Differentially methylated regions in bipolar disorder and suicide

Abstract

The addition of a methyl group to, typically, a cytosine-guanine dinucleotide (CpG) creates distinct DNA methylation patterns across the genome that can regulate gene expression. Aberrant DNA methylation of CpG sites has been associated with many psychiatric disorders including bipolar disorder (BD) and suicide. Using the SureSelect^XT system, Methyl-Seq, we investigated the DNA methylation status of CpG sites throughout the genome in 50 BD individuals (23 subjects who died by suicide and 27 subjects who died from other causes) and 31 nonpsychiatric controls. We identified differentially methylated regions (DMRs) from three analyses: (a) BD subjects compared to nonpsychiatric controls (BD-NC), (b) BD subjects who died by suicide compared to nonpsychiatric controls (BDS-NC), and (c) BDS subjects compared to BD subjects who died from other causes (BDS-BDNS). One DMR from the BDS-NC analysis, located in ARHGEF38, was significantly hypomethylated (23.4%) in BDS subjects. This finding remained significant after multiple testing (P_Bootstrapped = 9.0 × 10^-3 ), was validated using pyrosequencing, and was more significant in males. A secondary analysis utilized Ingenuity Pathway Analysis to identify enrichment in nominally significant DMRs. This identified an association with several pathways including axonal guidance signaling, calcium signaling, β-adrenergic signaling, and opioid signaling. Our comprehensive study provides further support that DNA methylation alterations influence the risk for BD and suicide. However, further investigation is required to confirm these associations and identify their functional consequences.

Keywords: ARHGEF38; opioid signaling; suicidal behavior; β-adrenergic signaling.

FIGURE 1

Schematic and pyrosequencing results for ARHGEF38. (a) A schematic of the two ARHGEF38 transcripts. Vertical black lines represent exons, black rectangles represent untranslated regions, and the arrows represent the direction of transcription. The differentially methylated region (DMR) identified in the BDS-NC analysis is highlighted by the blue box; it is located just downstream of the short transcript and in an intron of the long transcript. This DMR is also magnified (chr4:106553214–106553473; hg19 assembly) within the larger blue box with the CpG sites analyzed represented by blue circles. The DNase hypersensitivity site and transcription factor binding sites found in this region are represented by green or purple boxes, and all genomic locations are numbered. Pyrosequencing results for the ARHGEF38 DMR with standard error of the mean (SEM) for (b) all subjects, (c) male subjects, and (d) female subjects. ** denotes p ≤ .01 and *** denotes p < .001

FIGURE 2

A Venn diagram of pathways associated with bipolar disorder (BD) and suicide. Each analysis is labeled with the number of pathways identified from the top findings (p < .05) using Ingenuity Pathway Analysis. The top unique pathway found exclusively in only one of the analyses and the top pathway found in all analyses are annotated