load and virus genotyping. Original submitted 26 October 2010; Revision submitted 13 December 2010.

Aim: HLA-B*57:01 status needs to be determined before initiating abacavir therapy. We developed a pharmacogenetic real-time (Q)-PCR screening test using two sets of sequence specific primers. This test has been implemented into routine clinical practice. Materials & methods: HIV-infected patients admitted at our University Hospital were thus genotyped using the above mentioned test. A panel of 80 DNA samples with a known genotype were used to characterize Q-PCR conditions using different master mixes. Results: A total of 353 patients were genotyped, detecting 15 (4.25%) HLA-B*57:01 positive carriers. Among the negative patients, 17.2% were treated with abacavir without any hypersensitivity reaction. Using different Q-PCR master mixes, significantly lower cutoff Ct values were found, thus new analytical settings are provided. Conclusion: The pharmacogenetic test developed in our laboratory for the fast screening of HLA-B*57:01 can be successfully implemented into routine clinical practice. All 16 sequences (including an additional six) currently known for the HLA-B*57:01 allele are detected by sequence specific primers used in this test. The Brilliant II SYBR(®) Green QPCR MM (Stratagene) can safely replace the master mix originally used to develop the test.

To examine the safety profile of oseltamivir in children and evaluate the impact of P-glycoprotein polymorphisms on the incidence of neuropsychiatric adverse events (NPAE) in oseltamivir-treated children. This prospective cohort study was conducted in our tertiary care pediatric hospital (University Hospitals of Geneva, Switzerland) during the H1N1 pandemia, between 1 October 2009 and 31 January 2010. All newborn to 18 year-old patients presenting at the emergency department with a flu-like illness were eligible for inclusion. Adverse events were systematically recorded by pediatricians and/or by parents at home using a diary card, with a 30-day follow-up period. The causality assessment of oseltamivir in NPAE was performed by two clinical pharmacologists. After informed consent, enrolled patients were also genotyped for ABCB1 3435C>T (rs1045642) and 2677G>T/A (rs2032582) polymorphisms. Among the 42 H1N1-infected, oseltamivir-treated children who were genotyped for ABCB1 3435C>T and 2677G>T/A variants, 36% presented NPAE. When examining the association between the diplotype and the development of NPAE, we observed that the frequency of NPAE displayed a 'genotype-trend effect' with the variant and the wild-type subgroups at the two far ends. A total of 11% of the 2677GG-3435CC individuals (wild-type homozygous) presented NPAE, compared with 39% of the individuals being heterozygous for at least one variant allele and 67% of the 2677TT-3435TT individuals (homozygous variants) (p = 0.149, nonsignificant). These observations suggest a potential influence of ABCB1 polymorphisms in oseltamivir-related NPAE, maybe as a result of an enhanced permeability of the blood-brain barrier to oseltamivir

Revolutionary new technologies, capable of transforming the economics of sequencing, are providing an unparalleled opportunity to analyze human genetic variation comprehensively at the whole-genome level within a realistic timeframe and at affordable costs. Current estimates suggest that it would cost somewhere in the region of 30 million US dollars to sequence an entire human genome using Sanger-based sequencing, and on one machine it would take about 60 years. Solexa is widely regarded as a company with the necessary disruptive technology to be the first to achieve the ultimate goal of the so-called 1,000 dollars human genome - the conceptual cost-point needed for routine analysis of individual genomes. Solexa's technology is based on completely novel sequencing chemistry capable of sequencing billions of individual DNA molecules simultaneously, a base at a time, to enable highly accurate, low cost analysis of an entire human genome in a single experiment. When applied over a large enough genomic region, these new approaches to resequencing will enable the simultaneous detection and typing of known, as well as unknown, polymorphisms, and will also offer information about patterns of linkage disequilibrium in the population being studied. Technological progress, leading to the advent of single-molecule-based approaches, is beginning to dramatically drive down costs and increase throughput to unprecedented levels, each being several orders of magnitude better than that which is currently available. A new sequencing paradigm based on single molecules will be faster, cheaper and more sensitive, and will permit routine analysis at the whole-genome level.

The 1000 Genomes Project aims to provide detailed genetic variation data on over 1000 genomes from worldwide populations using the next-generation sequencing technologies. Some of the samples utilized for the 1000 Genomes Project are the International HapMap samples that are composed of lymphoblastoid cell lines derived from individuals of different world populations. These same samples have been used in pharmacogenomic discovery and validation. For example, a cell-based, genome-wide approach using the HapMap samples has been used to identify pharmacogenomic loci associated with chemotherapeutic-induced cytotoxicity with the goal to identify genetic markers for clinical evaluation. Although the coverage of the current HapMap data is generally high, the detailed map of human genetic variation promised by the 1000 Genomes Project will allow a more in-depth analysis of the contribution of genetic variation to drug response. Future studies utilizing this new resource may greatly enhance our understanding of the genetic basis of drug response and other complex traits (e.g., gene expression), therefore, help advance personalized medicine.

Hepatocellular carcinoma (HCC) is one of the most frequent solid tumors worldwide and represents the third cause of mortality among deaths from cancer. It has been extensively studied in terms of genetic alteration in the last 10 years and our knowledge has dramatically increased in this field, leading to the definition of different altered pathways in hepatocarcinogenesis. Recently, a comprehensive study of genetic and transcriptomic alterations in a large series of HCC tumors enabled the identification of a six-group molecular-based classification of HCC, defined by a simple 16-gene signature. This classification is closely related to specific alteration of WNT and AKT oncogenic pathways. Together with the analysis of defined oncogenic proteins, such global classifications could be useful in the prediction of future-targeted therapy efficiency.

Clozapine is still considered the gold standard for treatment-resistant schizophrenia patients; however, up to 40% of patients do not respond adequately. Identifying potential predictors of clinical response to this last-line antipsychotic could represent an important goal for treatment. Among these, functional polymorphisms involved in dopamine system modulation, known to be disrupted in schizophrenia, may play a role. We examined the COMT Val158Met polymorphism, which plays a key role in dopamine regulation at the prefrontal level, and the 5-HT1A-R -1019 C/G polymorphism, a target of clozapine activity involved in the interaction between the serotonin and dopamine systems. 107 neuroleptic-refractory, biologically unrelated Italian patients (70 males and 37 females) with a DSM-IV diagnosis of schizophrenia who were being treated with clozapine were recruited. Psychopathology was assessed by the Positive and Negative Symptoms Scale (PANSS) at the beginning of treatment, and at weeks 8 and 12. Genomic DNA was extracted from venous blood samples. COMT rs4680 (Val158Met) and 5-HT1A-R rs6295 (-1019 C/G) polymorphisms were analyzed by PCR-based restriction fragment length and direct sequencing, respectively. We found a significant effect of COMT and 5-HT1A-R on the PANSS Negative Subscale variation, with greater improvement among COMT Val/Val and 5-HT1A-R G/G subjects. The findings support the hypothesis that COMT rs4680 and 5-HT1A-R rs6295 polymorphisms could influence the negative symptom response to clozapine, probably through modulation of the dopaminergic system. Original submitted 26 February 2014; Revision submitted 15 October 2014.

Aim: To evaluate whether polymorphisms at -857, -1031, -308 and -238 positions of the TNF-α gene influence response to TNF-α-blocker therapy in Chinese Han patients with ankylosing spondylitis. Patients & methods: A total of 106 patients with ankylosing spondylitis were recruited and genotyped for -857, -1031, -308 and -238 TNF-α gene polymorphisms. In total, 32 received infliximab and 74 received a recombinant human TNF-α receptor II-IgG Fc fusion protein (rhTNFR-Fc). At the end of 12 weeks, patients were assessed using the Assessment of SpondyloArthritis International Society (ASAS) 20, 40, 50 and 70 criteria. Results: Polymorphisms at -308 and -238 did not affect therapeutic response. The -857 C/C genotype (p = 0.0021) responded better to therapy. The -1031 T/T genotype (p = 0.0004) showed better outcome. Conclusion: In Chinese Han ankylosing spondylitis patients, polymorphisms at the -308 and -238 positions of the TNF-α gene are unable to predict TNF-α-blocker response; however, -857 C/C and -1031 T/T genotypes have the ability to predict good response.

The identification of predictive markers of response to chemoradiotherapy treatment remains a promising approach for patient management in order to obtain the best response with minor side effects. Initially, we investigated whether the analysis of several markers previously studied and others not yet evaluated could predict response to 5-fluorouracil- and capecitabine-based neoadjuvant treatment in locally advanced rectal cancer. We studied germline and tumoral samples of 65 stage II/III rectal patients. A panel of pharmacogenetic markers was genotyped in paired peripheral blood samples and rectal cancer tumors. Our results seem to confirm the previously described association of thymidylate synthase and the prediction of chemoradiotherapy response in rectal cancer. However, it failed to confirm the clinical utility proposed for XRCC1, ERCC1, ERCC2, MTHFR and EGFR polymorphisms in blood/germline samples. Subsequently, with the aim of improving prediction of individual response and assessing the role of studied polymorphisms in response to treatment, we determined if changes in tumor response to these markers could predict clinical outcome. We found a high degree of changes between germline and tumor samples, mainly somatic mutations without microsatellite instability, and a minor frequency of loss-of-heterozygosity events. In tumoral samples, XRCC1 appeared to be significantly associated (p = 0.006) with downstaging of the tumor (odds ratio: 7.93; 95% CI: 1.03-60.83), but the increasing of TYMS low-expression alleles contradict the previous results observed in germline samples. The detection of somatic mutations in rectal cancer tumors led us to re-evaluate the utility of the tests performed in blood samples for these polymorphisms in rectal cancer. Furthermore, studies aimed at assessing the influence of pharmacogenetic markers in treatment response performed in blood samples should take into account the particular pattern of hypermutability present in each tumor type. We hypothesize that different patterns of hypermutability present in each tumor type would be related to the different results in association studies related to response to the treatment.

Recent developments in a number of molecular profiling technologies, including genomic/genetic testing, proteomic profiling and metabolomic analysis have allowed the development of 'personalized medicine'. Irinotecan is one of the models for personalized medicine based on pharmacogenetics, and a number of clinical studies have revealed significant associations between UGT1A1*28 and irinotecan toxicity. Based on this cumulative evidence, the US FDA and pharmaceutical companies revised the irinotecan label in June 2005. However, a recommended strategy for irinotecan-dose adjustments based on individual genetic factors has not yet been fully established. This article provides an overview of recent progress in irinotecan pharmacogenetics and discusses the clinical significance of the UGT1A1 genotype/haplotype with regard to severe irinotecan toxicity.

CYP2D6 is genotyped clinically for prediction of response to tamoxifen, psychotropic drugs and other medications. Phenotype prediction is dependent upon accurate genotyping. The CYP Allele Nomenclature Committee maintains the allelic nomenclature for CYP2D6; however, in some cases, the list of polymorphisms associated with a given allele is incomplete. Clinical laboratories and in vitro diagnostic manufacturers rely upon this nomenclature, in addition to the literature, to infer allelic function and haplotypes and when they design CYP2D6-testing platforms. This article provides more complete sequencing data for the CYP2D6*11 allele and describes the difficulties encountered in genotyping CYP2D6 when incomplete data are available. The CYP Allele Nomenclature Committee should provide clear information about the completeness of the original data used to define each allele.

Polymorphisms in the 5' regulatory region of the thymidylate synthase gene (TYMS) have been shown to modulate thymidylate synthase expression and are associated with resistance to fluoropyrimidine-based therapies. These polymorphisms include a two repeat (2R) or three repeat (3R) of a 28-bp sequence and a G>C SNP in the second repeat of the 3R allele (TSER*3 G>C). Genotyping methods for the TYMS 5'-UTR polymorphisms have typically involved visualizing PCR and RFLP products on agarose gels. This article describes the use of a robust capillary electrophoresis assay for TYMS 5'-UTR genotyping. As part of pharmacogenetic studies, we performed TYMS genotyping for the 5'-UTR polymorphisms in 314 colorectal cancer patients. A gel-based capillary electrophoresis method, employing a high-resolution gel cartridge on a QIAxcel(®) system, was developed to detect PCR products and RFLP fragment sizes. The high resolution of the capillary electrophoresis technique allowed identification of a 6-bp insertion in the second repeat of the 3R allele in three patients. The frequency of the insertion allele was 0.4% in Caucasians and 1.3% in African-Americans. We also found 3.3% of Caucasian patients were heterozygous for a G>C SNP in the first repeat of the 2R allele, but this allele was not observed in the African-American patients. We describe a robust RFLP genotyping technique that employs size discrimination by capillary electrophoresis to genotype the TYMS TSER*3 G>C SNP. The technique also allows identification of a 6-bp insertion in the 3R allele, and we report the allelic frequencies for two uncommon TSER alleles.

A recent study found that variation in camptothecin pharmacodynamic genes (TOP1, PARP1, TDP1 and XRCC1) correlated with efficacy and risk of neutropenia in irinotecan-treated cancer patients (median dose: 180 mg/m2), which suggests that these genes might predict outcomes to irinotecan-based therapies. The present study was conducted to evaluate previous gene associations using an independent sample of patients receiving irinotecan. DNA was isolated from 85 advanced cancer patients treated with 300 or 350 mg/m2 irinotecan and genotyped for haplotype-tag polymorphisms across TOP1, PARP1, TDP1 and XRCC1. Associations between genotypes and haplotypes and log(absolute neutrophil count nadirs) were assessed by linear regression. No associations were observed. Our findings suggest that the genes we tested do not influence toxicity of irinotecan when adminstered at 300-350 mg/m2.

Aim: Drug-drug interactions (DDIs) are a widely recognized major cause of adverse drug reactions, but two other newly described important types of interactions also exist: drug-gene interactions (DGIs) and drug-drug-gene interactions (DDGIs). A drug-gene interaction occurs when a patient's genetic CYP450 type (e.g., CYP2D6 poor metabolizer) affects that patient's ability to clear a drug. A drug-drug-gene interaction occurs when the patient's CYP450 genotype and another drug in the patient's regimen (e.g., a CYP2D6 inhibitor) affect that individual's ability to clear a drug. Their prevalence has not been previously described. This pilot study investigates the frequency of DDIs, DGIs and DDGIs in a sample of CYP450 tested individuals. Materials & methods: The investigators conducted a retrospective analysis of 1143 individuals with known CYP2D6, CYP2C19 and CYP2C9 genotypes. Using the individuals' medication lists and YouScript(®), a software tool to analyze cumulative DDIs and DGIs, the prevalence of DDI, DGI and DDGIs was analyzed. Results: A total of 1053 potential major or substantial interactions were identified in 501 individuals. DDIs accounted for 66.1% of the total interactions. The remaining 33.9% of interactions were DGIs (14.7%) and DDGIs (19.2%). When compared with DDIs alone, DGIs and DDGIs increased the total number of potentially clinically significant interactions by 51.3%. Conclusion: In the future, identifying DGIs and DDGIs may lead to a more comprehensive method of identifying individuals who are at risk for adverse drug reactions.

For decades it has been well known that elevated levels of homocysteine are harmful to humans on the basis of clinical observations derived from classical model diseases, such as inherited metabolic disorders. This group of diseases includes classical homocystinuria and several other inherited diseases affecting the so-called 'transsulfuration pathways'. Homocysteine lies in a metabolic checkpoint that interconnects one-carbon-transferring reactions with metabolism of sulfur-containing amino acids, since every molecule of 5-methyltetrahydrofolate derived either from plasma or generated from other folate species must be demethylated to liberate the reduced tetrahydrofolate. This unidirectional mechanism operates in every cell and has no alternative in eukaryotic cells. Antifolates are a group of anticancer agents targeting various metabolic steps within folate metabolism. They exert an indirect influence on the rate of appearance/disappearance of homocysteine from cellular and plasma compartments. Recently, it has been postulated that homocysteine may be a marker of the 'pharmacodynamic effect' of methotrexate, but studies attesting to this role are only now emerging. Here, we explore the genetic disease of folate and homocysteine metabolism and discuss the links between these model disorders with pharmacology and pharmacogenetics of folate antagonists used in the clinic. We outline possible ways of how homocysteine may be used as a biomarker of antifolate therapy.

The human µ-opioid receptor variant 118 A>G (rs1799971) has become one of the most analyzed genetic variants in the pain field. At the molecular level, the variant reduces opioid receptor signaling efficiency and expression, the latter probably via a genetic-epigenetic interaction. In experimental settings, the variant was reproducibly associated with decreased effects of exogenous opioids. However, this translates into very small clinical effects (meta-analysis of 14 studies: Cohen's d = 0.096; p = 0.008), consisting of slightly higher opioid dosing requirements in peri- and post-operative settings. An effect can neither be maintained for chronic analgesic therapy nor for opioid side effects. It seems unlikely that further studies will reveal larger effect sizes and, therefore, further analyses appear unwarranted. Thus, due to its small effect size, the SNP is without major clinical relevance as a solitary variant, but should be regarded as a part of complex genotypes underlying pain and analgesia.

The recent introduction of new microarray expression technologies and the further development of established platforms ensure that the researcher is presented with a range of options for performing an experiment. Whilst this has opened up the possibilities for future applications, such as exon-specific arrays, increased sample throughput and 'chromatin immunoprecipitation (ChIP) on chip' experiments, the initial decision processes and experiment planning are made more difficult. This review will give an overview of the various technologies that are available to perform a microarray expression experiment, from the initial planning stages through to the final data analysis. Both practical aspects and data analysis options will be considered. The relative advantages and disadvantages will be discussed with insights provided for future directions of the technology.

Schizophrenia is a severe disorder that significantly affects the quality of life and total functioning of patients and their caregivers. Clozapine is the first atypical antipsychotic with fewer adverse effects and established efficacy. As a rule of thumb, risperidone is one of the most reliable and effective antipsychotics for newly diagnosed and chronic schizophrenics. Pharmacogenetic studies have identified genomic variants of candidate genes that seem to be important in the way a patient responds to treatment. The recent progress made in pharmacogenomics will improve the quality of treatment, since drug doses will be tailored to the special needs of each patient. In this article, we review the available literature attempting to delineate the role of genomic variations in clozapine and risperidone response in schizophrenic patients of various ethnicities. We conclude that pharmacogenomics for these two drugs is still not ready for implementation in the clinic.

Manifestation of tardive dyskinesia (TD) among schizophrenia subjects on long-term antipsychotic treatment with typical drugs has been a clinical concern. Despite its association with extrapyramidal symptoms, typical drugs are still routinely prescribed globally though marginally superior atypical drugs have long been available. The genetic component in the etiology of TD is well documented. Search for these determinants has led to a few consensus associations of CYP2D6 *10, CYP1A2*1F, DRD2 Taq1A (rs1800497), DRD3 Ser9Gly (rs6280) and MnSOD Ala9Val (rs4880) variants with TD. However, translation of these observations into the clinic has not been achieved so far. This review discusses the salient features of TD etiopathology, current status of TD genetics, interactions between genetic and nongenetic factors, some major drawbacks, challenges and expected focus in TD research over the next decade, with emphasis on pharmacogenetics.

To investigate the -1291 C/G promoter polymorphism (rs1800544) of the adrenergic alpha-2A receptor (ADRA2A) with clozapine-/olanzapine-induced weight gain in European-Americans and African-Americans. The alpha-adrenergic receptors inhibit lipolysis in the adipose tissue and are involved in weight gain regulation. Moreover, two previous studies indicated an association with antipsychotic-induced weight gain with the same polymorphism in Asian populations. We analyzed a relatively large (n=129) and well-characterized group of patients and monitored them for a period of 6-14 weeks. Our refined sample consisted of 60 European-Americans and 39 African-Americans on clozapine or olanzapine, prospectively. In European-Americans, we observed a significant difference in weight gain across the genotypic categories (p=0.046). The carriers of the C allele gained more weight compared with the subjects homozygous for the GG allele (CC + CG vs GG; 3.73 +/- 4.13 kg vs 0.23 +/- 2.92 kg; p=0.013). We did not find a significant association in African-Americans, although the sample size was probably too small. Our observations suggest a possible role of ADRA2A polymorphisms in clozapine-/olanzpaine-induced weight gain in subjects of European descent.

Methotrexate (MTX), widely used in the treatment of rheumatoid arthritis (RA), inhibits dihydrofolate reductase and folate-dependent enzymes. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and has been shown to be polymorphic, affecting the enzyme activity. To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and MTX efficacy in the treatment of RA, a total of 174 RA patients, treated with MTX plus methylprednisone 4 mg and folic acid 5 mg were analyzed. In univariate regression analysis model, the MTHFR 677T allele was associated with significantly higher frequency of remission, whereas in the case of the 1298C allele, a tendency for higher remission rate was observed. In multivariate regression analysis, the presence of both 677T and 1298C alleles was associated with an increased frequency of remission. The results of our study suggest that the MTHFR 677T and 1298C alleles may be associated with an increased rate of RA remission in patients treated with MTX receiving high doses of folic acid supplementation.

To investigate intra- and inter-ethnic differences in three widespread (E158K, V257M and E308G) and two African-specific (D132H and L360P) flavin-containing monooxygenase 3 (FMO3) polymorphisms. Allele frequencies were determined by TaqMan allelic discrimination assay in 2152 healthy volunteers from Europe (Swedes, Italians and Turks), East Asia (Japanese) and sub-Saharan Africa (nine ethnic groups covering eastern, southern and western regions), followed by haplotype and linkage analysis. Significant subpopulation differences (p < 0.001) in allele frequencies were found for E158K, V257M and E308G in Europeans and regional differences (p < 0.01) for D132H among Africans. No carrier of P360 was identified. Cis-linkage between G308 and K158 was confirmed with the compound variant (K158/G308) being found in a high proportion (12.0-38.3%) of non-African subjects, but rarely (1.3%) among Africans. Distribution of functionally relevant FMO3 polymorphisms varies not only between ethnicities but also within. The K158/G308 variant may have potential clinical importance primarily in non-African populations due to its low prevalence in Africa.

Nonpharmacogenetic factors may play a key role in the success of oral endocrine therapy for breast cancer. Adherence, defined as following medical advice as well as persistence and duration of use for patients prescribed endocrine therapy, may impact significantly on recurrence and survival. Side effects from tamoxifen or aromatase inhibitors may lead to patients stopping or switching therapy, while comorbidities, consequent coprescribing and patient perceptions may also influence outcomes. Interventions to improve adherence and persistence are required and could have as great an effect on survival as applying pharmacogenetic principles to the endocrine management of breast cancer.

The confluence of biotechnology, computer sciences and the completion of genome sequencing efforts for sev-eral organisms have led to revolution-ary changes in biomedical research. Genomics and informatics are playing increasingly important roles as discov-ery tools in the basic biological sci-ences and as diagnostic and rational therapeutic aids in the clinical arena. In recent years, the synthesis or fabri-cation of high-density arrays of speci-fied DNA sequences comprising all known genes of an organism on a sin-gle glass slide or 'chip' have gained popularity. Labeled RNA or DNA tar-gets (such as messenger RNAs (mRNAs) obtained from cells, tissues or organisms under different condi-tions) can be analyzed by hybridiza-tion on DNA [1-4]. This technology allows several types of questions to be asked on a qualitatively different scale than has been previously possible and will have a dramatic impact on the pace of discovery in the field of molec-ular medicine and drug discovery. The drug discovery process seeks to develop a biological or chemical entity that, when administered to a patient, will improve the disease symptomatol-ogy or actually treat the underlying pathophysiological basis of the particu-lar disease state. A target is the biologi-cal entity, usually a gene, mRNA or protein, with which a pharmaceutical is designed to interact. The completion of the human genome sequence has defined the genomic relationships between drug targets. In addition, sequencing of pathogenic lower organ-isms has uncovered a host of novel microbial targets. Gene expression profiling has been a mainstay molecular biological approach for many years. The availability of com-plete genome sequence information along with improved technology has created a situation where these tech-niques can be applied in a much broader manner, thereby facilitating profiling of large sections of the tran-scriptome. The University of California San Diego Extension, Bioscience Microarray Technologies – An Over-view meeting, March 13–15, 2002 pro-vided an important forum for reviewing advances and future trends in this field (see Box 1).

The OPRM1 gene encodes the µ-opioid receptor, which is the primary site of action of most opioids. Several studies and three meta-analyses have examined a possible link between the exonic OPRM1 A118G (rs1799971) polymorphism and opioid dependence; however, results have been inconclusive. Therefore, a systematic review and meta-analysis have been carried out to examine whether this polymorphism is associated with opioid dependence. Thirteen studies (n = 9385), comprising 4601 opioid dependents and 4784 controls, which evaluated association of the OPRM1 rs1799971 polymorphism with susceptibility to opioids, were included in this study. Our meta-analysis showed significant association between this polymorphism and susceptibility to opioid dependence in overall studies under a codominant model, as well as susceptibility to opioid dependence or heroin dependence in Asians under an autosomal dominant model. The nonsynonymous OPRM1 rs1799971 might be a risk factor for addiction to opioids or heroin in an Asian population.

Aim: We examined whether HLA-DRB1*1501 and four VDR SNPs influence the macrophage response to infection with Mycobacterium tuberculosis (Mtb) via innate immune versus drug treatment or drug delivery mechanisms. Materials & methods: Monocyte-derived macrophages from 24 healthy donors were infected with Mtb in vitro. Survival of intracellular bacilli and secretion of cytokines and nitric oxide by the infected cells were monitored with and without exposure to isoniazid and rifabutin. Results: Haplotype analysis was conducted, and an arbitrary score of genetic 'susceptibility' (S ) score ranging from -3 to +3 was assigned to donors based on the presence or absence of genetic markers. S scores correlated more strongly with Mtb survival (r = 0.68) than TNF and nitric oxide (NO; r = ∼0.01-0.11). A specific haplotype was significantly associated with decreased Mtb survival (p < 0.05), increased NO and decreased IL-10/IL-4. Macrophages with S scores ≥ 2 secreted significantly (p < 0.05) more IL-10 and IL-4, and less NO upon infection, and supported Mtb survival. Microparticulate drugs showed higher bactericidal activity than free drugs, irrespective of S score. Conclusion: S score predicts colonization of macrophages by Mtb, as does haplotype analysis. Drug-containing microparticles are superior to free drugs across diverse genetic backgrounds.

Recently, the USA FDA has made a labeling change to the drug information contained in carbamazepine. Owing to recent data implicating the HLA allele B*1502 as a marker for carbamazepine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis in Han Chinese, the FDA recommends genotyping all Asians for the allele. This allele is seen in high frequency in many Asian populations other than Han Chinese, but there are few data on whether the allele is a marker for this severe outcome in anyone other than Han Chinese. In fact, the association has not been found in Caucasian patients. We review the data that prompted this recommendation, list data for other ethnic groups, both Asian and non-Asian, and briefly discuss the implication of this recommendation for clinical practice.

The CYP2D6 -1584C>G (rs1080985) polymorphism has been identified as another major factor for CYP2D6 function that is possibly associated with ultrarapid metabolism. The mutant -1584G promoter genotype seems to be consistently related to a higher protein expression than -1584C. However, the impact this SNP in the CYP2D6 promoter region has on plasma levels of patients taking CYP2D6 substrates, such as thioridazine, has not been studied. Previously, we showed the validity of the mesoridazine:thioridazine ratio to assess CYP2D6 activity in clinical settings. Therefore, the aim of this study was to analyze the relationship between the presence of the CYP2D6 -1584C>G polymorphism and the plasma concentrations of thioridazine and its metabolites in a previously studied population of patients in order to evaluate the implications for CYP2D6 hydroxylation capacity. The CYP2D6 -1584C>G polymorphism was determined by using a PCR-RFLP method in 61 Caucasian psychiatric patients receiving thioridazine monotherapy. Among patients with two active CYP2D6 genes, there were significant differences in the thioridazine:mesoridazine plasma concentrations ratio (p < 0.05) among the three CYP2D6 -1584C>G genotype groups. Moreover, in this group of patients the thioridazine:mesoridazine ratio was lower (p < 0.05) in carriers of CYP2D6 -1584G allele than in patients homozygous for CYP2D6 -1584C allele. However, no differences in thioridazine or its metabolite concentrations between homozygous CYP2D6 -1584C allele carriers and carriers of the -1584G allele were found. According to the present results the concentration ratio of thioridazine to mesoridazine was related to the CYP2D6 -1584C>G polymorphism. It is likely that individuals who carry CYP2D6 -1584G versus homozygotes for the -1584C allele may present an increased CYP2D6 activity.

Background & aim: The CYP2D6 -1584C>G polymorphism (rs1080985) has been identified as a major factor for CYP2D6 expression and function, with the mutant -1584G promoter type being consistently associated with significantly greater expression than -1584C. It may therefore be associated with ultrarapid metabolism. The objective of the present study was to explore the relationship between the CYP2D6 -1584C>G polymorphism and the debrisoquine metabolic ratio in healthy volunteers in order to evaluate its potential impact on the ultrarapid CYP2D6 hydroxylation capacity. Materials & methods: The CYP2D6 -1584C>G polymorphism was analyzed in 320 unrelated healthy individuals who were previously phenotyped for debrisoquine hydroxylation. Results: The metabolic ratio (log10 mean ± standard deviation) of individuals with the -1584G allele was lower than that of individuals with the -1584C allele for carriers of one active CYP2D6 gene (-0.13 ± 0.33 and 0.17 ± 0.52, respectively; p < 0.05) or two active CYP2D6 genes (-0.32 ± 0.39 and -0.20 ± 0.44, respectively; p < 0.05). Conclusion: The presence of the -1584G allele in the promoter region of the CYP2D6 gene was related to a high CYP2D6 hydroxylation capacity.

A major goal of pharmacogenomics is to identify the human genetic variation that influences susceptibility to complex diseases. Recently, theoretical statistical analyses have suggested that genes for complex diseases may be found by linkage disequilibrium (i.e., association). Single nucleotide polymorphism (SNP) susceptibility alleles for common diseases can occur at high frequencies in various populations and, thus, have a major impact on morbidity and mortality. To be successful, SNP mapping studies require successful teamwork, integrating clinicians, epidemiologists, molecular genetics experts, laboratory automation engineers, bioinformatics and database experts. New statistical methods are also developing rapidly and promise to further increase the power of these studies. A recent conference on human genetic variation provided an opportunity for experts in all of these disciplines to exchange ideas. At present, great technological challenges need to be overcome in order to increase the throughput greatly while lowering cost and still maintaining high accuracy for SNP genotyping. Although this approach is relatively new (at least on the scale now being contemplated), the large payoffs anticipated to accrue from the successful mapping of SNPs in disease genes has led the area to be very strongly supported by both public and private funding sources. The potential payoff for improving disease diagnosis and therapeutic efficacy, with better avoidance of adverse events based on SNP associations, is providing a tremendous incentive to move this effort forward at an ever-accelerating pace.

CYP2C19*17 is a novel variant allele causing ultrarapid metabolism of CYP2C19 substrates. In the present study we investigated the CYP2C19*17 allelic frequency and recalculated previously reported frequencies of the CYP2C19*1/*1 genotype and of all genotype-derived phenotypes for CYP2C19 in the Greek population. A total of 283 nonrelated healthy Greek ethnic subjects that had already been genotyped for CYP2C19*2 and *3 alleles as well as for CYP2D6 and CYP2C9 variant alleles participated in the study. The CYP2C19*17 allele was genotyped by the PCR-RFLP method. The CYP2C19*17 allele frequency was 19.61%. The prevalence of CYP2C19*17 carriers in the Greek population was estimated at 31.80%, while the frequency of CYP2C19*1/*1 genotype was recalculated to 44.17% from 75.97% in our previous study. Several subjects possessing both CYP2C19*17 and variant alleles of CYP2D6 and CYP2C9 were also identified. The CYP2C19*17 allele is present in Greeks at a high frequency similar to that found in other European populations of Caucasian origin. Our study highlights the need of reassessing and updating CYP2C19 allelic frequencies in various populations in view of the major role that CYP2C19*17 may have in predicting the clinical outcome of drugs metabolized by CYP2C19.

To determine the main clinical and genetic factors having impact on early coronary stent thrombosis. Genotyping of CYP2C19*2, *17 and CYP4F2*3 in patients with (n = 31) and without stent thrombosis (n = 456) was performed. Clinical and genetic data were analyzed by binary logistic regression. Smoking (OR: 0.317; 95% CI: 0.131-0.767), high-density lipoprotein level in mmol/l (OR: 0.142; 95% CI: 0.040-0.506), CYP2C19*2*2 versus *1*1 and *1*2 genotype (OR: 11.625; 95% CI: 3.498-38.633), CYP4F2 AA versus GA and GG genotype (OR: 3.532; 95% CI: 1.153-10.822) were associated with early stent thrombosis. For the first time we have identified a clinically important polymorphism (CYP4F2 G1347A) that was independently associated with early stent thrombosis. Original submitted 18 August 2014; Revision submitted 10 November 2014.

Breast cancer is an excellent disease paradigm for systems biology. At the time of writing, a simple PubMed search for 'breast cancer' returns nearly 99,000 hits, compared with 51,000 or 16,000 for lung and colon cancer respectively, even though in terms of mortality lung and colon cancers are responsible for four-times more deaths per annum in the UK. These figures reflect the effort and money invested in breast cancer research. It is because breast cancer research is data-rich, crowded and competitive (often perceived as a negative for clinical and basic scientific researchers) that it is such an appealing area of research for systems biologists. For systems biologists, data is currency, and they scavenge diverse and multilayered datasets, from biochemical through genomics and transcriptomics to proteomics, in order to populate computational models. We discuss how dynamic modeling can be used as a tool for predicting responses to new and existing drugs, and what needs to be done to make systems biology a useful tool in the clinic.

The CYP2C9 c.449G>A (p.R150H, rs7900194) polymorphism, which confers the CYP2C9*8 allele, is common in persons of African descent and results in reduced clearance of the narrow therapeutic index drugs, warfarin and phenytoin. Because of significant homology in DNA sequence at the 449G>A locus among CYP2C genes, the 449G>A variant cannot be reliably detected via PCR-based genotyping assays that require a short PCR product, such as pyrosequencing. Herein, we propose genotyping for the CYP2C9 c.-1766T>C polymorphism via pyrosequencing as an alternative and accurate means of identifying the CYP2C9*8 allele.

This study evaluated the relationships between IL-18 polymorphisms and tacrolimus elimination in Chinese liver transplant patients. Eighty-four liver transplant patients from Shanghai (training set) and 50 patients from Shandong (validating set) were inculded. IL-18 polymorphisms (rs5744247, rs7106524, rs549908, rs187238 and rs1946518) and CYP3A5 rs776746 were genotyped. In training set, daily drug dose, total bilirubin, donor CYP3A5 rs776746 and IL-18 rs5744247 genotypes were screened to construct prediction model for tacrolimus elimination. This model was confirmed in validating set (p < 0.001). Donor IL-18 rs5744247 polymorphism was an independent predictor of tacrolimus elimination in the first week after transplantation in both training (p = 0.008) and validating cohorts (p = 0.033). Donor IL-18 rs5744247 polymorphism may influence on tacrolimus elimination. Original submitted 16 July 2014; Revision submitted 12 November 2014.

Aim: Since previous functional studies of short haplotypes and polymorphic sites of SLC6A3 have shown variant-dependent and drug-sensitive promoter activity, this study aimed to understand whether a large SLC6A3 regulatory region, containing these small haplotypes and polymorphic sites, can display haplotype-dependent promoter activity in a drug-sensitive and pathway-related manner. Materials & methods: By creating and using a single copy number luciferase-reporter vector, we examined regulation of two different SLC6A3 haplotypes (A and B) of the 5´ 18-kb promoter and two known downstream regulatory variable number tandem repeats by 17 drugs in four different cellular models. Results: The two regulatory haplotypes displayed up to 3.2-fold difference in promoter activity. The regulations were drug selective (37.5% of the drugs showed effects), and both haplotype and cell type dependent. Pathway analysis revealed at least 13 main signaling hubs targeting SLC6A3, including histone deacetylation, AKT, PKC and CK2 α-chains. Conclusion: SLC6A3 may be regulated via either its promoter or the variable number tandem repeats independently by specific signaling pathways and in a haplotype-dependent manner. Furthermore, we have developed the first pathway map for SLC6A3 regulation. These findings provide a framework for understanding complex and variant-dependent regulations of SLC6A3.

Breast carcinogenesis involves genetic and epigenetic alterations that cause aberrant gene function. Recent progress in the knowledge of epigenomics has had a profound impact on the understanding of mechanisms leading to breast cancer, and consequently the development of new strategies for diagnosis and treatment of breast cancer. Epigenetic regulation has been known to involve three mutually interacting events--DNA methylation, histone modifications and nucleosomal remodeling. These processes modulate chromatin structure to form euchromatin or heterochromatin, and in turn activate or silence gene expression. Alteration in expression of key genes through aberrant epigenetic regulation in breast cells can lead to initiation, promotion and maintenance of carcinogenesis, and is even implicated in the generation of drug resistance. We currently review known roles of the epigenetic machinery in the development and recurrence of breast cancer. Furthermore, we highlight the significance of epigenetic alterations as predictive biomarkers and as new targets of anticancer therapy.

Aim: This study examined whether the ABCG2 421C>A polymorphism and variants in other genes potentially related to the pharmacokinetics of rosuvastatin influenced the plasma concentration of rosuvastatin in Chinese patients with hypercholesterolemia. Patients & methods: Overnight fasting blood samples were collected from 291 patients who had received a rosuvastatin 10 mg night-time dose for at least 4 weeks. Plasma concentrations of rosuvastatin and N-desmethyl rosuvastatin were quantified using liquid chromatography tandem mass spectrometry. Results: In subjects with the ABCG2 421AA genotype (n = 39), the mean plasma concentrations of rosuvastatin and its metabolite were 63 and 41% greater than the values in those with the 421CA genotype (n = 108) and 120 and 99% greater than in those with the 421CC genotype (n = 129). The plasma concentrations of rosuvastatin were associated (r = -0.194; p = 0.001) with the percentage reduction in low-density lipoprotein cholesterol with rosuvastatin, but the association was not significant after adjusting for the ABCG2 421C>A polymorphism. The SLCO1B1 521T>C polymorphism was associated with increased plasma concentrations of rosuvastatin and impaired N-demethylation of rosuvastatin, but had no impact on its lipid-lowering effect. Polymorphisms in CYP2C9, CYP2C19 and SLC10A1 had minimal effects. Conclusion: These findings suggest that the increased plasma concentrations of rosuvastatin in Chinese patients are associated with increased lipid-lowering effects and lower doses of rosuvastatin should be effective in subjects with the ABCG2 421C>A variant.

To investigate the correlation between the methylation status in the MDR1 promoter region and the MDR1 genetic polymorphism. A total of 194 unrelated subjects (105 men and 89 women) with a median age of 26 years were enrolled in this study. DNA was extracted and PCR-RFLP was performed for C1236T, C3435T and G2677T/A polymorphism genotyping. The combined bisulfite restriction analysis (COBRA) method was also performed to determine DNA methylation levels in the MDR1 promoter region. Genotype frequencies for the variants SNPs were assessed for deviation from Hardy-Weinberg equilibrium using the chi2 test. Nonparametric tests including Kruskal-Wallis method and the Mann-Whitney U test were used to compare the DNA methylation levels between different genotypes. The allelic frequency distribution of the C1236T, C3435T and G2677T/A was found to be in good agreement with previous reports. Our study revealed significant correlation between different genotypes of C3435T and G2677T/A, but there is no significant difference between the different genotypes of C1236T. A correlation between MDR1 genetic polymorphisms C3435T and G2677T/A, as well as haplotypes derived from C1236T, G2677T/A and C3435T, with methylation status of MDR1 promoter region was found in this study. Further investigations are needed to explore the molecular mechanism and clinical significance of this correlation.

Leflunomide is an isoxazole derivative structurally and functionally unrelated to other known immunomodulatory drugs. The main molecular target of leflunomide is dihydroorotate dehydrogenase (DHODH), a key enzyme of de novo pyrimidine synthesis. The human DHODH gene sequence is highly conserved and contains only one common missense polymorphism in the coding regions. This SNP (refSNP ID: rs3213422) is localized in the first exon of the DHODH gene (19C>A) and leads to Gln7Lys amino acid substitution in the cationic N-terminal region of the DHODH polypeptide, and it has not yet been investigated in relation to enzyme activity or DHODH inhibitor efficacy. The aim of the study was to examine the effect of this polymorphism on leflunomide treatment outcome in rheumatoid arthritis (RA) patients. The study was carried out on 147 patients (123 women, 24 men, mean age: 52.8 +/- 11.03 years) diagnosed with RA and treated with leflunomide 20 mg daily. Clinical improvement was evaluated according to the American College of Rheumatology 20% and 50% response criteria. The frequency of remission was increased in C allele carriers compared with patients with the A allele. The results of this study suggest that DHODH polymorphism may be associated with leflunomide treatment outcome in RA patients.

Results of several studies have indicated that the variation of c.-3279T>G in the UDP-glucuronosyltransferase (UGT)1A1 gene could be a further factor for the development of hyperbilirubinemia. However, this variant has not been reported in the Taiwanese population. PCR-restriction fragment length polymorphism was utilized to determine variants at nucleotides -3279 (*60), -53 (*28) and 211 (*6) in the UGT1A1 gene for 178 Taiwanese hyperbilirubinemic patients and 200 controls. A total of ten and nine diplotypes were observed in the hyperbilirubinemic patients and controls, respectively. Subjects possessing diplotypes of compound haplotypes (*60/*28, *60/*6, *1/*60 plus *1/*28 plus *1/*6); *60/*60; *60/*60 plus 1/*28 and *6/*6 were significantly related to hyperbilirubinemia development, with an odds ratio of 7.83-188.00 (p = 0.012 approximately <0.001). A subgroup possessing diplotypes of *60/*60 plus *28/*28 were only found in hyperbilirubinemic patients, not in the controls. Bilirubin concentration amongst these patients carrying a diplotype of *60/*60 plus *28/*28 (mean [SD]: 39.2 [10.77] micromol/l) was significantly higher than that in the diplotype subgroups of *60/*60 plus *1/*28 (30.4 [4.10] micromol/l) and *6/*6 (30.3 [3.08] micromol/l) (p = 0.046 and 0.034, respectively). The c.-3279T>G variant is a further factor for the development of hyperbilirubinemia. Our results also demonstrate that possessing the *60/*60 plus *28/*28 diplotype in the UGT1A1 gene is a determinant of relatively higher bilirubin values amongst hyperbilirubinemic patients.