TSPAN5, ERICH3 and selective serotonin reuptake inhibitors in major depressive disorder: pharmacometabolomics-informed pharmacogenomics.

Millions of patients suffer from major depressive disorder (MDD), but many do not respond to selective serotonin reuptake inhibitor (SSRI) therapy. We used a pharmacometabolomics-informed pharmacogenomics research strategy to identify genes associated with metabolites that were related to SSRI response. Specifically, 306 MDD patients were treated with citalopram or escitalopram and blood was drawn at baseline, 4 and 8 weeks for blood drug levels, genome-wide single nucleotide polymorphism (SNP) genotyping and metabolomic analyses. SSRI treatment decreased plasma serotonin concentrations (P<0.0001). Baseline and plasma serotonin concentration changes were associated with clinical outcomes (P<0.05). Therefore, baseline and serotonin concentration changes were used as phenotypes for genome-wide association studies (GWAS). GWAS for baseline plasma serotonin concentrations revealed a genome-wide significant (P=7.84E-09) SNP cluster on chromosome four 5' of TSPAN5 and a cluster across ERICH3 on chromosome one (P=9.28E-08) that were also observed during GWAS for change in serotonin at 4 (P=5.6E-08 and P=7.54E-07, respectively) and 8 weeks (P=1.25E-06 and P=3.99E-07, respectively). The SNPs on chromosome four were expression quantitative trait loci for TSPAN5. Knockdown (KD) and overexpression (OE) of TSPAN5 in a neuroblastoma cell line significantly altered the expression of serotonin pathway genes (TPH1, TPH2, DDC and MAOA). Chromosome one SNPs included two ERICH3 nonsynonymous SNPs that resulted in accelerated proteasome-mediated degradation. In addition, ERICH3 and TSPAN5 KD and OE altered media serotonin concentrations. Application of a pharmacometabolomics-informed pharmacogenomic research strategy, followed by functional validation, indicated that TSPAN5 and ERICH3 are associated with plasma serotonin concentrations and may have a role in SSRI treatment outcomes.

Pharmacogenomics of selective serotonin reuptake inhibitor treatment for major depressive disorder: genome-wide associations and functional genomics.

A genome-wide association (GWA) study of treatment outcomes (response and remission) of selective serotonin reuptake inhibitors (SSRIs) was conducted using 529 subjects with major depressive disorder. While no SNP associations reached the genome-wide level of significance, 14 SNPs of interest were identified for functional analysis. The rs11144870 SNP in the riboflavin kinase (RFK) gene on chromosome 9 was associated with 8-week treatment response (odds ratio (OR)=0.42, P=1.04 × 10⁻6). The rs915120 SNP in the G protein-coupled receptor kinase 5 (GRK5) gene on chromosome 10 was associated with 8-week remission (OR=0.50, P=1.15 × 10⁻5). Both SNPs were shown to influence transcription by a reporter gene assay and to alter nuclear protein binding using an electrophoretic mobility shift assay. This report represents an example of joining functional genomics with traditional GWA study results derived from a GWA analysis of SSRI treatment outcomes. The goal of this analytical strategy is to provide insights into the potential relevance of biologically plausible observed associations.

A genome-wide association study identifies an association of a common variant in TERT with susceptibility to idiopathic pulmonary fibrosis.

In order to identify a gene(s) susceptible to idiopathic pulmonary fibrosis (IPF), we conducted a genome-wide association (GWA) study by genotyping 159 patients with IPF and 934 controls for 214 508 tag single-nucleotide polymorphisms (SNPs). We further evaluated selected SNPs in a replication sample set (83 cases and 535 controls) and found a significant association of an SNP in intron 2 of the TERT gene (rs2736100), which encodes a reverse transcriptase that is a component of a telomerase, with IPF; a combination of two data sets revealed a p value of 2.9 x 10(-8) (GWA, 2.8 x 10(-6); replication, 3.6 x 10(-3)). Considering previous reports indicating that rare mutations of TERT are found in patients with familial IPF, we suggest that the common genetic variation within TERT may contribute to the risk of sporadic IFP in the Japanese population.

Strain-based HLA association analysis identified HLA-DRB1*09:01 associated with modern strain tuberculosis.

Tuberculosis (TB) occurs as a result of complex interactions between the host immune system and pathogen virulence factors. Human leukocyte antigen (HLA) class II molecules play an important role in the host immune system. However, no study has assessed the association between HLA class II genes and susceptibility to TB caused by specific strains. This study investigated the possible association of HLA class II genes with TB caused by modern and ancient Mycobacterium tuberculosis (MTB). The study included 682 patients with TB and 836 control subjects who were typed for HLA-DRB1 and HLA-DQB1 alleles. MTB strains were classified using a large sequence polymorphism typing method. Association analysis was performed using common HLA alleles and haplotypes in different MTB strains. HLA association analysis of patients infected with modern MTB strains showed significant association for HLA-DRB1*09:01 (odds ratio [OR] = 1.82; P-value = 9.88 × 10-4 ) and HLA-DQB1*03:03 alleles (OR = 1.76; P-value = 1.31 × 10-3 ) with susceptibility to TB. Haplotype analysis confirmed that these alleles were in strong linkage disequilibrium and did not exert an interactive effect. Thus, the results of this study showed an association between HLA class II genes and susceptibility to TB caused by modern MTB strains, suggesting the importance of strain-specific analysis to determine susceptibility genes associated with TB.

NAT2 slow acetylator associated with anti-tuberculosis drug-induced liver injury in Thai patients.

BACKGROUND: Anti-tuberculosis drug-induced liver injury (AT-DILI) is one of the most common forms of drug-induced liver injury (DILI) in high tuberculosis (TB) burden countries. Among anti-tuberculosis drugs, isoniazid is the main cause of hepatotoxicity in patients with AT-DILI. OBJECTIVE: To investigate the association of AT-DILI with N-acetyltransferase 2 (NAT2) genotype status in Thai TB patients. METHODS: We enrolled 53 patients diagnosed with AT-DILI and 85 patients who tolerated anti-tuberculosis treatment as controls. Acetylator status was determined based on the inferred NAT2 haplotypes from four common single-nucleotide polymorphisms (SNPs) in Thais using Sanger sequencing. RESULTS: Phenotype frequencies of the NAT2 acetylator in AT-DILI patients were respectively 71.7%, 22.6% and 5.7% for slow, intermediate and rapid acetylators. Among slow, intermediate, and rapid acetylators in treatment tolerant controls, phenotype frequencies were respectively 22.4%, 62.4% and 15.3%. Slow NAT2 acetylators demonstrated a significant association with risk of AT-DILI. The odds ratio of comparing slow NAT2 acetylator in DILI patients and tolerance was 8.80 (95%CI 4.01-19.31, P = 1.53 × 10-8). CONCLUSIONS: Slow acetylator status in the NAT2 genotype is a significant risk factor for DILI in Thai patients with TB. This evidence provides confirmatory data in support of the role of NAT2 in AT-DILI in the Thai population.

Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for human leukocyte antigen B (HLA-B) genotype and allopurinol dosing: 2015 update.

The Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for HLA-B*58:01 Genotype and Allopurinol Dosing was originally published in February 2013. We reviewed the recent literature and concluded that none of the evidence would change the therapeutic recommendations in the original guideline; therefore, the original publication remains clinically current. However, we have updated the Supplemental Material and included additional resources for applying CPIC guidelines into the electronic health record. Up-to-date information can be found at PharmGKB (http://www.pharmgkb.org).

The International SSRI Pharmacogenomics Consortium (ISPC): a genome-wide association study of antidepressant treatment response.

Response to treatment with selective serotonin reuptake inhibitors (SSRIs) varies considerably between patients. The International SSRI Pharmacogenomics Consortium (ISPC) was formed with the primary goal of identifying genetic variation that may contribute to response to SSRI treatment of major depressive disorder. A genome-wide association study of 4-week treatment outcomes, measured using the 17-item Hamilton Rating Scale for Depression (HRSD-17), was performed using data from 865 subjects from seven sites. The primary outcomes were percent change in HRSD-17 score and response, defined as at least 50% reduction in HRSD-17. Data from two prior studies, the Pharmacogenomics Research Network Antidepressant Medication Pharmacogenomics Study (PGRN-AMPS) and the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study, were used for replication, and a meta-analysis of the three studies was performed (N=2394). Although many top association signals in the ISPC analysis map to interesting candidate genes, none were significant at the genome-wide level and the associations were not replicated using PGRN-AMPS and STAR*D data. The top association result in the meta-analysis of response represents SNPs 5' upstream of the neuregulin-1 gene, NRG1 (P = 1.20E - 06). NRG1 is involved in many aspects of brain development, including neuronal maturation and variations in this gene have been shown to be associated with increased risk for mental disorders, particularly schizophrenia. Replication and functional studies of these findings are warranted.

Kinetic Interpretation of the Importance of OATP1B3 and MRP2 in Docetaxel-Induced Hematopoietic Toxicity.

Neutropenia is a lethal dose-limiting toxicity of docetaxel. Our previous report indicated that the prevalence of severe docetaxel-induced neutropenia is significantly associated with genetic polymorphisms in solute carrier organic anion transporter 1B3 (SLCO1B3) (encoding organic anion-transporting polypeptide 1B3 (OATP1B3)) and ATP-binding cassette subfamily C2 (ABCC2) (encoding multidrug-resistant-associated protein 2 (MRP2)). Therefore, we investigated their significance in docetaxel-induced neutropenia. In vitro experiments suggested their possible involvement in the hepatic uptake of docetaxel and its efflux from bone marrow cells. To further characterize a quantitative impact of OATP1B3 and MRP2 on neutropenia, we used an in silico simulation of the neutrophil count in docetaxel-treated subjects with functional changes in OATP1B3 and MRP2 in a pharmacokinetic/pharmacodynamic model. The clinically reported odds ratios for docetaxel-induced neutropenia risk were explained by the decreased function of OATP1B3 and MRP2 to 41 and 32%, respectively. These results suggest that reduced activities of OATP1B3 and MRP2 associated with systemic exposure and local accumulation in bone marrow cells, respectively, account for the docetaxel-induced neutropenia observed clinically.

CYP2D6 genotype and adjuvant tamoxifen: meta-analysis of heterogeneous study populations.

The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta-analysis on data from 4,973 tamoxifen-treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor-positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease-free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow-up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.

Clinical Pharmacogenetics Implementation Consortium guidelines for human leukocyte antigen-B genotype and allopurinol dosing.

Allopurinol is the most commonly used drug for the treatment of hyperuricemia and gout. However, allopurinol is also one of the most common causes of severe cutaneous adverse reactions (SCARs), which include drug hypersensitivity syndrome, Stevens­Johnson syndrome, and toxic epidermal necrolysis. A variant allele of the human leukocyte antigen (HLA)-B, HLA-B*58:01, associates strongly with allopurinolinduced SCAR. We have summarized the evidence from the published literature and developed peer-reviewed guidelines for allopurinol use based on HLA-B genotype.

Impact of the CYP4F2 p.V433M polymorphism on coumarin dose requirement: systematic review and meta-analysis.

A systematic review and a meta-analysis were performed to quantify the accumulated information from genetic association studies investigating the impact of the CYP4F2 rs2108622 (p.V433M) polymorphism on coumarin dose requirement. An additional aim was to explore the contribution of the CYP4F2 variant in comparison with, as well as after stratification for, the VKORC1 and CYP2C9 variants. Thirty studies involving 9,470 participants met prespecified inclusion criteria. As compared with CC-homozygotes, T-allele carriers required an 8.3% (95% confidence interval (CI): 5.6-11.1%; P < 0.0001) higher mean daily coumarin dose than CC homozygotes to reach a stable international normalized ratio (INR). There was no evidence of publication bias. Heterogeneity among studies was present (I(2) = 43%). Our results show that the CYP4F2 p.V433M polymorphism is associated with interindividual variability in response to coumarin drugs, but with a low effect size that is confirmed to be lower than those contributed by VKORC1 and CYP2C9 polymorphisms.

Genome-wide association study on bipolar disorder in the Bulgarian population.

Bipolar disorder is a severe psychiatric disorder influenced by environmental and genetic factors. Genetic studies have implicated many variants in the disease's etiology but only few have been successfully replicated. We conducted a genome-wide association study (GWAS) on bipolar disorder in the Bulgarian population followed by a replication study of the top 100 single nucleotide polymorphisms (SNPs) showing the smallest P values. The GWAS was performed on 188 bipolar disorder patients and 376 control subjects genotyped on the Illumina 550 platform. The replication study was conducted on 122 patients and 328 controls. Although our study did not show any association P value that achieved genome-wide significance, and none of the top 100 SNPs reached the Bonferroni-corrected P value in the replication study, the plausible involvement of some variants cannot be entirely discarded. Three polymorphisms, rs8099939 [P = 2.12 × 10(-6), odds ratio (OR) = 1.95, 95% confidence interval (CI) = 1.43-2.67] in GRIK5, rs6122972 (P = 3.11 × 10(-6), OR = 2.02, 95% CI = 1.46-2.80) in PARD6B and rs2289700 (P = 9.14 × 10(-6), OR = 2.13, 95% CI = 1.53-2.95) in CTSH remained associated at a similar level after Mantel-Haenszel test for combining the results from the genome-wide and replication studies. A modest association was also detected for SNP rs1012053 (GWAS P = 4.50 × 10(-2)) in DGKH, which has already been reported as the most significant variant in a previous genome-wide scan on bipolar disorder. However, further studies using larger datasets are needed to identify variants with smaller effects that contribute to the risk of bipolar disorder.

The involvement of flavin-containing monooxygenase but not CYP3A4 in metabolism of itopride hydrochloride, a gastroprokinetic agent: comparison with cisapride and mosapride citrate.

The goals of the present study were to identify the enzyme responsible for metabolism of itopride hydrochloride (itopride) and to evaluate the likelihood of drug interaction involving itopride. In human liver microsomes, the involvement of flavin-containing monooxygenase in N-oxygenation, the major metabolic pathway of itopride, was indicated by the following results: inhibition by methimazole and thiourea, heat inactivation, and protection against heat inactivation by NADPH. When the effects of ketoconazole on the metabolism of itopride, cisapride, and mosapride citrate (mosapride) were examined using human liver microsomes, ketoconazole strongly inhibited the formation of the primary metabolites of cisapride and mosapride, but not itopride. Other cytochrome P450 (CYP) 3A4 inhibitors, cimetidine, erythromycin, and clarithromycin, also inhibited the metabolism of cisapride and mosapride. In an in vivo study, itopride (30 mg/kg), cisapride (1.5 mg/kg), or mosapride (3 mg/kg) was orally administered to male rats with or without oral pretreatment with ketoconazole (120 mg/kg) twice daily for 2 days. The ketoconazole pretreatment significantly increased the area under the serum concentration curve and the maximum serum concentration of cisapride and mosapride but had no significant effect on the pharmacokinetics of itopride. In addition, itopride did not inhibit five specific CYP-mediated reactions of human liver microsomes. These results suggest that itopride is unlikely to alter the pharmacokinetics of other concomitantly administered drugs.

The suncus (Suncus murinus) shows poor metabolic phenotype for trimethylamine N-oxygenation.

In vitro and in vivo N-oxygenation of trimethylamine (TMA) in the suncus (Suncus murinus) was investigated. The N-oxygenation of TMA has been thought to be catalyzed by flavin-containing monooxygenase (FMO). In a previous study, we found that the levels of mRNAs for FMOs were extremely low in the suncus. Thus, we intended to evaluate the capacity of the suncus to N-oxygenate TMA compared to the rat. Eadie-Hofstee plots of the TMA N-oxygenation by suncus liver microsomes showed a biphasic pattern, suggesting that more than two enzymes were involved in this reaction. The low K(m) component in the suncus showed a twofold higher K(m) (55 vs. 31 microM) and a fourfold lower V(max) (0.61 vs 2.5 nmol/min/mg protein) values than those obtained using rat liver microsomes, resulting in a sevenfold lower V(max)/K(m) (11 vs 82 microl/min/mg protein) value. After an intraperitoneal administration of TMA (10 mg/kg body wt), the suncus excreted 39.6% of the dose in 24-h urine as TMA, whereas the rats excreted 6.3%. Metabolic ratio in the TMA N-oxygenation was 1.42 and 0.11 in the suncus and the rat, respectively. These results indicate that the suncus can be an animal model for a poor metabolizer phenotype in TMA metabolism.

Potent and non-specific inhibition of cytochrome P450 by JM216, a new oral platinum agent.

Bis-acetato-ammine-dichloro-cyclohexylamine-platinum (IV), JM216, is the first antineoplastic platinum compound that can be given to patients orally. Several phase II clinical trials of JM216 monotherapy have already been reported. However, no information on the potential drug interactions caused by JM216 is available. In this study, the capacity of JM216 to inhibit cytochrome P450 (CYP) in human liver microsomes was investigated by measuring the inhibition potential (IC50 and Ki) on prototype reactions. Specific substrates of CYP included testosterone (catalysed by CYP3A4), paclitaxel (CYP2C8), 7-ethoxyresorufin (CYP1A1, CYP1A2), coumarin (CYP2A6), aniline (CYP2E1) and (+/-)-bufuralol (CYP2D6). JM216 inhibited the catalytic activities of CYP isozymes. The IC50 values were between 0.3 microM and 10 microM, indicating strong and non-specific inhibitory effects of JM216. The inhibition occurred in a non-competitive manner, and the Ki value was 1.0 and 0.9 microM for metabolite formation of testosterone and paclitaxel respectively. Therefore, some in vivo studies should be conducted to determine whether or not there is a correlation between in vivo and in vitro results.

A high-level expression of CYP2A in the lung of the suncus (Suncus murinus) and its role in the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Northern blot analysis of mRNA prepared from the lung of Suncus murinus (suncus), which was classified as an ancestor of primates, revealed that the expression level of cytochrome P450 2A (CYP2A) mRNA was about 100-fold higher than in the lung from rats and mice. To confirm that the pulmonary CYP2A of the suncus had a catalytic activity, the metabolism of a specific substrate for CYP2A6, (+)-cis-3,5-dimethyl-2-(3-pyridyl) thiazolidin-4-one hydrochloride (SM-12502), was determined. The intrinsic clearance for SM-12502 S-oxidation by the suncus lung microsomes was calculated to be 99-fold higher than that by rat liver microsomes. The mutagen-producing activity of a 9,000 g supernatant fraction prepared from suncus lung was examined using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as a promutagen. The results showed that the suncus lung possessed 82-fold higher mutagen-producing activity than the rat lung, indicating that NNK was efficiently activated by the CYP2A isoform expressed in the suncus lung and that the suncus was a sensitive animal species to the genotoxicity of NNK contained in tobacco smoke.

Accumulation of the 1-methyl-4-phenylpyridinium ion in suncus (Suncus murinus) brain: implication for flavin-containing monooxygenase activity in brain microvessels.

The metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was examined in an effort to evaluate the role of flavin-containing monooxygenase (FMO) expressed in the brain of suncus (Suncus murinus) and rats. MPTP was metabolized to generate both 1-methyl-4-phenylpyridinium ion (MPP(+)) and MPTP N-oxide by brain homogenates from rats. Although the level of MPP(+)-producing activity was similar in suncus and rats, a remarkable difference was found between the animal species in MPTP N-oxygenase activity, which was not detectable in brain homogenates from suncus. The concentrations of MPP(+) in suncus brain after a single ip administration of MPTP were markedly higher than that in rats, probably because of the lack of FMO activity in the suncus brain. The MPTP N-oxygenase activity of microvessel homogenates of rat brain was 21-fold greater than that of whole brain homogenates. These results suggest that FMO(s) plays a significant role in the detoxification of MPTP in cerebral endothelial cells.

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection.

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 microM and the calibration curve was linear at least up to 5 microM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 microM and from 3.5 to 6.7 at 5 microM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 microM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.

The house musk shrew (Suncus murinus): a unique animal with extremely low level of expression of mRNAs for CYP3A and flavin-containing monooxygenase.

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.