Bipolar disorder (BD) is a severe and highly heritable neuropsychiatric disorder with a lifetime prevalence of 1%. Molecular genetic studies have identified the first BD susceptibility genes. However, the disease pathways remain largely unknown. Accumulating evidence suggests that microRNAs, a class of small noncoding RNAs, contribute to basic mechanisms underlying brain development and plasticity, suggesting their possible involvement in the pathogenesis of several psychiatric disorders, including BD. In the present study, gene-based analyses were performed for all known autosomal microRNAs using the largest genome-wide association data set of BD to date (9747 patients and 14 278 controls). Associated and brain-expressed microRNAs were then investigated in target gene and pathway analyses. Functional analyses of miR-499 and miR-708 were performed in rat hippocampal neurons. Ninety-eight of the six hundred nine investigated microRNAs showed nominally significant P -values, suggesting that BD-associated microRNAs might be enriched within known microRNA loci. After correction for multiple testing, nine microRNAs showed a significant association with BD. The most promising were miR-499 , miR-708 and miR-1908 . Target gene and pathway analyses revealed 18 significant canonical pathways, including brain development and neuron projection. For miR-499 , four Bonferroni-corrected significant target genes were identified, including the genome-wide risk gene for psychiatric disorder CACNB2 . First results of functional analyses in rat hippocampal neurons neither revealed nor excluded a major contribution of miR-499 or miR-708 to dendritic spine morphogenesis. The present results suggest that research is warranted to elucidate the precise involvement of microRNAs and their downstream pathways in BD.

Early life stress (ELS) is associated with increased vulnerability for diseases in later life, including psychiatric disorders. Animal models and human studies suggest that this effect is mediated by epigenetic mechanisms. In humans, epigenetic studies to investigate the influence of ELS on psychiatric phenotypes are limited by the inaccessibility of living brain tissue. Due to the tissue-specific nature of epigenetic signatures, it is impossible to determine whether ELS induced epigenetic changes in accessible peripheral cells, for example, blood lymphocytes, reflect epigenetic changes in the brain. To overcome these limitations, we applied a cross-species approach involving: (i) the analysis of CD34+ cells from human cord blood; (ii) the examination of blood-derived CD3+ T cells of newborn and adolescent nonhuman primates (Macaca mulatta); and (iii) the investigation of the prefrontal cortex of adult rats. Several regions in MORC1 (MORC family CW-type zinc finger 1; previously known as: microrchidia (mouse) homolog) were differentially methylated in response to ELS in CD34+ cells and CD3+ T cells derived from the blood of human and monkey neonates, as well as in CD3+ T cells derived from the blood of adolescent monkeys and in the prefrontal cortex of adult rats. MORC1 is thus the first identified epigenetic marker of ELS to be present in blood cell progenitors at birth and in the brain in adulthood. Interestingly, a gene-set-based analysis of data from a genome-wide association study of major depressive disorder (MDD) revealed an association of MORC1 with MDD.

Bipolar disorder (BD) is a highly heritable psychiatric disease characterized by recurrent episodes of mania and depression. To identify new BD genes and pathways, the present study employed a three-step approach. First, gene-expression profiles of BD patients were assessed during both a manic and an euthymic phase. These profiles were compared intra-individually and with the gene-expression profiles of controls. Second, those differentially expressed genes that were considered potential trait markers of BD were validated using data from the Psychiatric Genomics Consortiums' genome-wide association study (GWAS) of BD. Third, the implicated molecular mechanisms were investigated using pathway analytical methods. In the present patients, this novel approach identified: (i) sets of differentially expressed genes specific to mania and euthymia; and (ii) a set of differentially expressed genes that were common to both mood states. In the GWAS data integration analysis, one gene (STAB1) remained significant (P=1.9 × 10−4) after adjustment for multiple testing. STAB1 is located in close proximity to PBMR1 and the NEK4-ITIH1-ITIH3-ITIH4 region, which are the top findings from GWAS meta-analyses of mood disorder, and a combined BD and schizophrenia data set. Pathway analyses in the mania versus control comparison revealed three distinct clusters of pathways tagging molecular mechanisms implicated in BD, for example, energy metabolism, inflammation and the ubiquitin proteasome system. The present findings suggest that STAB1 is a new and highly promising candidate gene in this region. The combining of gene expression and GWAS data may provide valuable insights into the biological mechanisms of BD.

Schizophrenia (SCZ) is a severe and debilitating neuropsychiatric disorder with an estimated heritability of ~80%. Recently, de novo mutations, identified by next-generation sequencing (NGS) technology, have been suggested to contribute to the risk of developing SCZ. Although these studies show an overall excess of de novo mutations among patients compared with controls, it is not easy to pinpoint specific genes hit by de novo mutations as actually involved in the disease process. Importantly, support for a specific gene can be provided by the identification of additional alterations in several independent patients. We took advantage of existing genome-wide single-nucleotide polymorphism data sets to screen for deletions or duplications (copy number variations, CNVs) in genes previously implicated by NGS studies. Our approach was based on the observation that CNVs constitute part of the mutational spectrum in many human disease-associated genes. In a discovery step, we investigated whether CNVs in 55 candidate genes, suggested from NGS studies, were more frequent among 1637 patients compared with 1627 controls. Duplications in RB1CC1 were overrepresented among patients. This finding was followed-up in large, independent European sample sets. In the combined analysis, totaling 8461 patients and 112 871 controls, duplications in RB1CC1 were found to be associated with SCZ (P=1.29 × 10(-5); odds ratio=8.58). Our study provides evidence for rare duplications in RB1CC1 as a risk factor for SCZ.

Bipolar disorder (BIP) is a highly heritable disorder with complex patterns of genetic inheritance, and recent genetic findings highlight the role of numerous common variants each with subtle effects.1 The existence of Mendelian subtypes of BIP (rare variants of very strong effects) has been postulated, particularly as such variants could prove to be more tractable for subsequent biological investigation.2 One way to evaluate this hypothesis is via the study of pedigrees densely affected with BIP in which a genetic variant of strong effect inherited from a common ancestor may be more likely. With the advent of high-throughput technologies, we can now search densely-affected pedigrees for specific variants that may contribute to risk for BIP. We therefore evaluated a Spanish multi-generational pedigree with an exceptional prevalence of BIP using multiple complementary genomic techniques (Table S1). This pedigree contains 18 cases with BIP (including a sibship with six of 11 affected) and seven individuals with recurrent major depressive disorder (Figure 1a). The lifetime prevalence of mood disorders in this large pedigree (6 generations; 120 individuals, 30 with known mood disorders, 42 with DNA) makes it a strong candidate for identifying genetic risk factors of near-Mendelian effects. Our search strategy is depicted in Figure 1b. All protocols were IRB approved and all subjects provided written informed consent. Figure 1 (a) The pedigree was ascertained in Spain, and has a high prevalence of mood disorders, particularly bipolar disorder (type 1) and recurrent major depressive disorder. First, we used genome-wide linkage analysis and an affecteds-only approach to identify genomic regions sharing identity-by-descent. Linkage analysis was performed using microsatellite data from 13 individuals.3 We used data from Illumina HumanOmni Quad genotyping arrays to identify shared segments using Beagle,4 Germline,5 PedIBD,6 and runs of identity-by-state.7 Regions identified by linkage or with two sharing methods were considered candidate regions (Table S2, six regions totaling 93.7Mb). Second, we hypothesized that one of these regions contained a novel, rare functional single nucleotide variant (SNV) of high penetrance. We attempted to identify novel SNVs in these six genomic regions shared by BIP cases using exome sequencing (five BIP cases) and whole genome sequencing (three BIP cases). Whole genome sequence data was screened to identify high-quality homozygous or heterozygous SNVs that were within the candidate regions, novel, of predicted functional consequence, and present in all three BIP cases. This procedure identified 26 SNVs in three olfactory receptors (OR4C3, OR9G9, and OR4C45). Because multiple variants were present in each gene, given the existence of other highly similar members of the large olfactory gene family, and manual review of alignment patterns, we believed all to be due to misalignment. We repeated this process for the exome sequencing data, and identified 21 SNVs (17 were also identified by whole genome sequencing), and all were in olfactory genes (OR4C3, OR9G9, and OR8U8/OR8U1), and likely due to incorrect alignment. We next evaluated CNVs in these regions. Using PennCNV8 calls from Illumina arrays, no novel CNVs were identified in these candidate regions that were >1kb, present in ≥8 of 11 BIP cases, and confirmed via whole genome sequencing. Therefore, we were unable to identify any potential SNVs or CNVs within these candidate regions that were promising for follow-up. Third, we then extended our analysis genome-wide. From the whole genome sequencing of three individuals, we identified novel SNVs of predicted functionality that were present in all three BIP cases (Table S3). We excluded SNVs in olfactory receptors, with questionable alignments, or that were not confirmed in exome sequencing of five BIP cases. This resulted in eight SNVs that we sequenced in 11 affected individuals using Sanger sequencing. Five SNVs verified and had plausible inheritance patterns, and were then genotyped in 42 individuals in the pedigree. Two SNVs were potentially interesting (chr12:52452495 C>T in NR4A1 and chr18:47793974 G>C in MBD1). Neither exhibited unequivocal Mendelian inheritance (Figures S2–S3). The NR4A1 (nerve growth factor IB, NGFIB or Nur77) SNV is novel but common in this pedigree (homozygous or heterozygous in 30/34 individuals descended from the founders, excluding married-in individuals) and did not clearly segregate in BIP cases as the SNV was found in 92% of BIP cases and 86% of subjects without a mood disorder (although the number of unaffecteds is small). The MBD1 (methyl-CpG-binding domain protein 1) SNV tracked with BIP (66%), other mood disorders (88%), and less so in unaffecteds (21%). However, this SNV was recently identified by the 1000 Genomes Project in subjects from the United Kingdom. Therefore, this variant is not completely novel, but has an interesting pattern of segregation. Both variants warrant additional follow-up in a larger case-control samples although neither appears to be a strong novel Mendelian variant. Genome-wide expansion of the CNV search identified no novel CNVs that were present in ≥8 BIP cases, >1kb in size, overlapped an exon, and verified by whole genome sequencing. Fourth, given the absence of compelling results in support of a near-Mendelian variant, we evaluated the contribution of common variation in this pedigree. The presence of many common variant risk alleles in a pedigree is a potential explanation for a dense pedigree.9 This can be due to or exacerbated by assortative mating (in which mental illness in the family of a spouse is more likely to be tolerated if one has it in one’s own family) and result in an accumulation of common risk alleles. We therefore calculated risk profile scores, which are the weighted number of BIP risk alleles in each subject. Based on results from the Psychiatric GWAS Consortium10 (7,481 BIP cases and 9,250 controls), risk profile scores were computed for the GAIN BIP cases (N=1080) and controls (N=1058),11 and the 11 BIP cases in the densely-affected pedigree (Figure 1c). As anticipated, the GAIN BIP cases had significantly higher risk profile scores than the GAIN controls (p < 0.001). Risk profile scores for 11 BIP cases from the Spanish pedigree were also significantly greater than controls (p < 0.001) but not significantly different from GAIN BIP cases (p = 0.19). It is particularly notable that the BIP pedigree cases did not have markedly lower risk profile scores (e.g., low common variant profiles might be consistent with the present of a strong mutation). Rather, the BIP pedigree cases appeared to have common variant risk profile scores similar to European-American BIP cases ascertained clinically without regard to family history. In conclusion, we systematically assessed this large pedigree dense with BIP for genetic variants of strong effect. This comprehensive analysis did not conclusively identify any SNVs or CNVs of near-Mendelian effect. However, we cannot exclude the presence of risk variants with more complex inheritance patterns, variants with more cryptic functional effects, or variants missed due to coverage or individuals sequenced. However, the common variant risk profiles of BIP cases in this pedigree are similar to those of BIP cases ascertained without regard to family history. Therefore, it is possible that the etiology of BIP in this pedigree is more related to multiple common risk variants rather than one or a few variants of extremely strong effect.

Genome-wide association studies have reported an association between the A-allele of rs1006737 within CACNA1C and affective disorders and schizophrenia. The aim of the present study was to investigate the relationship between rs1006737 and established and potential endophenotypes for these disorders in a population-based cohort of 3793 subjects, using an analytical method designed to assess a previously reported sex-specific effect of CACNA1C. The investigated endophenotypes included personality traits and resilience factors. At 10-year follow-up, subjects were screened for depressive symptoms. All subjects were genotyped for rs1006737. The direction of the effect and mode of inheritance of rs1006737 differed between the sexes. In men, the A-allele was associated with higher emotional lability and lower resilience, that is, lower sense of coherence (P=0.021), lower perceived social support (P=0.018), lower dispositional optimism (P=0.032) and more depressive symptoms at follow-up (P=0.007). In women, the A-allele was associated with lower emotional lability and stronger resilience, that is, higher sense of coherence (P=0.00028), higher perceived social support (P=0.010), lower neuroticism (P=0.022) and fewer depressive symptoms at follow-up (P=0.035). After conservative Bonferroni correction for 32 tests, results only remained significant for sense of coherence in women (P=0.009). These results suggest that CACNA1C is involved in the genetic architecture of endophenotypes for affective disorders and schizophrenia, and that it shows a distinct sex-specific effect. Comprehensive phenotype characterization in case–control samples and the general population, as well as an adequate modeling of sex-specific genetic effects, may be warranted to elucidate the pathogenetic mechanisms conferred by robustly identified susceptibility genes.

Research suggests that clinical symptom dimensions may be more useful in delineating the genetics of bipolar disorder (BD) than standard diagnostic models. To date, no study has applied this concept to data from genome-wide association studies (GWAS). We performed a GWAS of factor dimensions in 927 clinically well-characterized BD patients of German ancestry. Rs9875793, which is located in an intergenic region of 3q26.1 and in the vicinity of the solute carrier family 2 (facilitated glucose transporter), member 2 gene (SLC2A2), was significantly associated with the factor analysis-derived dimension 'negative mood delusions' (n=927; P=4.65 × 10−8, odds ratio (OR)=2.66). This dimension was comprised of the symptoms delusions of poverty, delusions of guilt and nihilistic delusions. In case–control analyses, significant association with the G allele of rs9875793 was only observed in the subgroup of BD patients who displayed symptoms of 'negative mood delusions' (allelic χ2 model: PG=0.0001, OR=1.92; item present, n=89). Further support for the hypothesis that rs9875793 is associated with BD in patients displaying 'negative mood delusions' symptom, such as delusions of guilt, was obtained from an European American sample (GAIN/TGEN), which included 1247 BD patients and 1434 controls (PEA=0.028, OR=1.27).

Recent molecular studies have implicated common alleles of small to moderate effect and rare alleles with larger effect sizes in the genetic architecture of schizophrenia (SCZ). It is expected that the reliable detection of risk variants with very small effect sizes can only be achieved through the recruitment of very large samples of patients and controls (that is tens of thousands), or large, potentially more homogeneous samples that have been recruited from confined geographical areas using identical diagnostic criteria. Applying the latter strategy, we performed a genome-wide association study (GWAS) of 1169 clinically well characterized and ethnically homogeneous SCZ patients from a confined area of Western Europe (464 from Germany, 705 from The Netherlands) and 3714 ethnically matched controls (1272 and 2442, respectively). In a subsequent follow-up study of our top GWAS results, we included an additional 2569 SCZ patients and 4088 controls (from Germany, The Netherlands and Denmark). Genetic variation in a region on chromosome 11 that contains the candidate genes AMBRA1, DGKZ, CHRM4 and MDK was significantly associated with SCZ in the combined sample (n=11 540; P=3.89 × 10−9, odds ratio (OR)=1.25). This finding was replicated in 23 206 independent samples of European ancestry (P=0.0029, OR=1.11). In a subsequent imaging genetics study, healthy carriers of the risk allele exhibited altered activation in the cingulate cortex during a cognitive control task. The area of interest is a critical interface between emotion regulation and cognition that is structurally and functionally abnormal in SCZ and bipolar disorder.

Independent evidence for the selective influence of GABA A receptors on one component of the bipolar disorder phenotype