Common DNA methylation alterations in multiple brain regions in autism

Abstract

Autism spectrum disorders (ASD) are increasingly common neurodevelopmental disorders defined clinically by a triad of features including impairment in social interaction, impairment in communication in social situations and restricted and repetitive patterns of behavior and interests, with considerable phenotypic heterogeneity among individuals. Although heritability estimates for ASD are high, conventional genetic-based efforts to identify genes involved in ASD have yielded only few reproducible candidate genes that account for only a small proportion of ASDs. There is mounting evidence to suggest environmental and epigenetic factors play a stronger role in the etiology of ASD than previously thought. To begin to understand the contribution of epigenetics to ASD, we have examined DNA methylation (DNAm) in a pilot study of postmortem brain tissue from 19 autism cases and 21 unrelated controls, among three brain regions including dorsolateral prefrontal cortex, temporal cortex and cerebellum. We measured over 485,000 CpG loci across a diverse set of functionally relevant genomic regions using the Infinium HumanMethylation450 BeadChip and identified four genome-wide significant differentially methylated regions (DMRs) using a bump hunting approach and a permutation-based multiple testing correction method. We replicated 3/4 DMRs identified in our genome-wide screen in a different set of samples and across different brain regions. The DMRs identified in this study represent suggestive evidence for commonly altered methylation sites in ASD and provide several promising new candidate genes.

Figure 1

(a) Summary of post-mortem brain samples and analytic procedures utilized. (b and c) Modified region-based (“Dry”) Manhattan plots for epigenome-based analysis. Region-specific DNA methylation was calculated from 450K data, and p values were determined by permutation analysis to accommodate the genome-wide screen. (b) Three differentially methylated regions (DMRs) in the temporal cortex brain region are significantly associated with ASD. (c) One DMR in the cerebellum is significantly associated with ASD. Each red segment represents the adjusted p-value for a genomic region on the Illumina Infinium 450K Methylation BeadChip. The significance threshold was based on a family-wise error rate of 0.1.

Figure 2

Two genome-wide significant differentially methylated regions (DMRs) located at PRRT1 (a–c) and C11orf21/TSPAN32 (d–f) are relatively hypomethylated in temporal cortex tissue from autistic individuals. (a and d) DMR plots for temporal cortex brain samples. (b and e) DMR plots for prefrontal cortex brain samples. (c and f) DMR plots for cerebellum brain samples. For (a and d), methylation data is shown in the top panels. Smoothed lines denote mean methylation levels for individuals with (green) and without (purple) ASD across the DMR. Each point represents the methylation level of a particular individual, green for those with autism and purple for those without autism, at a specific genomic location. The methylation values range from 0 to 1 with 0 equal to 0% methylation and 1 equal to 100% methylation. The upper middle panels provide information about potential copy number changes. Each point, green for autistic and purple for normal individuals, represents the difference between total probe intensity for a given individual and the mean total intensity across all individuals for a given probe. The black points in the lower middle panel are individual probe-based nominal p-values for differences in methylation between the two groups (autism and normal). The dashed magenta line indicates the significance threshold used (nominal p < 0.05) such that points above the line are significant in replication analyses. The bottom panel displays relevant genome annotation information. For (b, c, e, and f), plots are as described above but blue and orange points represent individuals with and without autism, respectively. Dotted boxes describe the location of methylation values that correspond to probes with significant nominal p-values.

Figure 3

A genome-wide significant differentially methylated region (DMR) located 3.45 Kb upstream of ZNF57 is relatively hypermethylated in temporal cortex tissue from autistic individuals. (a) DMR plots for temporal cortex brain samples. (b) DMR plots for temporal cortex brain samples from male individuals. (c) DMR plots for temporal cortex brain samples from female individuals. For (a–c), methylation data is shown in the top panel. Lines denote mean methylation levels for individuals with (green) and without (purple) autism across the DMR. Each point represents the methylation level of a particular individual, green for those with autism and purple for those without autism, at a specific genomic location. The methylation values range from 0 to 1 with 0 equal to 0% methylation and 1 equal to 100% methylation. The upper middle panel provides information about potential copy number changes. Each point, green for autistic and purple for normal individuals, represents the difference between total probe intensity for a given individual and the mean total intensity across all individuals for a given probe. The black points in the lower middle panel are individual probe-based nominal p-values for differences in methylation between the two groups (autism and normal). The dashed magenta line indicates the significance threshold used (nominal p < 0.05) such that points above the line are significant in replication analyses. The bottom panel displays relevant genome annotation information. (d) DMR plots for all prefrontal cortex brain samples. (e) DMR plots for prefrontal cortex tissue samples from male individuals. (f) DMR plots for prefrontal cortex tissue samples from female individuals. (g) DMR plots for all cerebellum brain samples. (h) DMR plots for cerebellum samples from male individuals. (i) DMR plots for cerebellum samples from female individuals. For (d–h), plots are as described above but blue and orange points represent individuals with and without autism, respectively. Dotted boxes describe the location of methylation values that correspond to probes with significant nominal p-values.

Figure 4

A genome-wide significant differentially methylated region (DMR) at SDHAP3 is relatively hypermethylated in cerebellum tissue from autistic individuals. (a) DMR plots for cerebellum brain samples. (b) DMR plots for prefrontal cortex brain samples. (c) DMR plots for temporal cortex brain samples. For (a), methylation data is shown in the top panel. Lines denote mean methylation levels for individuals with (green) and without (purple) autism across the DMR. Each point represents the methylation level of a particular individual, green for those with autism and purple for those without autism, at a specific genomic location. The methylation values range from 0 to 1 with 0 equal to 0% methylation and 1 equal to 100% methylation. The upper middle panel provides information about potential copy number changes. Each point, green for autistic and purple for normal individuals, represents the difference between total probe intensity for a given individual and the mean total intensity across all individuals for a given probe. The black points in the lower middle panel are individual probe-based nominal p-values for differences in methylation between the two groups (autism and normal). The dashed magenta line indicates the significance threshold used (nominal p < 0.05) such that points above the line are significant in replication analyses. The bottom panel displays relevant genome annotation information. For (b and c), plots are as described above but blue and orange points represent individuals with and without autism, respectively. Dotted boxes describe the location of methylation values that correspond to probes with significant nominal p-values.