RNA-sequencing of the brain transcriptome implicates dysregulation of neuroplasticity, circadian rhythms and GTPase binding in bipolar disorder.

RNA-sequencing (RNA-seq) is a powerful technique to investigate the complexity of gene expression in the human brain. We used RNA-seq to survey the brain transcriptome in high-quality postmortem dorsolateral prefrontal cortex from 11 individuals diagnosed with bipolar disorder (BD) and from 11 age- and gender-matched controls. Deep sequencing was performed, with over 350 million reads per specimen. At a false discovery rate of <5%, we detected five differentially expressed (DE) genes and 12 DE transcripts, most of which have not been previously implicated in BD. Among these, Prominin 1/CD133 and ATP-binding cassette-sub-family G-member2 (ABCG2) have important roles in neuroplasticity. We also show for the first time differential expression of long noncoding RNAs (lncRNAs) in BD. DE transcripts include those of serine/arginine-rich splicing factor 5 (SRSF5) and regulatory factor X4 (RFX4), which along with lncRNAs have a role in mammalian circadian rhythms. The DE genes were significantly enriched for several Gene Ontology categories. Of these, genes involved with GTPase binding were also enriched for BD-associated SNPs from previous genome-wide association studies, suggesting that differential expression of these genes is not simply a consequence of BD or its treatment. Many of these findings were replicated by microarray in an independent sample of 60 cases and controls. These results highlight common pathways for inherited and non-inherited influences on disease risk that may constitute good targets for novel therapies.

Interaction networks of lithium and valproate molecular targets reveal a striking enrichment of apoptosis functional clusters and neurotrophin signaling.

The overall neurobiological mechanisms by which lithium and valproate stabilize mood in bipolar disorder patients have yet to be fully defined. The therapeutic efficacy and dissimilar chemical structures of these medications suggest that they perturb both shared and disparate cellular processes. To investigate key pathways and functional clusters involved in the global action of lithium and valproate, we generated interaction networks formed by well-supported drug targets. Striking functional similarities emerged. Intersecting nodes in lithium and valproate networks highlighted a strong enrichment of apoptosis clusters and neurotrophin signaling. Other enriched pathways included MAPK, ErbB, insulin, VEGF, Wnt and long-term potentiation indicating a widespread effect of both drugs on diverse signaling systems. MAPK1/3 and AKT1/2 were the most preponderant nodes across pathways suggesting a central role in mediating pathway interactions. The convergence of biological responses unveils a functional signature for lithium and valproate that could be key modulators of their therapeutic efficacy.

A systems approach to the biology of mood disorders through network analysis of candidate genes.

Meta analysis of association data of mood disorders has shown evidence for the role of particular genes in genetic risk. Integration of association data from meta analysis with differential expression data in brains of mood disorder patients could heighten the level of support for specific genes. To identify molecular mechanisms that may be disrupted in disease, a systems approach that involves analysis of biological networks created by these selected genes was employed.Interaction networks of hierarchical groupings of selected genes were generated using the Michigan Molecular Interactions (MiMI) software. Large networks were deconvoluted into subclusters of core complexes by using a community clustering program, GLay. Network nodes were functionally annotated in DAVID Bioinformatics Resource to identify enriched pathways and functional clusters. MAPK and beta adrenergic receptor signaling pathways were significantly enriched in the ANK3 and CACNA1C network. The PBRM1 network bolstered the enrichment of chromatin remodeling and transcription regulation functional clusters. Lowering the stringency for inclusion of other genes in network seeds increased network complexity and expanded the recruitment of enriched pathways to include signaling by neurotransmitter and hormone receptors, neurotrophin, ErbB and the cell cycle. We present a strategy to interrogate mechanisms in the cellular system that might be perturbed in disease. Network analysis of meta analysis- generated candidate genes that exhibited differential expression in mood disorder brains identified signaling pathways and functional clusters that may be involved in genetic risk for mood disorders.

Two variants in Ankyrin 3 (ANK3) are independent genetic risk factors for bipolar disorder.

Two recent reports have highlighted ANK3 as a susceptibility gene for bipolar disorder (BD). We first reported association between BD and the ANK3 marker rs9804190 in a genome-wide association study (GWAS) of two independent samples (Baum et al., 2008). Subsequently, a meta-analysis of GWAS data based on samples from the US and the UK reported association with a different ANK3 marker, rs10994336 (Ferreira et al., 2008). The markers lie about 340 kb apart in the gene. Here, we test both markers in additional samples and characterize the contribution of each marker to BD risk. Our previously reported findings at rs9804190, which had been based on DNA pooling, were confirmed by individual genotyping in the National Institute of Mental Health (NIMH) waves 1-4 (P=0.05; odds ratio (OR)=1.24) and German (P=0.0006; OR=1.34) samples. This association was replicated in an independent US sample known as NIMH wave 5 (466 cases, 212 controls; P=0.017; OR=1.38). A random-effects meta-analysis of all three samples was significant (P=3 x 10(-6); OR=1.32), with no heterogeneity. Individual genotyping of rs10994336 revealed a significant association in the German sample (P=0.0001; OR=1.70), and similar ORs in the NIMH 1-4 and NIMH 5 samples that were not significant at the P<0.05 level. Meta-analysis of all three samples supported an association with rs10994336 (P=1.7 x 10(-5); OR=1.54), again with no heterogeneity. There was little linkage disequilibrium between the two markers. Further analysis suggested that each marker contributed independently to BD, with no significant marker x marker interaction. Our findings strongly support ANK3 as a BD susceptibility gene and suggest true allelic heterogeneity.

Glucocorticoid receptor gene polymorphisms in premenopausal women with major depression.

Glucocorticoid receptor gene polymorphisms are associated with glucocorticoid hypersensitivity and visceral obesity. Perturbations in HPA axis sensitivity to glucocorticoids implicated in the pathogenesis of major depression may result from functional alterations in the glucocorticoid receptor gene. We 1) examined the prevalence of genotype distribution of specific polymorphisms of the glucocorticoid receptor gene (Bcl1, N363S, rs33388, rs33389) in a subset of women from the P.O.W.E.R. Study (which enrolled 21- to 45-year-old premenopausal women with major depression and healthy controls) and 2) explored whether such polymorphisms were associated with visceral obesity and insulin resistance. Women with major depression had a higher body mass index, a higher waist:hip ratio, and more body fat than did controls. No differences were observed in plasma and urinary cortisol or in insulin sensitivity. The G/G genotype of the Bcl1 polymorphism was significantly more common (p<0.03) in women with major depression (n=52) than in controls (n=29). In addition, GG homozygotes (depressed n=10; controls n=2) had higher waist:hip ratios than did non-GG carriers (p<0.02). N363S, rs33388, and rs33389 polymorphisms were not different between groups. In conclusion, premenopausal women with both major depression and the GG genotype of the Bcl1 polymorphism had greater abdominal obesity compared with non-GG carriers.

A genome-wide association study implicates diacylglycerol kinase eta (DGKH) and several other genes in the etiology of bipolar disorder.

The genetic basis of bipolar disorder has long been thought to be complex, with the potential involvement of multiple genes, but methods to analyze populations with respect to this complexity have only recently become available. We have carried out a genome-wide association study of bipolar disorder by genotyping over 550,000 single-nucleotide polymorphisms (SNPs) in two independent case-control samples of European origin. The initial association screen was performed using pooled DNA, and selected SNPs were confirmed by individual genotyping. While DNA pooling reduces power to detect genetic associations, there is a substantial cost saving and gain in efficiency. A total of 88 SNPs, representing 80 different genes, met the prior criteria for replication in both samples. Effect sizes were modest: no single SNP of large effect was detected. Of 37 SNPs selected for individual genotyping, the strongest association signal was detected at a marker within the first intron of diacylglycerol kinase eta (DGKH; P=1.5 x 10(-8), experiment-wide P<0.01, OR=1.59). This gene encodes DGKH, a key protein in the lithium-sensitive phosphatidyl inositol pathway. This first genome-wide association study of bipolar disorder shows that several genes, each of modest effect, reproducibly influence disease risk. Bipolar disorder may be a polygenic disease.

Fine mapping of a susceptibility locus for bipolar and genetically related unipolar affective disorders, to a region containing the C21ORF29 and TRPM2 genes on chromosome 21q22.3.

Linkage analyses of bipolar families have confirmed that there is a susceptibility locus near the telomere on chromosome 21q. To fine map this locus we carried out tests of allelic association using 30 genetic markers near the telomere at 21q22.3 in 600 bipolar research subjects and 450 ancestrally matched supernormal control subjects. We found significant allelic association with the microsatellite markers D21S171 (P=0.016) and two closely linked single-nucleotide polymorphisms, rs1556314 (P=0.008) and rs1785467 (P=0.025). A test of association with a three locus haplotype across the susceptibility region was significant with a permutation test of P=0.011. A two SNP haplotype was also significantly associated with bipolar disorder (P=0.01). Only two brain expressed genes, TRPM2 and C21ORF29 (TSPEAR), are present in the associated region. TRPM2 encodes a calcium channel receptor and TSPEAR encodes a peptide with repeats associated with epilepsy in the mouse. DNA from subjects who had inherited the associated marker alleles was sequenced. A base pair change (rs1556314) in exon 11 of TRPM2, which caused a change from an aspartic acid to a glutamic acid at peptide position 543 was found. This SNP showed the strongest association with bipolar disorder (P=0.008). Deletion of exon 11 of TRPM2 is known to cause dysregulation of cellular calcium homeostasis in response to oxidative stress. A second nonconservative change from arginine to cysteine at position 755 in TRPM2 (ss48297761) was also detected. A third nonconservative change from histidine to glutamic acid was found in exon 8 of TSPEAR. These changes need further investigation to establish any aetiological role in bipolar disorder.

Findings in an independent sample support an association between bipolar affective disorder and the G72/G30 locus on chromosome 13q33.

Markers near the nested genes G72 and G30 on chromosome 13q33 have been implicated in the etiology of schizophrenia and, recently, bipolar affective disorder (BPAD). Hattori et al (2003) reported that single-nucleotide polymorphisms (SNPs) near the G72/G30 locus were associated with BPAD in a sample of 22 pedigrees, and that SNP haplotypes were associated in a second, larger sample of triads. The present study attempts to replicate this finding in an independent case-control sample. Six SNPs near the G72/G30 locus, including the most strongly associated markers in the previous study, were tested in 139 cases and 113 ethnically matched controls. Significant association was detected between BPAD and two adjacent SNPs (smallest P=0.007; global P=0.024). Haplotype analysis produced additional support for association (smallest P=0.004; global P=0.004). Analysis of 31 unlinked microsatellite markers detected no population stratification in the cases or controls studied. Although the associated alleles and haplotypes differ from those previously reported, these new results provide further evidence, in an independent sample, for an association between BPAD and genetic variation in the vicinity of the genes G72 and G30.

Experimental gene interaction studies with SERT mutant mice as models for human polygenic and epistatic traits and disorders.

Current evidence indicates that virtually all neuropsychiatric disorders, like many other common medical disorders, are genetically complex, with combined influences from multiple interacting genes, as well as from the environment. However, additive or epistatic gene interactions have proved quite difficult to detect and evaluate in human studies. Mouse phenotypes, including behaviors and drug responses, can provide relevant models for human disorders. Studies of gene-gene interactions in mice could thus help efforts to understand the molecular genetic bases of complex human disorders. The serotonin transporter (SERT, 5-HTT, SLC6A4) provides a relevant model for studying such interactions for several reasons: human variants in SERT have been associated with several neuropsychiatric and other medical disorders and quantitative traits; SERT blockers are effective treatments for a number of neuropsychiatric disorders; there is a good initial understanding of the phenotypic features of heterozygous and homozygous SERT knockout mice; and there is an expanding understanding of the interactions between variations in SERT expression and variations in the expression of a number of other genes of interest for neuropsychiatry and neuropharmacology. This paper provides examples of experimentally-obtained interactions between quantitative variations in SERT gene expression and variations in the expression of five other mouse genes: DAT, NET, MAOA, 5-HT(1B) and BDNF. In humans, all six of these genes possess polymorphisms that have been independently investigated as candidates for neuropsychiatric and other disorders in a total of > 500 reports. In the experimental studies in mice reviewed here, gene-gene interactions resulted in either synergistic, antagonistic (including 'rescue' or 'complementation') or more complex, quantitative alterations. These were identified in comparisons of the behavioral, physiological and neurochemical phenotypes of wildtype mice vs. mice with single allele or single gene targeted disruptions and mice with partial or complete disruptions of multiple genes. Several of the descriptive phenotypes could be best understood on the basis of intermediate, quantitative alterations such as brain serotonin differences. We discuss the ways in which these interactions could provide models for studies of gene-gene interactions in complex human neuropsychiatric and other disorders to which SERT may contribute, including developmental disorders, obesity, polysubstance abuse and others.

Association between serotonin 4 receptor gene polymorphisms and bipolar disorder in Japanese case-control samples and the NIMH Genetics Initiative Bipolar Pedigrees.

Possible irregularities in serotonergic neurotransmission have been suggested as causes of a variety of neuropsychiatric diseases. We performed mutation and association analyses of the HTR4 gene, on 5q32, encoding the serotonin 4 receptor in mood disorders and schizophrenia. Mutation analysis was performed on the HTR4 exons and exon/intron boundaries in 48 Japanese patients with mood disorders and 48 patients with schizophrenia. Eight polymorphisms and four rare variants were identified. Of these, four polymorphisms at or in close proximity to exon d, g.83097C/T (HTR4-SVR (splice variant region) SNP1), g.83159G/A (HTR4-SVRSNP2), g.83164 (T)9-10 (HTR4-SVRSNP3), and g.83198A/G (HTR4-SVRSNP4), showed significant association with bipolar disorder with odds ratios of 1.5 to 2. These polymorphisms were in linkage disequilibrium, and only three common haplotypes were observed. One of the haplotypes showed significant association with bipolar disorder (P = 0.002). The genotypic and haplotypic associations with bipolar disorder were confirmed by transmission disequilibrium test in the NIMH Genetics Initiative Bipolar Pedigrees with ratios of transmitted to not transmitted alleles of 1.5 to 2.0 (P = 0.01). The same haplotype that showed association with bipolar disorder was suggested to be associated with schizophrenia in the case-control analysis (P = 0.003) but was not confirmed when Japanese schizophrenia families were tested. The polymorphisms associated with mood disorder were located within the region that encodes the divergent C-terminal tails of the 5-HT(4) receptor. These findings suggest that genomic variations in the HTR4 gene may confer susceptibility to mood disorder.

Mutation screening of two candidate genes from 13q32 in families affected with Bipolar disorder: human peptide transporter (SLC15A1) and human glypican5 (GPC5).

BACKGROUND: Multiple candidate regions as sites for Schizophrenia and Bipolar susceptibility genes have been reported, suggesting heterogeneity of susceptibility genes or oligogenic inheritance. Linkage analysis has suggested chromosome 13q32 as one of the regions with evidence of linkage to Schizophrenia and, separately, to Bipolar disorder (BP). SLC15A1 and GPC5 are two of the candidate genes within an approximately 10-cM region of linkage on chromosome 13q32. In order to identify a possible role for these candidates as susceptibility genes, we performed mutation screening on the coding regions of these two genes in 7 families (n-20) affected with Bipolar disorder showing linkage to 13q32. RESULTS: Genomic organization revealed 23 exons in SLC15A1 and 8 exons in GPC5 gene respectively. Sequencing of the exons did not reveal mutations in the GPC5 gene in the 7 families affected with BP. Two polymorphic variants were discovered in the SLC15A1 gene. One was T to C substitution in the third position of codon encoding alanine at 1403 position of mRNA in exon 17, and the other was A to G substitution in the untranslated region at position 2242 of mRNA in exon 23. CONCLUSIONS: Mutation analysis of 2 candidate genes for Bipolar disorder on chromosome 13q32 did not identify any potentially causative mutations within the coding regions or splice junctions of the SLC15A1 or GPC5 genes in 7 families showing linkage to 13q32. Further studies of the regulatory regions are needed to completely exclude these genes as causative for Bipolar disorder.

Expression of the smoothelin gene is mediated by alternative promoters.

OBJECTIVE: Two major isoforms of smoothelin have been reported, a 59-kDa smoothelin-A in visceral smooth muscle cells and a 110-kDa smoothelin-B in vascular smooth muscle cells. The present study was undertaken to investigate the expression of these smoothelin isoforms in different smooth muscle tissues and to determine how they are generated. METHODS: Western blotting with a new, well-defined, smoothelin antibody was used to confirm the existence of two major smoothelin isoforms. Northern blotting, RT-PCR, primer extension and 5'RACE were applied to analyse the expression of these isoforms in human and mouse. Promoter reporter assays were carried out to establish the existence of a dual promoter system governing the expression pattern of the gene. RESULTS: Antibody C6G confirmed the existence of two smoothelin proteins. Northern blotting showed that in vascular tissues a larger smoothelin transcript is generated than in visceral tissue. The cDNA of this larger smoothelin-B was cloned. Computer analysis of the open reading frame suggests an alpha-helical structure of 130 amino acids at the amino terminus of smoothelin-B. The smoothelin gene was cloned and sequenced. It comprises about 25 kb and contains 21 exons. The translational start of smoothelin-B is located in exon 2, whereas transcription and translation of the previously described smoothelin-A starts inside exon 10. Smoothelin-A and -B were demonstrated to be generated by two physically separated promoters. Splice variants within the calponin homology domain at the 3' end of the gene were found for both isoforms. CONCLUSIONS: Two major smoothelin isoforms are generated from a single gene by a dual promoter system in a tissue specific manner. Further variation in the smoothelin proteins is achieved by alternative splicing in the calponin homology domain.

A human glucocorticoid receptor gene variant that increases the stability of the glucocorticoid receptor beta-isoform mRNA is associated with rheumatoid arthritis.

OBJECTIVE: To study the occurrence and function of polymorphism in the human glucocorticoid receptor (hGR) gene in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). METHODS: We used single stranded conformation polymorphism (SSCP) and direct sequencing to study the hGR gene in 30 patients with RA, 40 with SLE, and 24 controls. A newly identified polymorphism was transfected in COS-1 cells and the stability of the mRNA containing the polymorphism was tested using real-time PCR. RESULTS: A polymorphism in the hGR gene in exon9beta, in an "ATTTA" motif, was found to be significantly associated with RA. Introduction of this polymorphism in the hGRb mRNA was found to significantly increase stability in vitro compared to the wild-type sequence. CONCLUSION: Our findings show an association between RA and a previously unreported polymorphism in the hGR gene. This polymorphism increased stability of hGRbeta mRNA, which could contribute to an altered glucocorticoid sensitivity since the hGRbeta is thought to function as an inhibitor of hGRalpha activity.

Lithium-related genetics of bipolar disorder.

Lithium is a potent prophylactic medication and mood stabilizer in bipolar disorder. However, clinical outcome is variable, and its therapeutic effect manifests after a period of chronic treatment, implying a progressive and complex biological response process. Signal transduction systems known to be perturbed by lithium involve phosphoinositide (PI) turnover, activation of the Wnt pathway via inhibition of glycogen synthase kinase-3beta (GSK-3beta), and a growth factor-induced, Akt-mediated signalling that promotes cell survival. These pathways, acting in synergy, probably prompt the amplification of lithium signal causing such immense impact on the neuronal network. The sequencing of the human genome presents an unparallelled opportunity to uncover the full molecular repertoire involved in lithium action. Interrogation of high-resolution expression microarrays and protein profiles represents a strategy that should help accomplish this goal. A recent microarray analysis on lithium-treated versus untreated PC12 cells identified multiple differentially altered transcripts. Lithium-perturbed genes, particularly those that map to susceptibility regions, could be candidate risk-conferring factors for mood disorders. Transcript and protein profiling in patients could reveal a lithium fingerprint for responsiveness or nonresponsiveness, and a signature motif that may be diagnostic of a specific phenotype. Similarly, lithium-sensitive gene products could provide a new generation of pharmacological targets.

Fine mapping supports previous linkage evidence for a bipolar disorder susceptibility locus on 13q32.

A region between D13S71 and D13S274 on 13q32 showed linkage to bipolar disorder (BP) based on a genome scan using markers with an average spacing of approximately 6 cM and an average heterozygosity of approximately 60% [Detera-Wadleigh et al., 1999: Proc Natl Acad Sci USA 96:5604-5609]. In an attempt to confirm this finding and achieve fine mapping of the susceptibility region, nine additional microsatellite markers with average heterozygosity of approximately 86%, located between D13S71 and D13S274, were typed in the same sample. The strongest linkage evidence was detected by multipoint linkage analysis (ASPEX program) around D13S779-D13S225 with maximum LOD score of 3.25 under Affection Status Model II (ASM II; P = 0.0000546). Data from additional nine markers resulted in a decrease of the 95% confidence interval of the linkage region. Association analyses with GASSOC TDT and ASPEX/sib_tdt detect potential linkage disequilibrium with several markers, including D13S280 (ASPEX TDT P = 0.0033, ASM I). These data generated using a higher marker density within the proposed susceptibility region strengthen the validity of our previous findings and suggest a finer localization of the susceptibility gene(s) on 13q32.

Evidence for association of the myo-inositol monophosphatase 2 (IMPA2) gene with schizophrenia in Japanese samples.

In our search for candidate genes for affective disorder on the short arm of chromosome 18, we cloned IMPA2, a previously unreported myo-inositol monophosphatase gene, that maps to 18p11.2. We determined its genomic structure and detected three new single nucleotide polymorphisms (SNPs). In the present study, we screened the gene further to search for additional polymorphisms in Japanese samples and identified seven other SNPs, including a novel missense mutation. These polymorphisms clustered into three regions of the gene. Three relatively informative SNPs, 58G>A, IVS1--15G>A and 800C>T from clusters 1, 2 and 3, respectively, were selected for association tests using a case-control design. The Japanese cohort included 302 schizophrenics, 205 patients with affective disorder and 308 controls. Genotyping was done either by melting curve analysis on the LightCycler or by sequencing. All three SNPs showed significant genotypic association (nominal P = 0.031--0.0001) with schizophrenia, but not with affective disorder. These findings increase the relevance of 18p11.2 to schizophrenia susceptibility because GNAL, which has been shown previously to be implicated in schizophrenia in an independent study, is in close physical proximity to IMPA2. Our findings suggest that IMPA2 or a gene nearby may contribute to the overall genetic risk for schizophrenia among Japanese.

Association study on the DUSP6 gene, an affective disorder candidate gene on 12q23, performed by using fluorescence resonance energy transfer-based melting curve analysis on the LightCycler.

We introduced a new genotyping method, fluorescence resonance energy transfer-based melting curve analysis on the LightCycler, for the analysis of the gene, DUSP6 (dual specificity MAP kinase phosphatase 6), in affective disorder patients. The DUSP6 gene is located on chromosome 12q22-23, which overlaps one of the reported bipolar disorder susceptibility loci. Because of its role in intracellular signalling pathways, the gene may be involved in the pathogenesis of affective disorders not only on the basis of its position but also of its function. We performed association analysis using a T>G polymorphism that gives rise to a missense mutation (Leu114Val). No evidence for a significant disease-causing effect was found in Japanese unipolars (n = 132) and bipolars (n = 122), when compared with controls (n = 299). More importantly, this study demonstrates that melting curve analysis on the LightCycler is an accurate, rapid and robust method for discriminating genotypes from biallelic markers. This strategy has the potential for use in high throughput scanning for and genotyping of single nucleotide polymorphisms (SNPs).