Autism Changes Global Gene Expression Regulation Figure 4. Down-regulated genes are enriched for biological pathways related to transcription and zinc. Traditional gene expression analyses were performed. Microarrays were processed and summarized using RMA. Post-summarization gene expression levels were quantile normalization because of the known group level differences at the level of the distribution. Probe sets were filtered for those that were called present in at least 50 of 146 samples. Figure 4a shows that there were more down-regulated than up-regulated genes in the blood of children with autism and children of older fathers when compared to children of younger fathers. 4b shows a very significant overlap (chi-squared = 2,085, p,.00001) of down-regulated genes in children with autism and children of older fathers. Figure 4c demonstrates that overlapping down-regulated genes are enriched for biological pathways associated with transcription and zinc. doi:10.1371/journal.pone.0016715.g004 To determine if these overlapping genes were enriched for particular biological pathways, DAVID was used to perform pathway analysis. As background, we used the 25,146 probe sets that were above our background cut-off requiring that they were called present in at least 50 out of 146 samples. Pathway analysis indicated that multiple pathways associated with transcriptional regulation and supported by multiple independent databases, e.g. GOTERM, BIOCARTA, OMIM disease, etc., were enriched PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e16715