Figure 3. Increased paternal age at birth is negatively associated with overall variance in gene expression in peripheral blood lymphocytes (PBL) of normal children. Paternal age at birth was found in multiple studies to be a risk factor for autism [26,28,34,35]. Previous work indicated that factors or interventions that modified mouse hippocampal-dependent behavior also modified the overall variance in gene expression in the predicted direction. In controls (figure 3a) but not in children with autism (not shown), overall variance in log-transformed measures of gene expression was significantly and negatively associated with paternal age at birth (r = -.283, R2 = .08, p = .03, number of subjects = 57). For the evaluation of paternal age effects, paternal ages were available for 78 children with autism and 57 control children. To directly compare overall microarray variance in children with autism to children of older fathers, we divided subjects by the median paternal age at birth in our study (31 years) and created 2 groups: 1) children from younger fathers (less than 31 years) (65 subjects: 30 controls and 35 children with autism); and 2) children from older fathers (31 years or older) (70 subjects: 27 controls and 43 children with autism). We compared mean levels of overall variance between children with autism and controls of older and younger fathers. As predicted, we found that overall variance was the same in children of older fathers and children with autism with fathers of any age (figure 3b). We found that the association of autism with decreased overall variance was only found in children of younger fathers and represented a difference of .78 standard deviations within our sample (p = .0007) (figure 3b). Error bars represent standard error. doi:10.1371/journal.pone.0016715.g003 Dev/year of paternal age) (figure 3a). Using an analysis of covariance (ANCOVA) results remained significant after control- ling for subject age (p = .046, parameter estimate = 2.052 Std Autism Changes Global Gene Expression Regulation Dev/year of paternal age) and scan batch (p = .03, parameter estimate = 2.055 Std Dev/year of paternal age). When both potential confounds were entered into the model the parameter estimate remained unchanged, but the p-value was slightly above .05 (p = .056, parameter estimate = 2.052 Std Dev/year of paternal age). Thus, paternal age-related changes in gene expression variance in control subjects were similar in direction to autism-related changes in variance. We also assessed the relationship between paternal age and variance in gene expression using Spearman rank correlation (Spearman rho = 2.274, rho corrected for ties = 2.281, p = .04, p corrected for ties = .036). The use of a non-parametric test addressed any potential concerns about the effects of possible outliers (though no values were greater than 3 standard deviations from the mean) and of problems arising from the use of non-normal data distributions with parametric tests. The measurement of variance in the distribution of gene expression levels was not normally distributed within the group of control children. Because increased paternal age and autism were both negatively related to overall variance in gene expression, it was predicted that the overall variance in gene expression for children of older fathers would be similar to that in children with autism. To confirm this hypothesis, we performed a median split of subjects by paternal age (median = 31 years) and assessed for differences in overall variance in gene expression between children with autism and controls in groups of children from older and younger fathers. As predicted, we found that the overall variance was the same in the blood of children from older fathers as in the blood of children with autism from fathers of any age (figure 3b). We found that the association of autism with decreased overall variance was only found in children of younger fathers (p = .0007) (figure 3b). As expected from the negative correlation, in the median split analysis, variance in gene expression in the blood of control children of older fathers was significantly decreased compared to control children of younger fathers (p = .03) (figure 3b). Decreased variance in gene expression is related to the down-regulation of genes involved in the regulation of transcription (figure 4 and tables S2–S4) To evaluate for a possible mechanism underlying decreased variance in gene expression in the blood of children with autism or from older fathers, we analyzed for specific gene expression differences between study groups. Gene expression patterns in the blood of children with autism and control children of older fathers were compared to gene expression patterns in the blood of control children of younger fathers. First, it was noted that in the blood of children with autism and children of older fathers, there were many more significantly down-regulated genes than up-regulated genes (figure 4a). In the comparison of children with autism to children of younger fathers, there 2,093 genes that were significantly down-regulated by at least 1.1-fold and only 641 genes that were up-regulated. In the blood of children of older fathers compared to children of younger fathers there were 1,476 down-regulated and 764 up-regulated genes. To determine if the same genes were up or down-regulated in children with autism and children of older fathers, we compared lists of genes and assessed the degree of overlap. The overlaps between genes that were down or up-regulated in children with autism and children of older fathers were highly significant. There were 593 genes that were down-regulated in both children with autism and children of older fathers (figure 4b) (chi square 2,071, p,.000001) and 145 genes that were both up-regulated in both comparisons (chi square 849, p,.000001). PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e16715