Comment[ArrayExpressAccession] E-GEOD-19738 Public Release Date 2011-10-04 Investigation Title Major Depressive Disorder blood gene expression Comment[Submitted Name] Major Depressive Disorder blood gene expression Experiment Description Background: Major Depressive Disorder (MDD) is a moderately heritable disorder with a high lifetime prevalence. At present, laboratory blood tests to support MDD diagnosis are not available. Methods: We used a classifier approach on blood gene expression profiles of a unique set of non-medicated subjects (MDD patients and controls) to select genes of which expression is predictive for disease status. To reveal blood gene expression changes related to MDD disease, we applied a powerful ex vivo stimulus to the blood, i.e. incubation with lipopolysaccharide (LPS; 10 ng/ml blood). Results: Based on LPS-stimulated blood gene expression using whole-genome microarrays in 42 subjects (primary cohort; 21 MDD patients (mean age 42.3 years), 21 healthy controls (mean age 41.9 years)), we identified a set of genes (CAPRIN1, CLEC4A, KRT23, MLC1, PLSCR1, PROK2, ZBTB16) that serves as a molecular signature of MDD. These findings were validated for the primary cohort using an independent quantitative PCR method (P = 0.007). The difference between depressive patients and controls was confirmed (P = 0.019) in a replication cohort of 13 patients with MDD (mean age 42.8 years) and 14 controls (mean age 45.6 years). The MDD-signature score comprised of expression levels of 7 genes could discriminate depressive patients from controls with sensitivity of 76.9% and specificity of 71.8%. Conclusions: We show for the first time that molecular analysis of stimulated blood cells can be used as an endophenotype for MDD diagnosis, which is a milestone in establishing biomarkers for neuropsychiatric disorders with moderate heritability in general. Our results may provide a new entry point for following and predicting treatment outcome, as well as prediction of severity and recurrence of MDD. In total, 33 MDD patients and 34 healthy controls were analyzed using basal gene expression in whole blood, and gene expression from whole blood that was stimulated with LPS for 5-6 h, using microarrays. Patients were arbitrarily selected from all patients to serve as primary cohort (nMDD = 21 (MDD01-MDD21); nControls = 21 (Con01-Con21)), or replication cohort (nMDD = 12 (MDD22-MDD35); nControls = 13 (Con22-Con37)) using microarrays. This submission does not include Samples CON21_LPS or CON30_LPS. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Spijker Spijker van Zanten de Jong Penninx van Dyck Zitman Smit Zitman Ylstra Smit Hoogendijk Person First Name sabine Sabine Jeroen Simone Brenda Richard Frans Jan Frans Bauke August Witte Person Mid Initials S W G H G B G Person Email geo@ncbi.nlm.nih.gov Person Affiliation Neuroscience Campus Amsterdam Person Phone Person Fax Person Address Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, De Boelelaan 1085, Amsterdam, Netherlands Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE19738-1 P-GSE19738-8 P-GSE19738-5 P-GSE19738-10 P-GSE19738-9 P-GSE19738-3 P-GSE19738-4 P-GSE19738-2 P-GSE19738-6 P-GSE19738-7 Protocol Description ID_REF =
VALUE = Loess and Aquantile normalized log2 signal intensity (Limma) From each subject, labeled cRNA (1.5 µg Cy3-labeled basal blood sample; 1.25 µg Cy-5 labeled LPS-stimulated blood sample) was hybridized for 15-17 h at 65 °C onto the 44K Human Agilent whole genome arrays according to the manufacturer’s protocol. After washing, the arrays were quick-dried using acetonitrile. NA. Signal intensities were extracted using Agilent Feature Extraction (v8.0). Data were analyzed with Limma (Bioconductor) using median signals and median background. Control spots were normalized, but were set not to influence normalization. Non-uniform signals were removed prior to further analysis. After background subtraction (Edwards, offset 30), within normalization (Loess) and between array normalization (Aquantile) data were exported to SPSS. Further data selection for inclusion consisted of the following criteria (in this order): 1) Signal intensities for any gene should be 1.3x background (> 6.8 (log2)), and have non-saturated levels (<15.7 (log2)); 2) In addition, for any gene the signal should be present in >80% of the control as well as in >80% of the MDD samples to avoid type-1 errors due to a limited number of participants in each group. Genes present in <80 % of MDD or control samples were given an absent-call. From the total of 41,675 spots, filtering resulted in 22,789 spots for the basal sample and 22,935 spots in the LPS-stimulated sample, with an overlap of 21,107 spots (>92%). Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using two color scan setting for 1x44k array slides. Scan resolution 10 µm. PMT set to 100%. Patients were arbitrarily selected from all patients to serve as primary cohort (nMDD = 21 (#MDD01-MDD21); nControls = 21 (#Con01-Con21)), or replication cohort (nMDD = 12 (#MDD22-35); nControls = 13 (#Con22-37)) using microarrays. Serial venous whole blood samples were obtained between 8:00 a.m. and 10:00 a.m., after overnight fasting, in one 7 ml heparin-coated tube (Greiner). Between 10-60 minutes after blood draw, 2.5 ml of blood was transferred into a PAXgene tube (Qiagen) and used as the basal (non-LPS stimulated) sample. This tube was kept at room temperature for a minimum of 2 h, and subsequently stored at –20 °C. The remaining blood (4.5 ml) was stimulated by addition of LPS (10 ng/ml blood; E. coli, Sigma). LPS-stimulated samples were laid flat and incubated at a slow rotation for 5-6 h at 37° C before a 2.5 ml sample of this LPS-stimulated blood was transferred into a PAXgene tube (Qiagen), and treated as described for the basal sample. RNA was prepared using PAX (PreAnalytiX; Qiagen) gene blood collection tubes following the manufacturer's recommendations (after thawing of the PAX tubes for 2 h at room temperature). The protocol includes lysis of blood cells, digestion of proteins (prot K treatment), and an on-column DNaseI digestion. The purity of RNA was first determined by spectrophotometry (NanoDrop ND–1000 UV-Vis Spectrophotometer, Nanodrop Technologies). Because of contaminants due to the isolation procedure, visible in the spectrophotogram at 200-230 nm, RNA samples were precipitated using ethanol, and NaAc with addition of 0.1 µg linear acryl-amide. Subsequently, RNA quality was determined by spectrophotometry and by using the RNA 6000 NanoChip kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only RNA passing quality control criteria (RIN values > 8) was used for further analysis. Labeling of RNA (1 µg) was according to the Agilent protocol (Agilent technologies, Palo Alto, USA). Using spectrophotometry, cRNA concentration and labeling efficiency were determined (Nanodrop ND-1000 UV-Vis Spectrophotometer, Nanodrop Technologies). For all subjects, the same set-up has been used with the basal sample labeled with Cy3 (green) and the LPS-stimulated sample labeled with Cy5 (red), except for one subject (MDD31). This excludes the possibility that differences between subjects are due to a color bias for which two-color arrays are known. Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation hybridization grow feature_extraction image_aquisition specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction labeling Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name SAMPLE TYPE TREATMENT SMOKING STATUS DISEASE AGE GENDER Experimental Factor Type sample type treatment smoking status disease age gender Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Stimulated gene expression profiles as a blood marker of major depressive disorder. Publication Author List Spijker S, Van Zanten JS, De Jong S, Penninx BW, van Dyck R, Zitman FG, Smit JH, Ylstra B, Smit AB, Hoogendijk WJ PubMed ID 20471630 Publication DOI 10.1016/j.biopsych.2010.03.017 Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE19738 Comment[GEOLastUpdateDate] 2011-10-04 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2011-10-03 Comment[ArrayExpressSubmissionDate] 2010-01-04 SDRF File E-GEOD-19738.sdrf.txt