[Show abstract][Hide abstract] ABSTRACT: Background
Epigenetic studies that utilize peripheral tissues to identify molecular substrates of neuropsychiatric disorders rely on the assumption that disease-relevant, cellular alterations that occur in the brain are mirrored and detectable in peripheral tissues such as blood. We sought to test this assumption by using a mouse model of Cushing's disease and asking whether epigenetic changes induced by glucocorticoids can be correlated between these tissue types.
Mice were treated with different doses of glucocorticoids in their drinking water for four weeks to assess gene expression and DNA methylation (DNAm) changes in the stress response gene Fkbp5.
Significant linear relationships were observed between DNAm and four-week mean plasma corticosterone levels for both blood (R2 = 0.68, P = 7.1 × 10−10) and brain (R2 = 0.33, P = 0.001). Further, degree of methylation change in blood correlated significantly with both methylation (R2 = 0.49, P = 2.7 × 10−5) and expression (R2 = 0.43, P = 3.5 × 10−5) changes in hippocampus, with the notable observation that methylation changes occurred at different intronic regions between blood and brain tissues.
Although our findings are limited to several intronic CpGs in a single gene, our results demonstrate that DNA from blood can be used to assess dynamic, glucocorticoid-induced changes occurring in the brain. However, for such correlation analyses to be effective, tissue-specific locations of these epigenetic changes may need to be considered when investigating brain-relevant changes in peripheral tissues.
[Show abstract][Hide abstract] ABSTRACT: Numerous genome-wide gene expression studies of bipolar disorder (BP) have been carried out. These studies are heterogeneous, underpowered and use overlapping samples. We conducted a systematic review of these studies to synthesize the current findings.
We identified all genome-wide gene expression studies on BP in humans. We then carried out a quantitative mega-analysis of studies done with post-mortem brain tissue. We obtained raw data from each study and used standardized procedures to process and analyze the data. We then combined the data and conducted three separate mega-analyses on samples from 1) any region of the brain (9 studies); 2) the prefrontal cortex (PFC) (6 studies); and 3) the hippocampus (2 studies). To minimize heterogeneity across studies, we focused primarily on the most numerous, recent and comprehensive studies.
A total of 30 genome-wide gene expression studies of BP done with blood or brain tissue were identified. We included 10 studies with data on 211 microarrays on 57 unique BP cases and 229 microarrays on 60 unique controls in the quantitative mega-analysis. A total of 382 genes were identified as significantly differentially expressed by the three analyses. Eleven genes survived correction for multiple testing with a q-value < 0.05 in the PFC. Among these were FKBP5 and WFS1, which have been previously implicated in mood disorders. Pathway analyses suggested a role for metallothionein proteins, MAP Kinase phosphotases, and neuropeptides.
We provided an up-to-date summary of results from gene expression studies of the brain in BP. Our analyses focused on the highest quality data available and provided results by brain region so that similarities and differences can be examined relative to disease status. The results are available for closer inspection on-line at Metamoodics [http://metamoodics.igm.jhmi.edu/], where investigators can look up any genes of interest and view the current results in their genomic context and in relation to leading findings from other genomic experiments in bipolar disorder.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Surrogate variable analysis (SVA) is a powerful method to identify, estimate, and utilize the components of gene expression heterogeneity due to unknown and/or unmeasured technical, genetic, environmental, or demographic factors. These sources of heterogeneity are common in gene expression studies, and failing to incorporate them into the analysis can obscure results. Using SVA increases the biological accuracy and reproducibility of gene expression studies by identifying these sources of heterogeneity and correctly accounting for them in the analysis. RESULTS: Here we have developed a web application called SVAw (Surrogate variable analysis Web app) that provides a user friendly interface for SVA analyses of genome-wide expression studies. The software has been developed based on open source bioconductor SVA package. In our software, we have extended the SVA program functionality in three aspects: (i) the SVAw performs a fully automated and user friendly analysis workflow; (ii) It calculates probe/gene Statistics for both pre and post SVA analysis and provides a table of results for the regression of gene expression on the primary variable of interest before and after correcting for surrogate variables; and (iii) it generates a comprehensive report file, including graphical comparison of the outcome for the user. CONCLUSIONS: SVAw is a web server freely accessible solution for the surrogate variant analysis of high-throughput datasets and facilitates removing all unwanted and unknown sources of variation. It is freely available for use at http://psychiatry.igm.jhmi.edu/sva. The executable packages for both web and standalone application and the instruction for installation can be downloaded from our web site.
Source Code for Biology and Medicine 03/2013; 8(1):8.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies (GWAS) have implicated ANK3 as a susceptibility gene for bipolar disorder (BP). We examined whether epistasis with ANK3 may contribute to the "missing heritability" in BP. We first identified via the STRING database 14 genes encoding proteins with prior biological evidence that they interact molecularly with ANK3. We then tested for statistical evidence of interactions between SNPs in these genes in association with BP in a discovery GWAS dataset and two replication GWAS datasets. The most significant interaction in the discovery GWAS was between SNPs in ANK3 and KCNQ2 (p = 3.18 × 10(-8)). A total of 31 pair-wise interactions involving combinations between two SNPs from KCNQ2 and 16 different SNPs in ANK3 were significant after permutation. Of these, 28 pair-wise interactions were significant in the first replication GWAS. None were significant in the second replication GWAS, but the two SNPs from KCNQ2 were found to significantly interact with five other SNPs in ANK3, suggesting possible allelic heterogeneity. KCNQ2 forms homo- and hetero-meric complexes with KCNQ3 that constitute voltage-gated potassium channels in neurons. ANK3 is an adaptor protein that, through its interaction with KCNQ2 and KCNQ3, directs the localization of this channel in the axon initial segment (AIS). At the AIS, the KCNQ2/3 complex gives rise to the M-current, which stabilizes the neuronal resting potential and inhibits repetitive firing of action potentials. Thus, these channels act as "dampening" components and prevent neuronal hyperactivity. The interactions between ANK3 and KCNQ2 merit further investigation, and if confirmed, may motivate a new line of research into a novel therapeutic target for BP.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Numerous candidate gene association studies of bipolar disorder (BP) have been carried out, but the results have been inconsistent. Individual studies are typically underpowered to detect associations with genes of small effect sizes. We conducted a meta-analysis of published candidate gene studies to evaluate the cumulative evidence. We systematically searched for all published candidate gene association studies of BP. We then carried out a random-effects meta-analysis on all polymorphisms that were reported on by three or more case-control studies. The results from meta-analyses of these genes were compared with the findings from a recent mega-analysis of eleven genome-wide association studies (GWAS) in BP performed by the Psychiatric GWAS Consortium (PGC). A total of 487 articles were included in our review. Among these, 33 polymorphisms in 18 genes were reported on by three or more case-control studies and included in the random-effects meta-analysis. Polymorphisms in BDNF, DRD4, DAOA, and TPH1, were found to be nominally significant with a P-value < 0.05. However, none of the findings were significant after correction for multiple testing. Moreover, none of these polymorphisms were nominally significant in the PGC-BP GWAS. A number of plausible candidate genes have been previously associated with BP. However, the lack of robust findings in our review of these candidate genes highlights the need for more atheoretical approaches to study the genetics of BP afforded by GWAS. The results of this meta-analysis and from other on-going genomic experiments in BP are available online at Metamoodics (http://metamoodics.igm.jhmi.edu).
American Journal of Medical Genetics Part B Neuropsychiatric Genetics 05/2012; 159B(5):508-18. · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
American Journal of Medical Genetics Part B Neuropsychiatric Genetics 01/2012; 159B(1):112-9. · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mood-incongruent psychotic features (MICP) are familial symptoms of bipolar disorder (BP) that also occur in schizophrenia (SZ), and may represent manifestations of shared etiology between the major psychoses. In this study we have analyzed three large samples of BP with imputed genome-wide association data and have performed a meta-analysis of 2196 cases with MICP and 8148 controls. We found several regions with suggestive evidence of association (P<10(-6)), although no marker met genome-wide significance criteria. The top associations were on chromosomes: 6q14.2 within the PRSS35/SNAP91 gene complex (rs1171113, P=9.67 × 10(-8)); 3p22.2 downstream of TRANK/LBA1 (rs9834970, P=9.71 × 10(-8)); and 14q24.2 in an intron of NUMB (rs2333194, P=7.03 × 10(-7)). These associations were present in all three samples, and both rs1171113 and rs2333194 were found to be overrepresented in an analysis of MICP cases compared with all other BP cases. To test the relationship of MICP with SZ, we performed polygenic analysis using the Psychiatric GWAS Consortium SZ results and found evidence of association between SZ polygenes and the presence of MICP in BP cases (meta-analysis P=0.003). In summary, our analysis of the MICP phenotype in BP has provided suggestive evidence for association of common variants in several genes expressed in the nervous system. The results of our polygenic analysis provides support for a modest degree of genetic overlap between BP with MICP and SZ, highlighting that phenotypic correlations across syndromes may be due to the influence of polygenic risk factors.
[Show abstract][Hide abstract] ABSTRACT: Mood disorders are highly heritable forms of major mental illness. A major breakthrough in elucidating the genetic architecture of mood disorders was anticipated with the advent of genome-wide association studies (GWAS). However, to date few susceptibility loci have been conclusively identified. The genetic etiology of mood disorders appears to be quite complex, and as a result, alternative approaches for analyzing GWAS data are needed. Recently, a polygenic scoring approach that captures the effects of alleles across multiple loci was successfully applied to the analysis of GWAS data in schizophrenia and bipolar disorder (BP). However, this method may be overly simplistic in its approach to the complexity of genetic effects. Data mining methods are available that may be applied to analyze the high dimensional data generated by GWAS of complex psychiatric disorders.
We sought to compare the performance of five data mining methods, namely, Bayesian networks, support vector machine, random forest, radial basis function network, and logistic regression, against the polygenic scoring approach in the analysis of GWAS data on BP. The different classification methods were trained on GWAS datasets from the Bipolar Genome Study (2191 cases with BP and 1434 controls) and their ability to accurately classify case/control status was tested on a GWAS dataset from the Wellcome Trust Case Control Consortium.
The performance of the classifiers in the test dataset was evaluated by comparing area under the receiver operating characteristic curves. Bayesian networks performed the best of all the data mining classifiers, but none of these did significantly better than the polygenic score approach. We further examined a subset of single-nucleotide polymorphisms (SNPs) in genes that are expressed in the brain, under the hypothesis that these might be most relevant to BP susceptibility, but all the classifiers performed worse with this reduced set of SNPs. The discriminative accuracy of all of these methods is unlikely to be of diagnostic or clinical utility at the present time. Further research is needed to develop strategies for selecting sets of SNPs likely to be relevant to disease susceptibility and to determine if other data mining classifiers that utilize other algorithms for inferring relationships among the sets of SNPs may perform better.
[Show abstract][Hide abstract] ABSTRACT: Comprehensive High-throughput Arrays for Relative Methylation (CHARM) was recently developed as an experimental platform and analytic approach to assess DNA methylation (DNAm) at a genome-wide level. Its initial implementation was for human and mouse. We adapted it for rat and sought to examine DNAm differences across tissues and brain regions in this model organism. We extracted DNA from liver, spleen, and three brain regions: cortex, hippocampus, and hypothalamus from adult Sprague Dawley rats. DNA was digested with McrBC, and the resulting methyl-depleted fraction was hybridized to the rat CHARM array along with a mock-treated fraction. Differentially methylated regions (DMRs) between tissue types were detected using normalized methylation log-ratios. In validating 24 of the most significant DMRs by bisulfite pyrosequencing, we detected large mean differences in DNAm, ranging from 33-59%, among the most significant DMRs in the across-tissue comparisons. The comparable figures for the hippocampus vs. hypothalamus DMRs were 14-40%, for the cortex vs. hippocampus DMRs, 12-29%, and for the cortex vs. hypothalamus DMRs, 5-35%, with a correlation of r(2) = 0.92 between the methylation differences in 24 DMRs predicted by CHARM and those validated by bisulfite pyrosequencing. Our adaptation of the CHARM array for the rat genome yielded highly robust results that demonstrate the value of this method in detecting substantial DNAm differences between tissues and across different brain regions. This platform should prove valuable in future studies aimed at examining DNAm differences in particular brain regions of rats exposed to environmental stimuli with potential epigenetic consequences.
Epigenetics: official journal of the DNA Methylation Society 11/2011; 6(11):1378-90. · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The heritable component to attempted and completed suicide is partly related to psychiatric disorders and also partly independent of them. Although attempted suicide linkage regions have been identified on 2p11-12 and 6q25-26, there are likely many more such loci, the discovery of which will require a much higher resolution approach, such as the genome-wide association study (GWAS). With this in mind, we conducted an attempted suicide GWAS that compared the single-nucleotide polymorphism (SNP) genotypes of 1201 bipolar (BP) subjects with a history of suicide attempts to the genotypes of 1497 BP subjects without a history of suicide attempts. In all, 2507 SNPs with evidence for association at P<0.001 were identified. These associated SNPs were subsequently tested for association in a large and independent BP sample set. None of these SNPs were significantly associated in the replication sample after correcting for multiple testing, but the combined analysis of the two sample sets produced an association signal on 2p25 (rs300774) at the threshold of genome-wide significance (P=5.07 × 10(-8)). The associated SNPs on 2p25 fall in a large linkage disequilibrium block containing the ACP1 (acid phosphatase 1) gene, a gene whose expression is significantly elevated in BP subjects who have completed suicide. Furthermore, the ACP1 protein is a tyrosine phosphatase that influences Wnt signaling, a pathway regulated by lithium, making ACP1 a functional candidate for involvement in the phenotype. Larger GWAS sample sets will be required to confirm the signal on 2p25 and to identify additional genetic risk factors increasing susceptibility for attempted suicide.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies (GWAS) have identified several susceptibility loci for bipolar disorder (BP), most notably ANK3. However, most of the inherited risk for BP remains unexplained. One reason for the limited success may be the genetic heterogeneity of BP. Clinical sub-phenotypes of BP may identify more etiologically homogeneous subsets of patients, which can be studied with increased power to detect genetic variation. Here, we report on a mega-analysis of two widely studied sub-phenotypes of BP, age at onset and psychotic symptoms, which are familial and clinically significant. We combined data from three GWAS: NIMH Bipolar Disorder Genetic Association Information Network (GAIN-BP), NIMH Bipolar Disorder Genome Study (BiGS), and a German sample. The combined sample consisted of 2,836 BP cases with information on sub-phenotypes and 2,744 controls. Imputation was performed, resulting in 2.3 million SNPs available for analysis. No SNP reached genome-wide significance for either sub-phenotype. In addition, no SNP reached genome-wide significance in a meta-analysis with an independent replication sample. We had 80% power to detect associations with a common SNP at an OR of 1.6 for psychotic symptoms and a mean difference of 1.8 years in age at onset. Age at onset and psychotic symptoms in BP may be influenced by many genes of smaller effect sizes or other variants not measured well by SNP arrays, such as rare alleles.
American Journal of Medical Genetics Part B Neuropsychiatric Genetics 02/2011; 156B(3):370-8. · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Family, twin, and adoption studies provide convincing evidence for a genetic contribution to suicidal behavior. The heritability for suicidal behavior depends in part on the transmission of psychiatric disorders, such as mood disorders and substance use disorders, but is also partly independent of them. Three linkage studies using the attempted suicide phenotype in pedigrees with bipolar disorder, major depression, or alcoholism have provided consistent evidence that 2p11-12 harbors a susceptibility gene for attempted suicide. A microarray expression study using postmortem brain samples has implicated a gene from the 2p11-12 candidate region, the trans-Golgi network protein 2 (TGOLN2) gene, as being consistently up-regulated in suicide cases as compared to controls. Here, we present a TGOLN2 case-control association study using nine single nucleotide polymorphisms (SNPs). These nine SNPs, which include seven tag SNPs and two coding SNPs, have been genotyped in 517 mood disorder subjects with a history of attempted suicide and 515 normal controls. Allelic and genotypic analyses of the case-control sample did not provide evidence for association with the attempted suicide phenotype. Eight of the nine SNPs provided supportive evidence for association (P-values ranging from 0.008 to 0.03) when we compared the attempted suicide cases with a history of alcoholism to the attempted suicide cases without a history of alcoholism. However, this association finding was not replicated in an independent sample. Taken together, these analyses do not provide support for the hypothesis that common genetic variation in TGOLN2 contributes significantly to the risk for attempted suicide in subjects with major mood disorders.
American Journal of Medical Genetics Part B Neuropsychiatric Genetics 03/2010; 153B(5):1016-23. · 3.23 Impact Factor