We screened ,61,000 individual twins from the Swedish Twin Registry for the symptoms of fatiguing illness [16–18]. All twins were born in Sweden of Scandinavian ancestry. Of 5,597 monozygotic twin pairs where both were alive and had provided usable responses to CFS screening questions, we identified 140 pairs of twins who met preliminary inclusion criteria: born 1935– 1985, classified as a monozygotic twin based on questionnaire responses [19], and discordant for chronic fatiguing illness (i.e., one twin reported substantial fatigue and the other twin was evidently well). A telephone interview using a standardized script was used to assess eligibility for participation. Twins who remained eligible attended a half-day clinical assessment by a specially trained physician at the Karolinska Institutet in Stockholm. At this visit, a CFS-focused medical assessment was conducted that included standardized medical history, physical examination, and screening biochemical, hormonal, and hematological studies in accordance with international recommendations [1]. Of 140 monozygotic and preliminarily discordant twin pairs, one or both twins declined participation in 23 pairs, 25 pairs were concordant for CFS-like illness, and inclusion criteria were not met in 35 pairs (e.g., chronic fatigue had resolved or an illness that could explain fatiguing symptoms such as neoplasia had emerged). After excluding these 83 pairs, 57 pairs of twins attended the clinical evaluation sessions, and 10 pairs were found not to meet inclusion criteria (9 pairs were concordant for the presence or absence of chronic fatigue or a medical explanation was detected – e.g., newly diagnosed type 2 diabetes mellitus – and 1 pair was dizygotic). Zygosity was confirmed by genotyping 46 single nucleotide polymorphisms using two Sequenom iPlex panels. In 3 pairs, microarray data of satisfactory quality could not be obtained. The analysis sample consisted of 44 pairs of rigorously discordant and genetically proven monozygotic twins. Discor- dance was defined as one twin meeting criteria for either idiopathic chronic fatigue (ICF, 12 pairs) or CFS (32 pairs) [1,2] and the co-twin was required never to have experienced impairing unusual fatigue or tiredness lasting more than one month. Thus, all affected twins were required to have current, long-standing ($6 months), medically unexplained fatigue associated with substantial impairment in social and occupational functioning and the unaffected co-twins were effectively well. A diagnosis of CFS adds a requirement for $4 of 8 specific symptoms (e.g., unrefreshing sleep, muscle pain) to that of ICF. We explain elsewhere the rationale for including ICF along with CFS based on phenotypic [17] and twin analyses [18]. Transient/situational factors can influence gene expression measurements. Biological sampling was standardized by having samples drawn from both members of a twin pair at the same place and time (,0900) after an overnight fast. We required that all subjects be in their usual state of health on the day of sampling (i.e., no acute illness or recent exacerbation of a chronic illness). It was neither practical nor ethical to study subjects medication-free, but we delayed assessment if there had been a recent significant dosage change. Peripheral venous blood was drawn using sterile technique into PAXgene tubes manufactured in the same batch (Qiagen, to protect RNA from degradation and to minimize ex vivo gene expression). Total RNA was purified using the PAXgene blood RNA kit following the manufacturer’s instructions (Qiagen). RNA quality was determined using the Agilent 2100 Bioanalyzer. Total RNA (5.0 mg) was labeled with the one-cycle cDNA synthesis kit (Invitrogen) and spiked with eukaryotic Poly-A RNA controls to check the target labeling process (Affymetrix). Synthesized cDNA was transcribed in vitro using the GeneChip IVT labeling kit Gene Expression in CFS (Affymetrix). The biotin labeled cRNA product (20 mg) was purified with a sample cleanup module (Qiagen) and samples were fragmented with the fragmentation buffer from Affymetrix at 94uC for 35 minutes. Fragmented and labeled targets (together with hybridization and oligo B2 controls) were hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays at 45uC for 16 hours. Washing and staining of the arrays were performed on the Affymetrix fluidics station using the EukGE-WS2v5_450 protocol. Imaging of the arrays and signal quantification were performed with the Affymetrix GeneChip Scanner 3000 and GeneChip Operating Software. For verification, we used qRT- PCR. RNA was converted to cDNA with Superscript III (Invitrogen) and qRT-PCR was run with ABI’s Taqman gene expression assays (with 18S rRNA as control). The DDCt method was used for the calculations. Array images were manually checked for defects using DChip [20,21] and then normalized using the RMA algorithm in Affymetrix Expression Console (v1.0). After normalization, the Bioconductor [22] Significance Analysis of Microarrays package [23] was used to compute modified paired t-tests that contrasted an affected twin with the unaffected co-twin for each transcript using R [24]. To adjust for multiple comparisons, the nominal permutation-based p-values from SAM were used to compute false discovery rate q-values [25–27]. Pathway analyses for KEGG pathways [28], GO keywords [29] (biological process, cellular component, and molecular function), and PFAM protein family groupings [30] were conducted using SAFE which performs array permutation to account for transcript correlation [31,32]. These expression data are available from GEO (http://www.ncbi.nlm. nih.gov/geo) under accession number GSE16059 and were prepared in accordance with MIAME 2.0 standards. Results The analytic data set consisted of microarray data from 44 pairs of monozygotic twins discordant for clinically-evaluated chronic fatiguing illness (Table 1). Most pairs were female (89%), and the median age at evaluation was 51 years. Of the affected twins, 32 met criteria for CFS and 12 for ICF with a median duration of chronic fatigue of 8 years with no significant difference between affected twins with CFS and ICF (paired t43 = 0.32, p = 0.75). Body mass index was similar between the affected and unaffected twins. Two affected individuals (4.5%) reported sudden onset of fatigue. Affected twins had significantly worse physical and mental functioning on the SF-36 [33] and reported significantly greater current fatigue. The mean functioning of affected twins was over a standard deviation worse than Swedish norms whereas the unaffected twins were similar to Swedish norms (http://www.sf- 36.org/nbscalc/index.shtml, accessed 12 December 2008). The main analyses contrasted gene expression in PBLs in 44 pairs of monozygotic twins affected with CFS or ICF to that of their unaffected co-twins. As inclusion of males could increase noise, the second planned analysis compared 39 female pairs with CFS or ICF and the third planned analysis compared the 28 pairs of female twins with CFS. For each of these three sets of statistical comparisons, the observed results did not deviate from those expected by chance (Figure S1). When we compared our findings to a list of 108 transcripts reported as differentially expressed in CFS [12–15], 107 were studied in our experiment, 101/107 had p.0.1 in our study, and only two had p,0.05 (CXCR4 p = 0.03 and RAP2C p = 0.04), a degree of overlap that does not depart from chance expectations. At the single transcript level, there was no biological evidence of altered gene expression in PBLs that correlated with chronic, impairing, and medically unexplained PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e5805