Gene Expression in Peripheral Blood Leukocytes in Monozygotic Twins Discordant for Chronic Fatigue: No Evidence of a Biomarker Andrea Byrnes1,2, Andreas Jacks3, Karin Dahlman-Wright4, Birgitta Evengard5, Fred A. Wright2, Nancy L. Pedersen3*, Patrick F. Sullivan1,3* 1 Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2 Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 3 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden, 4 Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden, 5 Department of Clinical Microbiology, University of Umea˚, Umea˚, Sweden Abstract Background: Chronic fatiguing illness remains a poorly understood syndrome of unknown pathogenesis. We attempted to identify biomarkers for chronic fatiguing illness using microarrays to query the transcriptome in peripheral blood leukocytes. Methods: Cases were 44 individuals who were clinically evaluated and found to meet standard international criteria for chronic fatigue syndrome or idiopathic chronic fatigue, and controls were their monozygotic co-twins who were clinically evaluated and never had even one month of impairing fatigue. Biological sampling conditions were standardized and RNA stabilizing media were used. These methodological features provide rigorous control for bias resulting from case-control mismatched ancestry and experimental error. Individual gene expression profiles were assessed using Affymetrix Human Genome U133 Plus 2.0 arrays. Findings: There were no significant differences in gene expression for any transcript. Conclusions: Contrary to our expectations, we were unable to identify a biomarker for chronic fatiguing illness in the transcriptome of peripheral blood leukocytes suggesting that positive findings in prior studies may have resulted from experimental bias. Citation: Byrnes A, Jacks A, Dahlman-Wright K, Evengard B, Wright FA, et al. (2009) Gene Expression in Peripheral Blood Leukocytes in Monozygotic Twins Discordant for Chronic Fatigue: No Evidence of a Biomarker. PLoS ONE 4(6): e5805. doi:10.1371/journal.pone.0005805 Editor: Etienne Joly, Universite´ de Toulouse, France Received March 2, 2009; Accepted April 21, 2009; Published June 5, 2009 Copyright: 2009 Byrnes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was funded by R01 AI056014 (PFS) from the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: In the interests of full disclosure, Dr. Sullivan reports receiving unrestricted research funding from Eli Lilly for genetic research in schizophrenia. The other authors report no conflicts. * E-mail: nancy.pedersen@ki.se (NLP); pfsulliv@med.unc.edu (PFS) Introduction The etiology of chronic fatigue syndrome (CFS) is unknown [1–3]. Many theories of the pathophysiology of CFS have been suggested [4–9], often based on suspicions of the role of an acute viral illness or immune dysfunction. The availability of a biomarker for CFS would be of particular benefit for clinical and basic research. Gene expression studies of peripheral blood leukocytes (PBLs) are a potentially promising source of biomarkers for CFS [10]. PBLs are both accessible and salient for CFS given the prominence of immune and infectious theories of its etiology. Moreover, gene expression patterns in human PBLs are not unrelated to less accessible tissues like brain [11]. We are aware of four published non-overlapping studies that compared gene expression in PBLs in cases with CFS in comparison to controls [12–15]. These small studies included a total of only 45 cases (5, 7, 8, and 25) and, of the 108 transcripts reported to have altered expression, only one transcript was altered in more than one study (MSN, moesin). [13,15] Given the lack of clarity in existing studies of CFS, we undertook an ‘‘unbiased’’, transcriptome-wide search for gene expression changes associated with CFS in PBLs. Our study had two notable design features. First, we used a control group optimal for detecting state-related gene expression changes and minimized false-positive findings due to genetic mismatching between cases and controls as we contrasted 44 individuals with clinically- evaluated chronic fatiguing illness with their 44 unaffected monozygotic co-twins. Use of rigorously discordant monozygotic twins provides the best control for genetic background currently possible in humans and allows use of paired statistics with greater statistical power. Second, we carefully standardized sampling conditions so that PBL samples were drawn into RNA-stabilizing media and taken from both members of a twin pair at the same time and place. Materials and Methods Ethics Statement The protocol was approved in advance by the ethical review board at UNC-CH and the Karolinska Institutet and all subjects provided written informed consent. PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5805