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Gene-Expression Differences in Peripheral Blood Between Lithium Responders and Non-Responders in the Lithium Treatment-Moderate Dose Use Study (Litmus)

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The Pharmacogenomics Journal (2014) 14, 182–191 & 2014 Macmillan Publishers Limited All rights reserved 1470-269X/14 www.nature.com/tpj ORIGINAL ARTICLE Gene-expression differences in peripheral blood between lithium responders and non-responders in the Lithium Treatment-Moderate dose Use Study (LiTMUS) RD Beech1, JJ Leffert1, A Lin2, LG Sylvia3, S Umlauf4, S Mane4, H Zhao5, C Bowden6, JR Calabrese7, ES Friedman8, TA Ketter9, DV Iosifescu10, NA Reilly-Harrington3, M Ostacher9, ME Thase11 and A Nierenberg3 This study was designed to identify genes whose expression in peripheral blood may serve as early markers for treatment response to lithium (Li) in patients with bipolar disorder. Although changes in peripheral blood gene-expression may not relate directly to mood symptoms, differences in treatment response at the biochemical level may underlie some of the heterogeneity in clinical response to Li. Subjects were randomized to treatment with (n ¼ 28) or without (n ¼ 32) Li. Peripheral blood gene-expression was measured before and 1month after treatment initiation, and treatment response was assessed after 6 months. In subjects treated with Li, 62 genes were differentially regulated in treatment responders and non-responders. Of these, BCL2L1 showed the greatest difference between Li responders and non-responders. These changes were specific to Li responders (n ¼ 9), and were not seen in Li non-responders or patients treated without Li, suggesting that they may have specific roles in treatment response to Li. The Pharmacogenomics Journal (2014) 14, 182–191; doi:10.1038/tpj.2013.16; published online 14 May 2013 Keywords: BCL2L1; bipolar disorder; gene expression; lithium-response; microarray INTRODUCTION significance. Thus, despite intensive work by multiple groups Lithium (Li) is a well-established treatment in the management spanning several decades, the need for biomarkers that can be of bipolar disorder. It was the first medication to effectively used to monitor and predict response to treatment with Li 18 treat mania1 and, 460 years later, it is still considered first line remains as great as ever. treatment for acute and maintenance phase treatment of bipolar Treatment with Li has been shown to affect the transcription of disorder.2,3 It is also the only medication to be consistently a large number of genes in both neuronal and non-neuronal 19 associated with a reduction in suicidal ideation or attempts in cells. These changes may influence a variety of processes patients with bipolar disorder.4–6 However, clinical response to including neuroplasticity and neurogenesis, which are 20,21 Li is heterogeneous, and the molecular basis for this variability is hypothesized to be responsible for the clinical effects of Li. unknown. Similarities in gene expression between central nervous system Clinical factors, including the pattern of manic and depressive and peripheral lymphoid tissue, combined with the inability episodes, age of illness onset, number of previous hospitalizations to directly sample gene expression in the living brain, have and continuous cycling between episodes, have been studied in led investigators to examine the feasibility of using blood as a attempts to identify subsets of patients who might respond more surrogate tissue to identify biomarkers related to specific 22 or less favorably to treatment with Li. However, a metaanalysis7 psychiatric disorders. A microarray study evaluating the 23 concluded that none of the potential predictors had a very comparability of gene expression in blood and brain found strong impact on response. DNA polymorphisms associated with a that whole blood shares significant gene expression similarities number of candidate genes have also been proposed as possible with multiple regions of the central nervous system. However, predictors of response to treatment with Li.8–12 However, these to our knowledge, there are no studies where effects of Li on results have been difficult to reproduce and other studies have gene-expression on brain and blood have been directly compared. 24 found no association between polymorphisms at these same sites, In a previous study, we examined gene-expression changes in and response to Li.13–16 More recently, genome-wide association 20 patients with bipolar depression who were treated with Li. We studies have been conducted to identify common gene variants found that in Li responders (subjects with an at least 50% decrease 25 that may be associated with Li response.17 Although some loci in Hamilton Depression Rating Scale ), 1 month after starting with suggestive evidence for linkage were found, no single- treatment with Li several antiapoptotic genes including BCL2L1 nucleotide polymorphisms met the threshold for genome-wide (BCL2-like 1; also known as B-cell lymphoma-like X) were 1Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA; 2Keck Foundation Biotechnology Biostatistics Resource, Yale University School of Medicine, New Haven, CT, USA; 3Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA; 4Center for Genome Analysis, Yale University School of Medicine, New Haven, CT, USA; 5Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT, USA; 6Departments of Psychiatry and Pharmacology, University of Texas Health Science, San Antonio, TX, USA; 7Department of Psychiatry, Case Western Reserve University, Cleveland, OH, USA; 8Department of Psychiatry, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 9Department of Psychiatry and Behavioral Science, Stanford University School of Medicine, Stanford, CA, USA; 10Departments of Psychiatry and Neuroscience, Mount Sinai Medical Center, New York, NY, USA and 11Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA. Correspondence: Dr RD Beech, Department of Psychiatry, Yale University School of Medicine, 34 Park St., 4th Floor, New Haven, CT 06519, USA. E-mail: [email protected] Received 13 November 2012; revised 15 February 2013; accepted 18 March 2013; published online 14 May 2013 Gene-expression differences in peripheral blood RD Beech et al 183 upregulated, and pro-apoptotic genes were downregulated, while Collection of blood samples and storage before RNA isolation. Blood the reverse pattern was seen in Li non-responders. However, as all samples were collected directly into PAXgene blood RNA tubes (QIAGEN, subjects had received Li, it could not be determined whether Valencia, CA, USA) and shipped by an overnight delivery service (FedEx) to these changes were specifically associated with response to Li. Dr Beech’s laboratory at Yale University. To minimize technical variability In the present study, we recruited subjects who had already due to batch-related variation in processing condition, blood samples were stored frozen at À 80 1C until all samples had been collected, and were agreed to participate in the Lithium Treatment-Moderate dose processed as close in time as possible. Use Study (LiTMUS).26,27 The LiTMUS study was a multisite clinical trial comparing the effectiveness of optimized treatment (OPT) Sample preparation and microarray analysis. Total RNA was isolated from versus OPT þ Li for the treatment of bipolar disorder. Patients 10 cc whole blood using the PAXgene Blood RNA Isolation kit per the participating in LiTMUS were randomized to OPT or OPT þ Li and manufacturer’s instructions, and depleted of globin mRNA message using followed clinically for 6 months. The goal of the present study was GLOBINclear hybridization capture technology (Ambion, Austin, TX, USA). to identify changes in the expression of specific genes occurring 1 Globin-reduced total RNA underwent complementary DNA synthesis and month after treatment initiation, which were predictive of clinical overnight in vitro transcription utilizing the Illumina TotalPrep RNA Amplification Kit (Ambion). Biotinylated complementary RNA (1.5 mg) was outcomes at the 6-month time point (study exit). hybridized onto an Illumina Sentrix Beadchip (HumanHT-12 v3), and then scanned on a BeadArray Reader, Illumina, Inc. (San Diego, CA, USA). Microarray hybridization and scanning were carried out at the Yale Center SUBJECTS AND METHODS for Genome Analysis. At the time of publication, all data will be deposited into the NCBI-GEO repository. Subjects All procedures involving human subjects were approved by the Yale Data analysis. Illumina BeadStudio software, Illumina, Inc. (San Diego, CA, Human Investigation Committee and the relevant institutional review USA) was used to generate probe and gene expression profiles of each board at each of the clinical sites participating in LiTMUS. Subjects sample. Quantile normalization was carried out using the package incor- included in the current study had already provided informed consent to porated in the Illumina BeadStudio software package.30 Further statistical participate in LiTMUS.26,27 For this study, a separate and optional genetic analysis was carried out on genes with a detection P-valueo0.01, as consent was presented to subjects allowing additional tubes of blood to determined using the Illumina BeadStudio software (that is, a 99% be drawn for this study. Inclusion and exclusion criteria for subjects with probability that expression was above background), in 90% of the samples. bipolar disorder were identical to those being used in LiTMUS. Fold-changes were calculated for each gene by dividing the level Participating sites included: Case Western Reserve University (Joseph R of expression
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