Transcriptomic and Epigenetic Findings in Autism Figure 3. No differential methylation of genomic DNA was identified between control and autism cerebellar cortex or BA19 cortex. (A) Unsupervised correspondence analysis of genome-wide CpG methylation in control and autism cerebellar cortex and BA19 cerebral cortex demonstrates that brain region and age are associated with the largest two sources of variability, respectively, in the data. The first two principal components are listed on the x- and y-axis respectively, with percent of variance explained in parentheses. Bubbles correspond to individual samples with area proportional to age of subject, bubble outline to region, and color to phenotype. (B) Percent of genome-wide methylated probes in cerebellar cortex or BA19 cortex in control and autism samples. Error bars represent standard deviations. There was no significant difference in global methylation between autism vs control cerebellum or BA19 cortex. There was a significant difference between brain regions, as expected (alpha = 0.05; Wilcoxon rank-sum test). (C) Pyrosequencing of bisulfite-treated DNA. Percent methylation at individual CpG dinucleotides is reported in cerebellar cortex samples (n $7 per group) for a priori candidate genes suspected to exhibit differential methylation. X-axis represents locus relative to transcription start site. Error bars represent standard deviations. doi:10.1371/journal.pone.0044736.g003 Fifth, we evaluated whole tissue extracts and might have missed epigenetic or transcriptional dysregulation specific to a subpop- ulation of clinically relevant cells. This study has several strengths. More than 100 uncommon or rare causes of autism are currently described yet autistic individuals with defined syndromic, metabolic or chromosomal etiologies comprise a minority of all individuals with autism. The pathophysiologic processes operative in some, or perhaps most, persons with these uncommon causes of autism may not necessarily be the same as the disease processes present in persons with idiopathic autism. Our subject selection process was, therefore, rigorous and included evaluation of clinical and molecular biologic data to produce a set of stringently defined idiopathic autistic male brains. We used high resolution micro- arrays to provide a detailed map of the epigenetic and transcriptional profiles of the analyzed brain regions. We also used complementary confirmatory techniques to evaluate candi- date genes. Conclusions Taken together, our analyses demonstrate significant transcrip- tional heterogeneity in autistic brains with decreased expression of mitochondrial oxidative phosphorylation and protein synthesis- related genes. The latter suggests that convergence of pathobiology in autism may occur at the level of fundamental cell processes such as mitochondrial energy metabolism and protein translation. Nonetheless, the transcriptional heterogeneity noted here also raises the possibility there may be diverse sets of biological pathways that are also dysregulated in different autistic individuals and this, in turn, may be relevant in subtyping individuals for clinical trials. Additionally, our data suggest that there are particular biological pathways associated with different symptom domains in autism. Perhaps clinical heterogeneity in autism is the result of differences in these specific pathways which act to control domain severities semi-independently. We found that DNA methylation of gene promoters is not widely disrupted in the autistic cerebellum or BA19 brain regions, suggesting that transcriptional changes are largely the result of other regulatory mechanisms. Supporting Information Figure S1 Top genes differentially expressed between autism and control brain accounting for region are enriched for regulation by HNF4A. Genes colored green are down-regulated and those colored red are up-regulated. Color intensity is proportional to log2-fold change between autistic and control groups. Transcription factor enrichment was carried out using Ingenuity Pathway Analysis transcription factor analysis, which showed a strong enrichment for HNF4A (p = 1.4E-8; Fisher’s exact test). (DOCM) PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44736