Autism and Increased Paternal Age Related Changes in Global Levels of Gene Expression Regulation Mark D. Alter1*, Rutwik Kharkar1, Keri E. Ramsey2, David W. Craig2, Raun D. Melmed3, Theresa A. Grebe3, R. Curtis Bay3, Sharman Ober-Reynolds3, Janet Kirwan3, Josh J. Jones3, J. Blake Turner4, Rene Hen 5 , Dietrich A. Stephan2 1 Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Neurogenomics Division, Translational Genomics Research Institute, Phoenix, Arizona, United States of America, 3 Southwest Autism Research and Resource Center, Phoenix, Arizona, United States of America, 4 Division of Child and Adolescent Psychiatry, Department of Psychiatry, Columbia University, New York, New York, United States of America, 5 Departments of Psychiatry and Neuroscience, Columbia University, New York, New York, United States of America Abstract A causal role of mutations in multiple general transcription factors in neurodevelopmental disorders including autism suggested that alterations in global levels of gene expression regulation might also relate to disease risk in sporadic cases of autism. This premise can be tested by evaluating for changes in the overall distribution of gene expression levels. For instance, in mice, variability in hippocampal-dependent behaviors was associated with variability in the pattern of the overall distribution of gene expression levels, as assessed by variance in the distribution of gene expression levels in the hippocampus. We hypothesized that a similar change in variance might be found in children with autism. Gene expression microarrays covering greater than 47,000 unique RNA transcripts were done on RNA from peripheral blood lymphocytes (PBL) of children with autism (n = 82) and controls (n = 64). Variance in the distribution of gene expression levels from each microarray was compared between groups of children. Also tested was whether a risk factor for autism, increased paternal age, was associated with variance. A decrease in the variance in the distribution of gene expression levels in PBL was associated with the diagnosis of autism and a risk factor for autism, increased paternal age. Traditional approaches to microarray analysis of gene expression suggested a possible mechanism for decreased variance in gene expression. Gene expression pathways involved in transcriptional regulation were down-regulated in the blood of children with autism and children of older fathers. Thus, results from global and gene specific approaches to studying microarray data were complimentary and supported the hypothesis that alterations at the global level of gene expression regulation are related to autism and increased paternal age. Global regulation of transcription, thus, represents a possible point of convergence for multiple etiologies of autism and other neurodevelopmental disorders. Citation: Alter MD, Kharkar R, Ramsey KE, Craig DW, Melmed RD, et al. (2011) Autism and Increased Paternal Age Related Changes in Global Levels of Gene Expression Regulation. PLoS ONE 6(2): e16715. doi:10.1371/journal.pone.0016715 Editor: Joanna Bridger, Brunel University, United Kingdom Received October 6, 2010; Accepted December 20, 2010; Published February 17, 2011 Copyright: 2011 Alter 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: Research support: Dr. Alter’s salary was paid in part through a NARSAD Young Investigator Award (http://www.narsad.org/?q = node/124/ apply_for_grants/124); sample collection and processing, microarrays, and salary support provided through a grant from the state of Arizona. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: markalter1968@gmail.com Introduction Autism is a severe neurodevelopmental disorder with character- istic social and communication deficits and ritualistic or repetitive behaviors that appear by age three. Many etiologies have been suggested and numerous risk factors have been identified [1]. Though autism is associated with a high degree of heritability, few specific genetic mutations have been identified accounting for a minority of cases [2,3,4,5,6], while the majority of cases are considered sporadic. The failure to identify specific gene variants for most cases of autism has been attributed to many potential factors including complex interactions of multiple genes, a heterogeneous disorder with multiple causes converging on the autistic phenotype, or epigenetic factors not related to specific genetic mutations or polymorphisms [2,3]. None of these hypotheses has been confirmed and they are not mutually exclusive. Research on gene expression in autism has previously focused on identifying specific or a limited group of genes related to disease [7,8,9]. The idea that alterations at the global level of gene expression regulation might be important in mediating the risk for autism or other disease states has been largely underexplored. Supporting the possible importance of global regulation of gene expression in neurodevelopmental disorders, genetic studies found that mutations in genes encoding for global regulators of gene expression were linked to neurodevelopmental disorders including autism [5,6]. Pharmacological studies also suggested that targeting global levels of gene expression regulation could impact neurode- velopment. For instance, valproate, a histone deacetylase inhibitor (HDACi), is a commonly used medication in the treatment of seizures, mental health disorders, and cancer that impacts global levels of gene expression regulation through chromatin based mechanisms. When given during gestation, valproate can adversely impact neurodevelopment in rodents and cause autism in humans [10,11,12,13]. Thus, both genetic and pharmacological studies suggest alterations in global levels of gene expression regulation can interfere with normal neurodevelopment. Addi- PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e16715