tional studies of various HDAC inhibitors in rodents have shown that HDAC inhibitors may act by altering levels of synaptic plasticity and in this context HDAC inhibitors have been used to modify learning, memory, and emotional behavior underscoring the potentially pleiotropic effects of targeting global levels of gene expression regulation [14,15,16,17,18,19]. Addressing the impact of variability in global levels of gene expression regulation on neurodevelopment in mice, we recently reported that the pattern of the distribution of gene expression levels, as assessed by variance in the distribution, accurately predicted mouse behavior in genetically identical animals. Specifically, we found that increased variance across the total distribution of gene expression levels in the hippocampus predicted increased levels of open-field exploration, a hippocampal dependent behavior. Developmental epigenetic interventions that modified the variance in the gene expression distribution in the hippocampus also modified mouse behavior in tandem [20]. In the current study, we used our previously established strategy of studying the overall pattern of the gene expression distribution to test the hypothesis that autism would be associated with alterations in global levels of gene expression regulation. To do this, we compared the pattern of the gene expression distributions, as assessed by variance across the total gene expression distribution, in the blood of children with autism and controls (schematic in figure 1). Further, since multiple studies associated increased paternal age with increased rates of autism and other neurodevelopmental disorders [21,22,23,24,25,26,27,28], we per- formed a secondary analysis to address the hypothesis that paternal age would be associated with changes in the pattern of the gene expression distribution. Finally, we used traditional Figure 1. Schematic for determining the large-scale organiza- tion of gene expression in the blood of children with autism and controls. The figure displays the steps used to evaluate the large- scale organization of gene expression in the blood of children with autism and controls. Peripheral blood lymphocytes were purified from the blood of children with autism (n = 82) or control subjects (n = 64). Total RNA was extracted for microarray experiments with Affymetrix Human U133 Plus 2.0 39 Expression Arrays. Array data was pre- processed and summarized using Affymetrix Microarray Analysis Suite 5.0 (MAS 5.0) to obtain 54,675 expression levels for approximately 47,000 unique transcripts including approximately 38,500 human genes. Expression levels were log2 transformed and the variance of log2 transformed expression levels was determined. doi:10.1371/journal.pone.0016715.g001 Autism Changes Global Gene Expression Regulation approaches to gene expression analysis to examine for biological pathways that might be causally related to changes global levels of gene expression regulation. Materials and Methods Subjects The Western Institutional Review Board approved all aspects of the current protocol related to human subjects including written informed consent and found that the research was conducted according to the principles expressed in the Declaration of Helsinki. All subjects were male. Probands and controls were all recruited from the Phoenix area via database mailing for probands and via flyers to scout, church, and sports groups for controls. Blood draws for all subjects were done between the spring and summer of 2004. Children with autism had a diagnosis of autism by a medical professional (developmental pediatrician, psycholo- gist, or child psychiatrist) according to the DSM-IV criteria, and confirmation of the diagnosis by ADOS [29] and ADI-R criteria [30]. Exclusion criteria included a significant prenatal history (prematurity ,35 weeks, intraventricular hemorrhage, severe asphyxia, or cerebral palsy), serious CNS abnormality, or known genetic or metabolic disorder. In an attempt to obtain a homogenous population of children with autism, non-classic forms of autism were excluded, including autism with regression and Asperger’s syndrome, a higher functioning form of autism where individuals have language skills within the normal range. In addition, each subject had a normal high-resolution chromosome analysis, and a negative Fragile X DNA test. 82 children with autism and 64 control children were included in the study. IQ scores were not done for this study, but all individuals did have a demonstrated impairment in language as part of the diagnostic criteria. For our secondary analysis, paternal ages were available for 78 children with autism and 57 controls. The group of children with autism was significantly younger than the control group (autism: mean - 5.5 years SD - 2.1; control: mean - 7.9 SD - 2.2, p,.0001). Paternal age was similar between groups. The study population was primarily Caucasian and there were no group level differences in ethnicity. Expression profiling Expression profiling was performed at Translational Genomics (TGen), a member of the NIMH Neuroscience Microarray Consortium. Total RNA was extracted from peripheral blood lymphocytes (PBL) within 30 minutes of the blood draw using the Qiagen Qiaquick kit (Germantown, MD). Isolated total RNA was double round amplified, cleaned, and biotin-labeled using Affymetrix’s GeneChip Two-Cycle Target Labeling kit (Santa Clara, CA) with a T7 promoter and Ambion’s MEGAscript T7 High Yield Transcription kit (Austin, TX) as per manufacturer’s protocol. Amplified and labeled cRNA was quantified on a spectrophotometer and run on a 1% TAE gel to check for an evenly distributed range of transcript sizes. Twenty micrograms of cRNA was fragmented to approximately 35–200 bp by alkaline treatment (200 mM Tris-acetate, pH 8.2, 500 mM KOAc, 150 mM MgOAc) and run on a 1% TAE gell to verify fragmentation. Separate hybridization cocktails were made using 15 micrograms of fragmented cRNA from each sample as per Affymetrix’s protocol. Two hundred microliters (containing 10 micrograms of fragmented cRNA) of each cocktail was separately hybridized to an Affymetrix Human Genome U133 Plus 2.0 Array for 16 h at 45 degree Celsius in the Hybridization Oven 640. The Affymetrix Human Genome Arrays measure the expression of over 47,000 PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e16715