Figure S2 Pathways enriched for differentially ex- pressed genes in autistic outliers. A. The top gene network for differentially expressed genes in cerebellar outlier samples vs. non-outlier control samples is associated with NF-kB signaling. B. The second gene network for differentially expressed genes in cerebellar outlier samples vs. non-outlier controls is associated with cell cycle regulation. Gene network analysis was carried out using Ingenuity Pathway Analysis. (DOCM) Figure S3 Results of reverse transcriptase real-time quantitative PCR. A. GAPDH-normalized fold changes of autistic vs. control cerebellar cortex gene expression for genes differentially expressed on microarrays. Direction of expression change on the microarray and in rtPCR assays is shown with p- values for Wilcoxon rank sum test. B. GAPDH-normalized fold changes of autistic vs. control BA19 cortex gene expression for candidate genes from literature or cerebellar cortex results. C. GAPDH-normalized fold changes of autistic vs. control cerebellar cortex gene expression in candidate genes from literature. Significant findings at a threshold value of 0.05 are denoted with an asterisk in the figure. Data were obtained from all cerebellar samples and at least 5 BA19 samples per group. (DOCM) Figure S4 Pyrosequencing of bisulfite-converted DNA in CpG islands for OXTR and CEBPD. Percent methylation at individual CpG dinucleotides is reported in cerebellar cortex samples (n $7 per group). X-axis represents locus relative to transcription start site. Human lymphocyte genomic DNA and enzymatically methylated human lymphocyte genomic DNA were used as controls. Error bars represent standard deviations. (DOCM) Table S1 Characteristics of study subjects. All subjects were male and cases were matched to controls for age within one year. {All other ethnicity values were ‘‘white’’. (DOC) Table S2 Individual subject characteristics. Ethnicity was obtained from ATP database when available. COD, cause of death; A, Social interaction impairments domain; B, Communi- cation and language impairments domain; C, Repetitive and stereotyped behaviors domain; D = Symptom onset before 36 months of age; ADI-R total = sum of scales A through D. All subjects were represented in DNA methylation experiments. FMR1 repeat length refers to the number of CGG trinucleotide repeats in the 59 untranslated region of the FMR1 gene. (DOC) Table S3 Top differentially expressed probes between autistic and control brain after controlling for brain region. The top differentially expressed genes between autistic and control brain are listed at a FDR ,5% and a log2-fold change of .0.7. P-values were adjusted by the method of Benjamini and Hochberg. OMIM mendelian disorders were listed if applicable. Gene description was from Illumina annotation. FC, fold change; OMIM, Online Mendelian Inheritance in Man. (DOC) Table S4 Probes involved in oxidative phosphorylation differentially expressed between autistic and control brain after controlling for brain region. The top 300 differentially expressed probes between autistic and control brain at a FDR ,5% included 17 oxidative phosphorylation genes identified in Ingenuity Pathway Analysis (pathway enrichment for oxidative phosphorylation p = 5.8E-14; Fisher’s exact test). P- Transcriptomic and Epigenetic Findings in Autism values were adjusted by the method of Benjamini and Hochberg. OMIM mendelian disorders were listed if applicable. FC, fold change; OMIM, Online Mendelian Inheritance in Man. (DOC) Table S5 Probes associated with ribosomes differen- tially expressed between autistic and control brain after controlling for brain region. The top 300 differentially expressed probes between autistic and control brain at a FDR ,5% included 12 ribosomal/protein translation genes identified in Ingenuity Pathway Analysis (pathway enrichment for EIF2 signaling p = 1.0E-6; Fisher’s exact test). P-values were adjusted by the method of Benjamini and Hochberg. OMIM mendelian disorders were listed if applicable. FC, fold change; OMIM, Online Mendelian Inheritance in Man. (DOC) Table S6 Differentially expressed genes in autism outlier cerebellar vs. age-matched controls. The top 300 DE probes at a FDR of ,5% were consolidated into a non- redundant, mappable list of 75 genes. Log2-fold change in autism is reported for our dataset. There were six directly overlapping DE genes from Voineagu et al. and Garbett et al. top gene lists from temporal cortex, which was statistically significant (p = 1.4E-9, OR = 67 [95%CI: 23–170]; Fisher’s exact test). (DOC) Table S7 Previously implicated genes for DNA methyl- ation abnormalities were not differentially methylated between autistic and control BA19 or cerebellar cortex by microarray analysis. Probes for BCL2, MECP2, OXTR, RORA, and UBE3A were compared between 9 matched autistic and control BA19 or cerebellar cortical samples. P-values are for tests analogous to two-tailed, paired t-tests. Differences are for group mean M-values (M-values are logit transformed beta-values, where beta-value is approximately the proportion of methylation at a particular locus). No probes were statistically significant, controlling for multiple comparisons (adjusted p-value = 0.002). All nucleotide positions are for the NCBI36/hg18 genome build. (DOC) Table S8 Primers used for real-time reverse-transcrip- tase quantitative PCR. Primers were designed to span introns or lie on an exon-exon junction. See Methods for description of assay conditions. (DOCX) Table S9 Primers used for pyrosequencing of bisulfite- converted DNA. (DOC) Acknowledgments We are grateful to the families who provided specimens from their loved ones for research and for the efforts of the Autism Tissue Program, Harvard Brain Tissue Resource Center and the National Institute for Child Health and Human Development Brain and Tissue Bank. We also thank K. Drake, P. Faber, P. Hanna, D. Klinkebiel and F. Mularo for advice regarding analytic procedures and B. Triggs-Raine for helpful discussions and review of the manuscript. Author Contributions Conceived and designed the experiments: MRG MRN. Performed the experiments: MRG. Analyzed the data: MRG RAR TF AHT MRN. Wrote the paper: MRG MRN. Designed subject selection criteria and all experiments: MRG MRN. Provided input on subject selection: TF. Provided input on methylation analysis: AHT. Performed tissue handling: MRG. Performed statistical analyses: MRG RAR. Involvment in data PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44736