Whole-genome sequencing analysis of suicide deaths integrating brain-regulatory eQTLs data to identify risk loci and genes.

Recent large-scale genome-wide association studies (GWAS) have started to identify potential genetic risk loci associated with risk of suicide; however, a large portion of suicide-associated genetic factors affecting gene expression remain elusive. Dysregulated gene expression, not assessed by GWAS, may play a significant role in increasing the risk of suicide death. We performed the first comprehensive genomic association analysis prioritizing brain expression quantitative trait loci (eQTLs) within regulatory regions in suicide deaths from the Utah Suicide Genetic Risk Study (USGRS). 440,324 brain-regulatory eQTLs were obtained by integrating brain eQTLs, histone modification ChIP-seq, ATAC-seq, DNase-seq, and Hi-C results from publicly available data. Subsequent genomic analyses were conducted in whole-genome sequencing (WGS) data from 986 suicide deaths of non-Finnish European (NFE) ancestry and 415 ancestrally matched controls. Additional independent USGRS suicide deaths with genotyping array data (n = 4657) and controls from the Genome Aggregation Database were explored for WGS result replication. One significant eQTL locus, rs926308 (p = 3.24e-06), was identified. The rs926308-T is associated with lower expression of RFPL3S, a gene important for neocortex development and implicated in arousal. Gene-based analyses performed using Sherlock Bayesian statistical integrative analysis also detected 20 genes with expression changes that may contribute to suicide risk. From analyzing publicly available transcriptomic data, ten of these genes have previous evidence of differential expression in suicide death or in psychiatric disorders that may be associated with suicide, including schizophrenia and autism (ZNF501, ZNF502, CNN3, IGF1R, KLHL36, NBL1, PDCD6IP, SNX19, BCAP29, and ARSA). Electronic health records (EHR) data was further merged to evaluate if there were clinically relevant subsets of suicide deaths associated with genetic variants. In summary, our study identified one risk locus and ten genes associated with suicide risk via gene expression, providing new insight into possible genetic and molecular mechanisms leading to suicide.

Distinct patterns of altered quantitative T1ρ and functional BOLD response associated with history of suicide attempts in bipolar disorder.

Despite the high risk for suicide, relatively few studies have explored the relationship between suicide and brain imaging measures in bipolar disorder. In addition, fewer studies have explored the possibility that altered brain metabolism may be associated with suicide attempt. To begin to fill in these gaps, we evaluated functional (task based fMRI) and metabolic (quantitative T1ρ) differences associated with suicide attempt in participants with bipolar disorder. Thirty-nine participants with bipolar disorder underwent fMRI during a flashing checkerboard task and 27 also underwent quantitative T1ρ. The relationship between neuroimaging and history of suicide attempt was tested using multiple regression while adjusting for age, sex, and current mood state. Differences between two measures of suicide attempt (binary: yes/no and continuous: number of attempts) were quantified using the corrected Akaike Information Criterion. Participants who had attempted suicide had greater fMRI task-related activation in visual areas and the cerebellum. The number of suicide attempts was associated with a difference in BOLD response in the amygdala, prefrontal cortex, and cerebellum. Increased quantitative T1ρ was associated with number of suicide attempts in limbic, basal ganglia, and prefrontal cortex regions. This study is a secondary analysis with a modest sample size. Differences between measures of suicide history may be due to differences in statistical power. History of suicide was associated with limbic, prefrontal, and cerebellar alterations. Results comparing those with and without suicide attempts differed from results using number of suicide attempts, suggesting that these variables have different neurobiological underpinnings.

Assessment of suicide attempt and death in bipolar affective disorder: a combined clinical and genetic approach.

Bipolar disorder (BP) suicide death rates are 10-30 times greater than the general population, likely arising from environmental and genetic risk factors. Though suicidal behavior in BP has been investigated, studies have not addressed combined clinical and genetic factors specific to suicide death. To address this gap, a large, harmonized BP cohort was assessed to identify clinical risk factors for suicide death and attempt which then directed testing of underlying polygenic risks. 5901 individuals of European ancestry were assessed: 353 individuals with BP and 2498 without BP who died from suicide (BPS and NBPS, respectively) from a population-derived sample along with a volunteer-derived sample of 799 individuals with BP and a history of suicide attempt (BPSA), 824 individuals with BP and no prior attempts (BPNSA), and 1427 individuals without several common psychiatric illnesses per self-report (C). Clinical and subsequent directed genetic analyses utilized multivariable logistic models accounting for critical covariates and multiple testing. There was overrepresentation of diagnosis of PTSD (OR = 4.9, 95%CI: 3.1-7.6) in BPS versus BPSA, driven by female subjects. PRS assessments showed elevations in BPS including PTSD (OR = 1.3, 95%CI:1.1-1.5, versus C), female-derived ADHD (OR = 1.2, 95%CI:1.1-1.4, versus C), and male insomnia (OR = 1.4, 95%CI: 1.1-1.7, versus BPSA). The results provide support from genetic and clinical standpoints for dysregulated traumatic response particularly increasing risk of suicide death among individuals with BP of Northern European ancestry. Such findings may direct more aggressive treatment and prevention of trauma sequelae within at-risk bipolar individuals.

Investigating rare pathogenic/likely pathogenic exonic variation in bipolar disorder.

Bipolar disorder (BD) is a serious mental illness with substantial common variant heritability. However, the role of rare coding variation in BD is not well established. We examined the protein-coding (exonic) sequences of 3,987 unrelated individuals with BD and 5,322 controls of predominantly European ancestry across four cohorts from the Bipolar Sequencing Consortium (BSC). We assessed the burden of rare, protein-altering, single nucleotide variants classified as pathogenic or likely pathogenic (P-LP) both exome-wide and within several groups of genes with phenotypic or biologic plausibility in BD. While we observed an increased burden of rare coding P-LP variants within 165 genes identified as BD GWAS regions in 3,987 BD cases (meta-analysis OR = 1.9, 95% CI = 1.3-2.8, one-sided p = 6.0 × 10-4), this enrichment did not replicate in an additional 9,929 BD cases and 14,018 controls (OR = 0.9, one-side p = 0.70). Although BD shares common variant heritability with schizophrenia, in the BSC sample we did not observe a significant enrichment of P-LP variants in SCZ GWAS genes, in two classes of neuronal synaptic genes (RBFOX2 and FMRP) associated with SCZ or in loss-of-function intolerant genes. In this study, the largest analysis of exonic variation in BD, individuals with BD do not carry a replicable enrichment of rare P-LP variants across the exome or in any of several groups of genes with biologic plausibility. Moreover, despite a strong shared susceptibility between BD and SCZ through common genetic variation, we do not observe an association between BD risk and rare P-LP coding variants in genes known to modulate risk for SCZ.

Male-specific association of the 2p25 region with suicide attempt in bipolar disorder.

We previously conducted a genome-wide association study (GWAS) of attempted suicide within bipolar disorder, which implicated common variation in the 2p25 region primarily in males. The top association signal from our GWAS occurred in an intergenic region of 2p25 (p = 5.07 × 10-8) and was supported by two independent studies. In the current study, to better characterize the association of the 2p25 region with attempted suicide, we sequenced the entire 350kb 2p25 region in 476 bipolar suicide attempters and 473 bipolar non-attempters using targeted next-generation sequencing. This fine-mapping project identified 4,681 variants in the 2p25 region. We performed both gene-level and individual-variant tests on our sequencing results and identified 375 variants which were nominally significant (p < 0.05) and three common variants that were significantly associated with attempted suicide in males (corrected p = 0.035, odds ratio (OR) = 2.13). These three variants are in strong linkage disequilibrium with the top variant from our GWAS. Our top five variants are also predicted expression quantitative trait loci (eQTL) for three genes in the 2p25 region based on publicly available brain expression databases. Our sequencing and eQTL data implicate these three genes - SH3YL1, ACP1, and FAM150B - and three additional pathways - androgen receptor, Wnt signaling, and glutamatergic/GABAergic signaling - in the association of the 2p25 region with suicide. The current study provides additional support for an association of the 2p25 region with attempted suicide in males and identifies several candidate genes and pathways that warrant further investigation to understand their role in suicidal behavior.

Targeted sequencing of the LRRTM gene family in suicide attempters with bipolar disorder.

Glutamatergic signaling is the primary excitatory neurotransmission pathway in the brain, and its relationship to neuropsychiatric disorders is of considerable interest. Our previous attempted suicide genome-wide association study, and numerous studies investigating gene expression, genetic variation, and DNA methylation have implicated aberrant glutamatergic signaling in suicide risk. The glutamatergic pathway gene LRRTM4 was an associated gene identified in our attempted suicide genome-wide association study, with association support seen primarily in females. Recent evidence has also shown that glutamatergic signaling is partly regulated by sex-related hormones. The LRRTM gene family encodes neuronal leucine-rich transmembrane proteins that localize to and promote glutamatergic synapse development. In this study, we sequenced the coding and regulatory regions of all four LRRTM gene members plus a large intronic region of LRRTM4 in 476 bipolar disorder suicide attempters and 473 bipolar disorder nonattempters. We identified two male-specific variants, one female- and five male-specific haplotypes significantly associated with attempted suicide in LRRTM4. Furthermore, variants within significant haplotypes may be brain expression quantitative trait loci for LRRTM4 and some of these variants overlap with predicted hormone response elements. Overall, these results provide supporting evidence for a sex-specific association of genetic variation in LRRTM4 with attempted suicide.

Differentially methylated regions in bipolar disorder and suicide.

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 SureSelectXT 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 (PBootstrapped = 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, ß-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.

Chronic social stress induces DNA methylation changes at an evolutionary conserved intergenic region in chromosome X.

Chronic stress resulting from prolonged exposure to negative life events increases the risk of mood and anxiety disorders. Although chronic stress can change gene expression relevant for behavior, molecular regulators of this change have not been fully determined. One process that could play a role is DNA methylation, an epigenetic process whereby a methyl group is added onto nucleotides, predominantly cytosine in the CpG context, and which can be induced by chronic stress. It is unknown to what extent chronic social defeat, a model of human social stress, influences DNA methylation patterns across the genome. Our study addressed this question by using a targeted-capture approach called Methyl-Seq to investigate DNA methylation patterns of the dentate gyrus at putative regulatory regions across the mouse genome from mice exposed to 14 days of social defeat. Findings were replicated in independent cohorts by bisulfite-pyrosequencing. Two differentially methylated regions (DMRs) were identified. One DMR was located at intron 9 of Drosha, and it showed reduced methylation in stressed mice. This observation replicated in one of two independent cohorts. A second DMR was identified at an intergenic region of chromosome X, and methylation in this region was increased in stressed mice. This methylation difference replicated in two independent cohorts and in Major Depressive Disorder (MDD) postmortem brains. These results highlight a region not previously known to be differentially methylated by chronic social defeat stress and which may be involved in MDD.

Assessment of Whole-Exome Sequence Data in Attempted Suicide within a Bipolar Disorder Cohort.

Suicidal behavior is a complex and devastating phenotype with a heritable component that has not been fully explained by existing common genetic variant analyses. This study represents the first large-scale DNA sequencing project designed to assess the role of rare functional genetic variation in suicidal behavior risk. To accomplish this, whole-exome sequencing data for ∼19,000 genes were generated for 387 bipolar disorder subjects with a history of suicide attempt and 631 bipolar disorder subjects with no prior suicide attempts. Rare functional variants were assessed in all exome genes as well as pathways hypothesized to contribute to suicidal behavior risk. No result survived conservative Bonferroni correction, though many suggestive findings have arisen that merit additional attention. In addition, nominal support for past associations in genes, such as BDNF, and pathways, such as the hypothalamic-pituitary-adrenal axis, was also observed. Finally, a novel pathway was identified that is driven by aldehyde dehydrogenase genes. Ultimately, this investigation explores variation left largely untouched by existing efforts in suicidal behavior, providing a wealth of novel information to add to future investigations, such as meta-analyses.

Targeted Sequencing of FKBP5 in Suicide Attempters with Bipolar Disorder.

FKBP5 is a critical component of the Hypothalamic-Pituitary-Adrenal (HPA) axis, a system which regulates our response to stress. It forms part of a complex of chaperones, which inhibits binding of cortisol and glucocorticoid receptor translocation to the nucleus. Variations in both the HPA axis and FKBP5 have been associated with suicidal behavior. We developed a systematic, targeted sequencing approach to investigate coding and regulatory regions in or near FKBP5 in 476 bipolar disorder suicide attempters and 473 bipolar disorder non-attempters. Following stringent quality control checks, we performed single-variant, gene-level and haplotype tests on the resulting 481 variants. Secondary analyses investigated whether sex-specific variations in FKBP5 increased the risk of attempted suicide. One variant, rs141713011, showed an excess of minor alleles in suicide attempters that was statistically significant following correction for multiple testing (Odds Ratio = 6.65, P-value = 7.5 x 10-4, Permuted P-value = 0.038). However, this result could not be replicated in an independent cohort (Odds Ratio = 0.90, P-value = 0.78). Three female-specific and four male-specific variants of nominal significance were also identified (P-value < 0.05). The gene-level and haplotype association tests did not produce any significant results. This comprehensive study of common and rare variants in FKBP5 focused on both regulatory and coding regions in relation to attempted suicide. One rare variant remained significant following correction for multiple testing but could not be replicated. Further investigation is required in larger sample sets to fully elucidate the association of this variant with suicidal behavior.

A targeted sequencing study of glutamatergic candidate genes in suicide attempters with bipolar disorder.

Suicidal behavior has been shown to have a heritable component that is partly driven by psychiatric disorders [Brent and Mann, 2005]. However, there is also an independent factor contributing to the heritability of suicidal behavior. We previously conducted a genome-wide association study (GWAS) of bipolar suicide attempters and bipolar non-attempters to assess this independent factor [Willour et al., 2012]. This GWAS implicated glutamatergic neurotransmission in attempted suicide. In the current study, we have conducted a targeted next-generation sequencing study of the glutamatergic N-methyl-D-aspartate (NMDA) receptor, neurexin, and neuroligin gene families in 476 bipolar suicide attempters and 473 bipolar non-attempters. The goal of this study was to gather sequence information from coding and regulatory regions of these glutamatergic genes to identify variants associated with attempted suicide. We identified 186 coding variants and 4,298 regulatory variants predicted to be functional in these genes. No individual variants were overrepresented in cases or controls to a degree that was statistically significant after correction for multiple testing. Additionally, none of the gene-level results were statistically significant following correction. While this study provides no direct support for a role of the examined glutamatergic candidate genes, further sequencing in expanded gene sets and datasets will be required to ultimately determine whether genetic variation in glutamatergic signaling influences suicidal behavior. © 2016 Wiley Periodicals, Inc.

Whole-gene sequencing investigation of SAT1 in attempted suicide.

Suicidal behavior imposes a tremendous cost, with current US estimates reporting approximately 1.3 million suicide attempts and more than 40,000 suicide deaths each year. Several recent research efforts have identified an association between suicidal behavior and the expression level of the spermidine/spermine N1-acetyltransferase 1 (SAT1) gene. To date, several SAT1 genetic variants have been inconsistently associated with altered gene expression and/or directly with suicidal behavior. To clarify the role SAT1 genetic variation plays in suicidal behavior risk, we present a whole-gene sequencing effort of SAT1 in 476 bipolar disorder subjects with a history of suicide attempt and 473 subjects with bipolar disorder but no suicide attempts. Agilent SureSelect target enrichment was used to sequence all exons, introns, promoter regions, and putative regulatory regions identified from the ENCODE project within 10 kb of SAT1. Individual variant, haplotype, and collapsing variant tests were performed. Our results identified no variant or assessed region of SAT1 that showed a significant association with attempted suicide, nor did any assessment show evidence for replication of previously reported associations. Overall, no evidence for SAT1 sequence variation contributing to the risk for attempted suicide could be identified. It is possible that past associations of SAT1 expression with suicidal behavior arise from variation not captured in this study, or that causal variants in the region are too rare to be detected within our sample. Larger sample sizes and broader sequencing efforts will likely be required to identify the source of SAT1 expression level associations with suicidal behavior. © 2016 Wiley Periodicals, Inc.

Exome Sequencing of Familial Bipolar Disorder.

IMPORTANCE: Complex disorders, such as bipolar disorder (BD), likely result from the influence of both common and rare susceptibility alleles. While common variation has been widely studied, rare variant discovery has only recently become feasible with next-generation sequencing. OBJECTIVE: To utilize a combined family-based and case-control approach to exome sequencing in BD using multiplex families as an initial discovery strategy, followed by association testing in a large case-control meta-analysis. DESIGN, SETTING, AND PARTICIPANTS: We performed exome sequencing of 36 affected members with BD from 8 multiplex families and tested rare, segregating variants in 3 independent case-control samples consisting of 3541 BD cases and 4774 controls. MAIN OUTCOMES AND MEASURES: We used penalized logistic regression and 1-sided gene-burden analyses to test for association of rare, segregating damaging variants with BD. Permutation-based analyses were performed to test for overall enrichment with previously identified gene sets. RESULTS: We found 84 rare (frequency <1%), segregating variants that were bioinformatically predicted to be damaging. These variants were found in 82 genes that were enriched for gene sets previously identified in de novo studies of autism (19 observed vs. 10.9 expected, P = .0066) and schizophrenia (11 observed vs. 5.1 expected, P = .0062) and for targets of the fragile X mental retardation protein (FMRP) pathway (10 observed vs. 4.4 expected, P = .0076). The case-control meta-analyses yielded 19 genes that were nominally associated with BD based either on individual variants or a gene-burden approach. Although no gene was individually significant after correction for multiple testing, this group of genes continued to show evidence for significant enrichment of de novo autism genes (6 observed vs 2.6 expected, P = .028). CONCLUSIONS AND RELEVANCE: Our results are consistent with the presence of prominent locus and allelic heterogeneity in BD and suggest that very large samples will be required to definitively identify individual rare variants or genes conferring risk for this disorder. However, we also identify significant associations with gene sets composed of previously discovered de novo variants in autism and schizophrenia, as well as targets of the FRMP pathway, providing preliminary support for the overlap of potential autism and schizophrenia risk genes with rare, segregating variants in families with BD.

Investigating the role of early childhood abuse and HPA axis genes in suicide attempters with bipolar disorder.

OBJECTIVE: Genes and the environment both play a major role in the risk for attempted suicide, and environments harboring stressors, such as early childhood abuse, have been linked to suicidal behavior. Such environments also disrupt the hypothalamic-pituitary-adrenal (HPA) axis pathway, which has been hypothesized to play a role in suicidal behavior. We investigated whether the risk for attempted suicide was attributable in part to the interaction between childhood physical and/or sexual abuse and genetic variation in 19 genes (±5 kb) integral to the HPA axis pathway. MATERIALS AND METHODS: Using the Genetic Association Information Network Bipolar Disorder and Translational Genomics Research Institute cohorts, we implemented PLINK's logistic regression-based 'interaction' approach to search for evidence of an interaction between 235 genotyped HPA axis single-nucleotide polymorphisms and early childhood abuse. Our study included 631 bipolar disorder suicide attempters and 657 bipolar disorder nonattempters with information on abuse. RESULTS: After correction for multiple testing, no significant interaction between the 235 HPA axis single-nucleotide polymorphisms and early childhood abuse was found. In our study, the strongest interaction was found with rs2664008 in the corticotropin-releasing hormone receptor 1 (CRHR1) gene, with a nominal interaction P-value of 1.22×10 and an interaction odds ratio of 0.47. CONCLUSION: Our findings suggest that further work and larger sample sizes are required to elucidate the link between early childhood abuse and the HPA axis in suicidal behavior.

Association study of X chromosome SNPs in attempted suicide.

We report the results of a high-density attempted suicide association study of the X chromosome, which genotyped 23,141 SNPs on 983 attempters and 1143 non-attempters and generated modest evidence for association for SH3KBP1 (P=1.07×10(-4)) and GRIA3 (P=4.01×10(-4)). These findings highlight the need for larger sample sets and meta-analytic approaches.

Association study of serotonin pathway genes in attempted suicide.

Epidemiological studies, such as family, twin, and adoption studies, demonstrate the presence of a heritable component to both attempted and completed suicide. Some of this heritability is accounted for by the presence of comorbid psychiatric disorders, but the evidence also indicates that a portion of this heritability is specific to suicidality. The serotonergic system has been studied extensively in this phenotype, but findings have been inconsistent, possibly due to the presence of multiple susceptibility variants and/or gene-gene interactions. In this study, we genotyped 174 tag and coding single nucleotide polymorphisms (SNPs) from 17 genes within the serotonin pathway on 516 subjects with a major mood disorder and a history of a suicide attempt (cases) and 515 healthy controls, with the goal of capturing the common genetic variation across each of these candidate genes. We tested the 174 markers in single-SNP, haplotype, gene-based, and epistasis analyses. While these association analyses identified multiple marginally significant SNPs, haplotypes, genes, and interactions, none of them survived correction for multiple testing. Additional studies, including assessment in larger sample sets and deep resequencing to identify rare causal variants, may be required to fully understand the role that the serotonin pathway plays in suicidal behavior.

Monoamine oxidase A regulates antisocial personality in whites with no history of physical abuse.

OBJECTIVE: Preclinical and human family studies clearly link monoamine oxidase A (MAOA) to aggression and antisocial personality (ASP). The 30-base pair variable number tandem repeat in the MAOA promoter regulates MAOA levels, but its effects on ASP in humans are unclear. METHODS: We evaluated the association of the variable number tandem repeat of the MAOA promoter with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, ASP disorder (ASPD) traits in a community sample of 435 participants from the Hopkins Epidemiology of Personality Disorders Study. RESULTS: We did not find an association between the activity of the MAOA allele and ASPD traits; however, among whites, when subjects with a history of childhood physical abuse were excluded, the remaining subjects with low-activity alleles had ASPD trait counts that were 41% greater than those with high-activity alleles (P < .05). CONCLUSION: The high-activity MAOA allele is protective against ASP among whites with no history of physical abuse, lending support to a link between MAOA expression and antisocial behavior.

Genome-wide association study of suicide attempts in mood disorder patients.

OBJECTIVE: Family and twin studies suggest that liability for suicide attempts is heritable and distinct from mood disorder susceptibility. The authors therefore examined the association between common genomewide variation and lifetime suicide attempts. METHOD: The authors analyzed data on lifetime suicide attempts from genomewide association studies of bipolar I and II disorder as well as major depressive disorder. Bipolar disorder subjects were drawn from the Systematic Treatment Enhancement Program for Bipolar Disorder cohort, the Wellcome Trust Case Control Consortium bipolar cohort, and the University College London cohort. Replication was pursued in the NIMH Genetic Association Information Network bipolar disorder project and a German clinical cohort. Depression subjects were drawn from the Sequential Treatment Alternatives to Relieve Depression cohort, with replication in the Netherlands Study of Depression and Anxiety/Netherlands Twin Register depression cohort. RESULTS: Strongest evidence of association for suicide attempt in bipolar disorder was observed in a region without identified genes (rs1466846); five loci also showed suggestive evidence of association. In major depression, strongest evidence of association was observed for a single nucleotide polymorphism in ABI3BP, with six loci also showing suggestive association. Replication cohorts did not provide further support for these loci. However, meta-analysis incorporating approximately 8,700 mood disorder subjects identified four additional regions that met the threshold for suggestive association, including the locus containing the gene coding for protein kinase C-epsilon, previously implicated in models of mood and anxiety. CONCLUSIONS: The results suggest that inherited risk for suicide among mood disorder patients is unlikely to be the result of individual common variants of large effect. They nonetheless provide suggestive evidence for multiple loci, which merit further investigation.

Chronic corticosterone exposure increases expression and decreases deoxyribonucleic acid methylation of Fkbp5 in mice.

There is evidence for hypercortisolemia playing a role in the generation of psychiatric symptoms and for epigenetic variation within hypothalamic-pituitary-adrenal (HPA) axis genes mediating behavioral changes. We tested the hypothesis that expression changes would be induced in Fkbp5 and other HPA axis genes by chronic exposure to corticosterone and that these changes would occur through the epigenetic mechanism of loss or gain of DNA methylation (DNAm). We administered corticosterone (CORT) to C57BL/6J mice via their drinking water for 4 wk and tested for behavioral and physiological changes and changes in gene expression levels using RNA extracted from hippocampus, hypothalamus, and blood for the following HPA genes: Fkbp5, Nr3c1, Hsp90, Crh, and Crhr1. The CORT mice exhibited anxiety-like behavior in the elevated plus maze test. Chronic exposure to CORT also caused a significant decrease in the hippocampal and blood mRNA levels of Nr3c1 and a decrease in Hsp90 in blood and caused an increase in Fkbp5 for all tissues. Differences were seen in Fkbp5 methylation in hippocampus and hypothalamus. To isolate a single-cell type, we followed up with an HT-22 mouse hippocampal neuronal cell line exposed to CORT. After 7 d, we observed a 2.4-fold increase in Fkbp5 expression and a decrease in DNAm. In the CORT-treated mice, we also observed changes in blood DNAm in Fkbp5. Our results suggest DNAm plays a role in mediating effects of glucocorticoid exposure on Fkbp5 function, with potential consequences for behavior.