Genome-wide association study of 40,000 individuals identifies two novel loci associated with bipolar disorder.

Bipolar disorder (BD) is a genetically complex mental illness characterized by severe oscillations of mood and behaviour. Genome-wide association studies (GWAS) have identified several risk loci that together account for a small portion of the heritability. To identify additional risk loci, we performed a two-stage meta-analysis of >9 million genetic variants in 9,784 bipolar disorder patients and 30,471 controls, the largest GWAS of BD to date. In this study, to increase power we used ∼2,000 lithium-treated cases with a long-term diagnosis of BD from the Consortium on Lithium Genetics, excess controls, and analytic methods optimized for markers on the X-chromosome. In addition to four known loci, results revealed genome-wide significant associations at two novel loci: an intergenic region on 9p21.3 (rs12553324, P = 5.87 × 10 - 9; odds ratio (OR) = 1.12) and markers within ERBB2 (rs2517959, P = 4.53 × 10 - 9; OR = 1.13). No significant X-chromosome associations were detected and X-linked markers explained very little BD heritability. The results add to a growing list of common autosomal variants involved in BD and illustrate the power of comparing well-characterized cases to an excess of controls in GWAS.

Genome-wide association of bipolar disorder suggests an enrichment of replicable associations in regions near genes.

Although a highly heritable and disabling disease, bipolar disorder's (BD) genetic variants have been challenging to identify. We present new genotype data for 1,190 cases and 401 controls and perform a genome-wide association study including additional samples for a total of 2,191 cases and 1,434 controls. We do not detect genome-wide significant associations for individual loci; however, across all SNPs, we show an association between the power to detect effects calculated from a previous genome-wide association study and evidence for replication (P = 1.5×10(-7)). To demonstrate that this result is not likely to be a false positive, we analyze replication rates in a large meta-analysis of height and show that, in a large enough study, associations replicate as a function of power, approaching a linear relationship. Within BD, SNPs near exons exhibit a greater probability of replication, supporting an enrichment of reproducible associations near functional regions of genes. These results indicate that there is likely common genetic variation associated with BD near exons (±10 kb) that could be identified in larger studies and, further, provide a framework for assessing the potential for replication when combining results from multiple studies.

Evidence for association of bipolar disorder to haplotypes in the 22q12.3 region near the genes stargazin, IFT27 and parvalbumin.

We have previously reported genome-wide significant linkage of bipolar disorder to a region on 22q12.3 near the marker D22S278. Towards identifying the susceptibility gene, we have conducted a fine-mapping association study of the region in two independent family samples, an independent case-control sample and a genome-wide association dataset. Two hundred SNPs were first examined in a 5 Mb region surrounding the D22S278 marker in a sample of 169 families and analyzed using PLINK. The peak of association was a haplotype near the genes stargazin (CACNG2), intraflagellar transport protein homolog 27 (IFT27) and parvalbumin (PVALB; P = 4.69 × 10(-4)). This peak overlapped a significant haplotype in a family based association study of a second independent sample of 294 families (P = 1.42 × 10(-5)). Analysis of the combined family sample yielded statistically significant evidence of association to a rare three SNP haplotype in the gene IFT27 (P = 8.89 × 10(-6)). Twelve SNPs comprising these haplotypes were genotyped in an independent sample of 574 bipolar I cases and 550 controls. Statistically significant association was found for a haplotype window that overlapped the region from the first two family samples (P = 3.43 × 10(-4)). However, analyses of the two family samples using the program LAMP, found no evidence for association in this region, but did yield significant evidence for association to a haplotype 3' of CACNG2 (P = 1.76 × 10(-6)). Furthermore, no evidence for association was found in a large genome-wide association dataset. The replication of association to overlapping haplotypes in three independent datasets suggests the presence of a bipolar disorder susceptibility gene in this region.

Further evidence for association of GRK3 to bipolar disorder suggests a second disease mutation.

OBJECTIVES: Two genome-wide linkage surveys suggest chromosome 22q12 may contain a susceptibility locus for bipolar disorder (BPD) in the immediate region of the gene G protein receptor kinase-3 (GRK3). We previously published evidence that a single nucleotide polymorphism (SNP) in the promoter region of GRK3, designated P5, was associated with BPD. This SNP, however, was too rare (allele frequency 0.007) to explain the evidence for linkage. METHODS: To identify other SNPs or haplotypes associated with illness, we have now sequenced an additional 28-kb genomic segment of GRK3 and tested an additional 35 SNPs for association with BPD in 181 Caucasian nuclear families. RESULTS: Transmission disequilibrium test analyses identified two closely related disease-associated haplotypes defined by four SNPs located upstream of the promoter region: transmission to nontransmission ratios=54:22 and 20:9, odds ratios=2.50 and 2.36, and P values=0.0009 and 0.05. The best P value remained significant after correction for multiple testing. These two haplotypes were found on an entirely different set of chromosomes from the previously identified SNP P5. They had a combined frequency of approximately 0.10 and, therefore, a much greater population attributable risk for disease than the previously identified P5 haplotype. CONCLUSIONS: These data provide evidence that at least two distinct haplotypes, and possibly two or more different underlying mutations, in GRK3 might be associated with BPD. These new findings add support for the hypothesis that a dysregulation in GRK3 expression alters signaling desensitization and thereby predisposes to the development of BPD.

Differential regulation of immediate-early gene expression in the prefrontal cortex of rats with a high vs low behavioral response to methamphetamine.

Methamphetamine (METH) administration mimics many of the symptoms of mania and can produce psychosis after chronic use. Both rodents and man display interindividual variation in response to METH. The molecular mechanisms underlying these differences might be relevant to both stimulant addiction and endogenous psychosis. We treated 50 Sprague-Dawley rats acutely with METH (4.0 mg/kg) and 10 control rats with saline, and measured their behavior for 3 h after drug administration. Animals were divided into high responders (HR) (top 20%) and low responders (LR) (lowest 20%) based on their stereotypy response. They were killed 24 h after injection. Total RNA was extracted from the prefrontal cortex (PFC) and the expression of approximately 30 000 transcripts were analyzed using Affymetrix 230 2.0 GeneChips. Real-time reverse transcription-polymerase chain reaction was used to validate the expression of a select group of genes. Forty-three genes exhibited significant differences in expression in HR vs LR 24 h after METH treatment including a group of immediate-early genes (IEGs) (eg, c-fos, junB, NGFI-B, serum-regulated glucocorticoid kinase). These IEG expression differences were accompanied by the significant downregulation of many of these genes compared to saline in the HR but not LR, suggesting a differential responsiveness of signal transduction pathways in these two groups of rats. In addition, the expression of other transcription factors in the PFC was significantly different in HR compared to LR. These gene expression changes may contribute to individual differences in responsiveness to stimulants and the development of mania and psychosis.

Genetic relationship between five psychiatric disorders estimated from genome-wide SNPs.

Most psychiatric disorders are moderately to highly heritable. The degree to which genetic variation is unique to individual disorders or shared across disorders is unclear. To examine shared genetic etiology, we use genome-wide genotype data from the Psychiatric Genomics Consortium (PGC) for cases and controls in schizophrenia, bipolar disorder, major depressive disorder, autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD). We apply univariate and bivariate methods for the estimation of genetic variation within and covariation between disorders. SNPs explained 17-29% of the variance in liability. The genetic correlation calculated using common SNPs was high between schizophrenia and bipolar disorder (0.68 ± 0.04 s.e.), moderate between schizophrenia and major depressive disorder (0.43 ± 0.06 s.e.), bipolar disorder and major depressive disorder (0.47 ± 0.06 s.e.), and ADHD and major depressive disorder (0.32 ± 0.07 s.e.), low between schizophrenia and ASD (0.16 ± 0.06 s.e.) and non-significant for other pairs of disorders as well as between psychiatric disorders and the negative control of Crohn's disease. This empirical evidence of shared genetic etiology for psychiatric disorders can inform nosology and encourages the investigation of common pathophysiologies for related disorders.

Genome-wide significant association between a 'negative mood delusions' dimension in bipolar disorder and genetic variation on chromosome 3q26.1.

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 χ(2) model: P(G)=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 (P(EA)=0.028, OR=1.27).

Allele specific analysis of the ADRBK2 gene in lymphoblastoid cells from bipolar disorder patients.

G-protein coupled receptor kinase-3 (GRK3), translated from the gene, ADRBK2 has been implicated as a candidate molecule for bipolar disorder through multiple, converging lines of evidence. In some individuals, the ADRBK2 gene harbors the A-haplotype, a collection of single nucleotide polymorphisms (SNPs) previously associated with an increased risk for bipolar disorder. Because the A-haplotype encompasses the ADRBK2 promoter, we hypothesized that it may alter the regulation of gene expression. Using histone H3 acetylation to infer promoter activity in lymphoblastoid cells from patients with bipolar disorder, we examined the A-haplotype within its genomic context and determined that at least four of its SNPs are present in transcriptionally active portions of the promoter. However, using chromatin immunoprecipitation followed by allele-specific PCR in samples heterozygous for the A-haplotype, we found no evidence of altered levels of acetylated histone H3 at the affected allele compared to the common allele. Similarly, using a transcribed SNP to discriminate expressed ADRBK2 mRNA strands by allele of origin; we found that the A-haplotype did not confer an allelic-expression imbalance. Our data suggest that while the A-haplotype is situated in active regulatory sequence, the risk-associated SNPs do not appear to affect ADRBK2 gene regulation at the level of histone H3 acetylation nor do they confer measurable changes in transcription in lymphoblastoid cells. However, tissue-specific mechanisms by which the A-haplotype could affect ADRBK2 in the central nervous system cannot be excluded.

Promoter variant in the GRK3 gene associated with bipolar disorder alters gene expression.

BACKGROUND: We have previously reported a single nucleotide polymorphism (P-5, G-384A) in the proximal promoter of the gene for G protein receptor kinase 3 (GRK3) that was associated with bipolar disorder in two independent samples. In this study, we examined whether the G-384A variant has a functional effect on GRK3 transcription. METHODS: Electrophoretic mobility shift assays were conducted using nuclear extracts from both Hela cells and adult mouse cortex. Transcriptional function was also examined using a dual luciferase reporter system transfected into in vitro human neuroblastoma cells and cultured mouse cortical neurons. RESULTS: The G-384A variant abolished or reduced the formation of DNA-protein complexes using nuclear extract from both HeLa cells and adult mouse cortical neuron cells. However, gene expression was significantly enhanced by G-384A in both in vitro human neuroblastoma cells and cultured mouse cortical neurons. CONCLUSIONS: These data suggest that the G-384A SNP in the promoter of human GRK3 gene represents an important functional variant. The G-384A variant may alter binding of Sp1/Sp4 transcription factors resulting in an increase in gene transcription and an increase in vulnerability to bipolar disorder.

The pharmacogenetics of lithium response depends upon clinical co-morbidity.

BACKGROUND: Based on results from randomized, controlled clinical trials, lithium monotherapy or lithium with the addition of an antipsychotic remains a first-line treatment option for both acute and long-term mood stabilization in bipolar mania. However, response to lithium is poor in bipolar patients who exhibit clinical characteristics such as rapid cycling and mixed manic states, suggesting that they may have a biologically and genetically distinct form of bipolar disorder. A test that could predict response to lithium based upon genetic factors would have significant clinical value. METHODS: Eight clinical characteristics were assessed in 92 lithium responders and 92 nonresponders; all probands were from families recruited for linkage studies. Lithium response was rated retrospectively from a standardized interviews and medical records. Eight candidate genes were selected from those reported to be associated with susceptibility to illness, lithium response, or lithium mechanism of action. Sixty-seven single nucleotide polymorphisms (SNPs) were genotyped in these subjects and analyzed for association with the defined clinical characteristics. RESULTS: Using q-value analysis for multiplicity correction, we found significant interactions between lithium response and SNPs (rs1387923 and rs1565445) in the gene encoding neurotrophic tyrosine kinase receptor type 2 (NTRK2) and suicidal ideation, and between SNP rs2064721 in the gene encoding inositol polyphosphate-1-phosphatase (INPP1) and post-traumatic stress disorder. CONCLUSION: These data support the idea that response to lithium has a multi-genetic etiology dependent upon manifestations of other clinical co-diagnoses.

Suggestive evidence for association of the circadian genes PERIOD3 and ARNTL with bipolar disorder.

Bipolar affective disorder (BPAD) is suspected to arise in part from malfunctions of the circadian system, a system that enables adaptation to a daily and seasonally cycling environment. Genetic variations altering functions of genes involved with the input to the circadian clock, in the molecular feedback loops constituting the circadian oscillatory mechanism itself, or in the regulatory output systems could influence BPAD as a result. Several human circadian system genes have been identified and localized recently, and a comparison with linkage hotspots for BPAD has revealed some correspondences. We have assessed evidence for linkage and association involving polymorphisms in 10 circadian clock genes (ARNTL, CLOCK, CRY2, CSNK1epsilon, DBP, GSK3beta, NPAS2, PER1, PER2, and PER3) to BPAD. Linkage analysis in 52 affected families showed suggestive evidence for linkage to CSNK1epsilon. This finding was not substantiated in the association study. Fifty-two SNPs in 10 clock genes were genotyped in 185 parent proband triads. Single SNP TDT analyses showed no evidence for association to BPAD. However, more powerful haplotype analyses suggest two candidates deserving further studies. Haplotypes in ARNTL and PER3 were found to be significantly associated with BPAD via single-gene permutation tests (PG = 0.025 and 0.008, respectively). The most suggestive haplotypes in PER3 showed a Bonferroni-corrected P-value of PGC = 0.07. These two genes have previously been implicated in circadian rhythm sleep disorders and affective disorders. With correction for the number of genes considered and tests conducted, these data do not provide statistically significant evidence for association. However, the trends for ARNTL and PER3 are suggestive of their involvement in bipolar disorder and warrant further study in a larger sample.

Combined analysis from eleven linkage studies of bipolar disorder provides strong evidence of susceptibility loci on chromosomes 6q and 8q.

Several independent studies and meta-analyses aimed at identifying genomic regions linked to bipolar disorder (BP) have failed to find clear and consistent evidence of linkage regions. Our hypothesis is that combining the original genotype data provides benefits of increased power and control over sources of heterogeneity that outweigh the difficulty and potential pitfalls of the implementation. We conducted a combined analysis using the original genotype data from 11 BP genomewide linkage scans comprising 5,179 individuals from 1,067 families. Heterogeneity among studies was minimized in our analyses by using uniform methods of analysis and a common, standardized marker map and was assessed using novel methods developed for meta-analysis of genome scans. To date, this collaboration is the largest and most comprehensive analysis of linkage samples involving a psychiatric disorder. We demonstrate that combining original genome-scan data is a powerful approach for the elucidation of linkage regions underlying complex disease. Our results establish genomewide significant linkage to BP on chromosomes 6q and 8q, which provides solid information to guide future gene-finding efforts that rely on fine-mapping and association approaches.