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. 2014 Sep 2;4(9):e433.
doi: 10.1038/tp.2014.70.

DNA methylation analysis of the autistic brain reveals multiple dysregulated biological pathways

S Nardone  1 , D Sharan Sams  1 , E Reuveni  1 , D Getselter  1 , O Oron  1 , M Karpuj  1 , E Elliott  1
Affiliations

DNA methylation analysis of the autistic brain reveals multiple dysregulated biological pathways

S Nardone et al. Transl Psychiatry. .

Abstract

Autism spectrum disorders (ASD) are a group of neurodevelopmental conditions characterized by dysfunction in social interaction, communication and stereotypic behavior. Genetic and environmental factors have been implicated in the development of ASD, but the molecular mechanisms underlying their interaction are not clear. Epigenetic modifications have been suggested as molecular mechanism that can mediate the interaction between the environment and the genome to produce adaptive or maladaptive behaviors. Here, using the Illumina 450 K methylation array we have determined the existence of many dysregulated CpGs in two cortical regions, Brodmann area 10 (BA10) and Brodmann area 24 (BA24), of individuals who had ASD. In BA10 we found a very significant enrichment for genomic areas responsible for immune functions among the hypomethylated CpGs, whereas genes related to synaptic membrane were enriched among hypermethylated CpGs. By comparing our methylome data with previously published transcriptome data, and by performing real-time PCR on selected genes that were dysregulated in our study, we show that hypomethylated genes are often overexpressed, and that there is an inverse correlation between gene expression and DNA methylation within the individuals. Among these genes there were C1Q, C3, ITGB2 (C3R), TNF-α, IRF8 and SPI1, which have recently been implicated in synaptic pruning and microglial cell specification. Finally, we determined the epigenetic dysregulation of the gene HDAC4, and we confirm that the locus encompassing C11orf21/TSPAN32 has multiple hypomethylated CpGs in the autistic brain, as previously demonstrated. Our data suggest a possible role for epigenetic processes in the etiology of ASD.

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Figures

Figure 1
Figure 1
DNA methylation changes in autistic cerebral cortex regions. (a, b) Heat map of differentially methylated CpG sites between autism and control cohorts in prefrontal cortex (a) and cingulate gyrus (b). Scaled methylation values are color-coded according to the legend on the left. The dendrogram delineates the hierarchical clustering based on the >5% differentially methylated CpG sites after FDR (<0.05) correction. The top bar indicates the disease status: red, autism; black, control. The bottom bars evidence further variables for each sample: sex (gray, male; black, female), comorbidity of seizures (green, autism case with seizure disorder; red, autism case without seizure disorder; black, control), age, pH assessment and postmortem interval (PMI). The corresponding scale for the quantitative variables is shown on the left. (c, d) Pie charts depicting the neighborhood and genomic locations of CpGs represented in the microarray (middle) and those differentially methylated between controls and autism in BA10 (left) and BA24 (right). Only CpG sites >5% differentially methylated at an FDR <0.05 were plotted. (e) Diagram depicting the number of CpGs showing significant methylation differences between BA10 (blue) and BA24 (red) in control samples (top) and autism samples (bottom) at FDR <0.05 and difference of methylation >5%. (f) Scatter plot illustrating the second principle component (PC2; y axis) describing the divergence in methylation between two cortical areas, BA10 and BA24, in autism and control groups. BA, Brodmann area; FDR, false discovery rate.
Figure 2
Figure 2
Gene Ontology analysis of differentially methylated CpGs in autistic prefrontal cortex. (a) Gene Ontology analysis for biological process and InterPro categories for CpG sites that were hypomethylated by >5% in the autism group at FDR <0.05. (b) Gene Ontology analysis for biological process and InterPro categories for CpG sites that were hypermethylated by >5% in the autism group at FDR <0.05. The analysis was performed using GREAT. (c) Venn diagram depicting the overlap between hypomethylated genes and hyperexpressed genes (top) and between hypermethylated genes and hypoexpressed genes (bottom), respectively from our methylation data (BA10) and Voineagu et al. transcriptome data (BA9). BA, Brodmann area; FDR, false discovery rate.
Figure 3
Figure 3
Directional association between gene expression and methylation in prefrontal cortex. Gene expression analysis of eight immune system-related genes that displayed multiple hypomethylated CpGs in the autism group (C3, C1qA, ITGB2, TNF-α, IRF8, SPI1, PTPN6 and HLA-DMB). For each gene, we show the gene expression analysis (left) and Spearman's ρ correlation coefficient (right) between individuals' gene expression and DNA methylation levels. All of these genes are significantly overexpressed in the autism group and display significant inverse correlations between DNA methylation levels and gene expression levels.
Figure 4
Figure 4
Dysregulated DNA methylation in the genes HDAC4 and TSPAN32. (a) Gene expression and directional association with β methylation values are depicted for HDAC4. The position of the significant CpGs in relation to the gene, CpG islands and the Illumina 450 K methylation array are shown on the top of the three box plots. Gene expression (left) and inverse (center) and direct (right) correlations between gene expression and CpG methylation levels are shown below. P-values for both expression and Spearman's ρ correlation coefficient were <0.05. (b) Differences in methylation between autistic and control cohorts are described for C11orf21/TSPAN32. The position of significant CpGs in relation to the gene, CpG islands and the Illumina 450 K methylation array are shown above. A bar chart provides a detailed description of each single CpG site for C11orf21/TSPAN32 for both autistic and control cohorts (below) with relative P-value indicated above each pair of bars (*P<0.05; **P<0.01; ***P<0.001). For one CpG site (highlighted in red) is shown a box plot describing the β methylation value in autism and control cohorts, and a Spearman's ρ correlation coefficient between the methylation β values (microarray) and the pyrosequencing methylation values. P-values for both methylation and Spearman's ρ correlation coefficient were <0.05.
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