Lithium-responsive genes and gene networks in bipolar disorder patient-derived lymphoblastoid cell lines.

Lithium (Li) is the mainstay mood stabilizer for the treatment of bipolar disorder (BD), although its mode of action is not yet fully understood nor is it effective in every patient. We sought to elucidate the mechanism of action of Li and to identify surrogate outcome markers that can be used to better understand its therapeutic effects in BD patients classified as good (responders) and poor responders (nonresponders) to Li treatment. To accomplish these goals, RNA-sequencing gene expression profiles of lymphoblastoid cell lines (LCLs) were compared between BD Li responders and nonresponders with healthy controls before and after treatment. Several Li-responsive gene coexpression networks were discovered indicating widespread effects of Li on diverse cellular signaling systems including apoptosis and defense response pathways, protein processing and response to endoplasmic reticulum stress. Individual gene markers were also identified, differing in response to Li between BD responders and nonresponders, involved in processes of cell cycle and nucleotide excision repair that may explain part of the heterogeneity in clinical response to treatment. Results further indicated a Li gene expression signature similar to that observed with clonidine treatment, an α2-adrenoceptor agonist. These findings provide a detailed mechanism of Li in LCLs and highlight putative surrogate outcome markers that may permit for advanced treatment decisions to be made and for facilitating recovery in BD patients.

Genetic pleiotropy between multiple sclerosis and schizophrenia but not bipolar disorder: differential involvement of immune-related gene loci.

Converging evidence implicates immune abnormalities in schizophrenia (SCZ), and recent genome-wide association studies (GWAS) have identified immune-related single-nucleotide polymorphisms (SNPs) associated with SCZ. Using the conditional false discovery rate (FDR) approach, we evaluated pleiotropy in SNPs associated with SCZ (n=21,856) and multiple sclerosis (MS) (n=43,879), an inflammatory, demyelinating disease of the central nervous system. Because SCZ and bipolar disorder (BD) show substantial clinical and genetic overlap, we also investigated pleiotropy between BD (n=16,731) and MS. We found significant genetic overlap between SCZ and MS and identified 21 independent loci associated with SCZ, conditioned on association with MS. This enrichment was driven by the major histocompatibility complex (MHC). Importantly, we detected the involvement of the same human leukocyte antigen (HLA) alleles in both SCZ and MS, but with an opposite directionality of effect of associated HLA alleles (that is, MS risk alleles were associated with decreased SCZ risk). In contrast, we found no genetic overlap between BD and MS. Considered together, our findings demonstrate genetic pleiotropy between SCZ and MS and suggest that the MHC signals may differentiate SCZ from BD susceptibility.

Clinical Pharmacogenetics Implementation Consortium guidelines for HLA-B genotype and carbamazepine dosing.

Human leukocyte antigen B (HLA-B) is a gene that encodes a cell surface protein involved in presenting antigens to the immune system. The variant allele HLA-B*15:02 is associated with an increased risk of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) in response to carbamazepine treatment. We summarize evidence from the published literature supporting this association and provide recommendations for the use of carbamazepine based on HLA-B genotype (also available on PharmGKB: http://www.pharmgkb.org). The purpose of this article is to provide information to allow the interpretation of clinical HLA-B*15:02 genotype tests so that the results can be used to guide the use of carbamazepine. The guideline provides recommendations for the use of carbamazepine when HLA-B*15:02 genotype results are available. Detailed guidelines regarding the selection of alternative therapies, the use of phenotypic tests, when to conduct genotype testing, and cost-effectiveness analyses are beyond the scope of this document. Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines are published and updated periodically on the PharmGKB website at (http://www.pharmgkb.org).

Enrichment of cis-regulatory gene expression SNPs and methylation quantitative trait loci among bipolar disorder susceptibility variants.

We conducted a systematic study of top susceptibility variants from a genome-wide association (GWA) study of bipolar disorder to gain insight into the functional consequences of genetic variation influencing disease risk. We report here the results of experiments to explore the effects of these susceptibility variants on DNA methylation and mRNA expression in human cerebellum samples. Among the top susceptibility variants, we identified an enrichment of cis regulatory loci on mRNA expression (eQTLs), and a significant excess of quantitative trait loci for DNA CpG methylation, hereafter referred to as methylation quantitative trait loci (mQTLs). Bipolar disorder susceptibility variants that cis regulate both cerebellar expression and methylation of the same gene are a very small proportion of bipolar disorder susceptibility variants. This finding suggests that mQTLs and eQTLs provide orthogonal ways of functionally annotating genetic variation within the context of studies of pathophysiology in brain. No lymphocyte mQTL enrichment was found, suggesting that mQTL enrichment was specific to the cerebellum, in contrast to eQTLs. Separately, we found that using mQTL information to restrict the number of single-nucleotide polymorphisms studied enhances our ability to detect a significant association. With this restriction a priori informed by the observed functional enrichment, we identified a significant association (rs12618769, P(bonferroni)<0.05) from two other GWA studies (TGen+GAIN; 2191 cases and 1434 controls) of bipolar disorder, which we replicated in an independent GWA study (WTCCC). Collectively, our findings highlight the importance of integrating functional annotation of genetic variants for gene expression and DNA methylation to advance the biological understanding of bipolar disorder.

A genome-wide association study of attempted suicide.

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.

Functional genetic variation in the Rev-Erbα pathway and lithium response in the treatment of bipolar disorder.

Bipolar disorder (BD) is characterized by disruptions in circadian rhythms such as sleep and daily activity that often normalize after lithium treatment in responsive patients. As lithium is known to interact with the circadian clock, we hypothesized that variation in circadian 'clock genes' would be associated with lithium response in BD. We determined genotype for 16 variants in seven circadian clock genes and conducted a candidate gene association study of these in 282 Caucasian patients with BD who were previously treated with lithium. We found that a variant in the promoter of NR1D1 encoding Rev-Erbα (rs2071427) and a second variant in CRY1 (rs8192440) were nominally associated with good treatment response. Previous studies have shown that lithium regulates Rev-Erbα protein stability by inhibiting glycogen synthase kinase 3ß (GSK3ß). We found that GSK3ß genotype was also suggestive of a lithium response association, but not statistically significant. However, when GSK3ß and NR1D1 genotypes were considered together, they predicted lithium response robustly and additively in proportion to the number of response-associated alleles. Using lymphoblastoid cell lines from patients with BD, we found that both the NR1D1 and GSK3ß variants are associated with functional differences in gene expression. Our findings support a role for Rev-Erbα in the therapeutic mechanism of lithium and suggest that the interaction between Rev-Erbα and GSK3ß may warrant further study.

A genome-wide association study of bipolar disorder and comorbid migraine.

Both migraine and bipolar affective disorder (BPAD) are complex phenotypes with significant genetic and nongenetic components. Epidemiological and clinical studies have showed a high degree of comorbidity between migraine and BPAD, and overlapping regions of linkage have been shown in numerous genome-wide linkage studies. To identify susceptibility factors for the BPAD/migraine phenotype, we conducted a genome-wide association study (GWAS) in 1001 cases with bipolar disorder collected through the NIMH Genetics Initiative for Bipolar Disorder and genotyped at 1 m single-nucleotide polymorphisms (SNPs) as part of the Genetic Association Information Network (GAIN). We compared BPAD patients without any headache (n = 699) with BPAD patients with doctor diagnosed migraine (n = 56). The strongest evidence for association was found for several SNPs in a 317-kb region encompassing the uncharacterized geneKIAA0564 {e.g. rs9566845 [OR = 4.98 (95% CI: 2.6-9.48), P = 7.7 × 10(-8)] and rs9566867 (P = 8.2 × 10(-8))}. Although the level of significance was significantly reduced when using the Fisher's exact test (as a result of the low count of cases with migraine), rs9566845 P = 1.4 × 10(-5) and rs9566867 P = 1.5 × 10(-5), this region remained the most prominent finding. Furthermore, marker rs9566845 was genotyped and found associated with migraine in an independent Norwegian sample of adult attention deficit hyperactivity disorder (ADHD) patients with and without comorbid migraine (n = 131 and n = 324, respectively), OR = 2.42 (1.18-4.97), P = 0.013. This is the first GWAS examining patients with bipolar disorder and comorbid migraine. These data suggest that genetic variants in the KIAA0564 gene region may predispose to migraine headaches in subgroups of patients with both BPAD and ADHD.

A genome-wide linkage study of bipolar disorder and co-morbid migraine: replication of migraine linkage on chromosome 4q24, and suggestion of an overlapping susceptibility region for both disorders on chromosome 20p11.

Migraine and Bipolar Disorder (BPAD) are clinically heterogeneous disorders of the brain with a significant, but complex, genetic component. Epidemiological and clinical studies have demonstrated a high degree of co-morbidity between migraine and BPAD. Several genome-wide linkage studies in BPAD and migraine have shown overlapping regions of linkage on chromosomes, and two functionally similar voltage-dependent calcium channels CACNA1A and CACNA1C have been identified in familial hemiplegic migraine and recently implicated in two whole genome BPAD association studies, respectively. We hypothesized that using migraine co-morbidity to look at subsets of BPAD families in a genetic linkage analysis would prove useful in identifying genetic susceptibility regions in both of these disorders. We used BPAD with co-morbid migraine as an alternative phenotype definition in a re-analysis of the NIMH Bipolar Genetics Initiative wave 4 data set. In this analysis we selected only those families in which at least two members were diagnosed with migraine by a doctor according to patients' reports. Nonparametric linkage analysis performed on 31 families segregating both BPAD and migraine identified a linkage signal on chromosome 4q24 for migraine (but not BPAD) with a peak LOD of 2.26. This region has previously been implicated in two independent migraine linkage studies. In addition we identified a locus on chromosome 20p11 with overlapping elevated LOD scores for both migraine (LOD=1.95) and BPAD (LOD=1.67) phenotypes. This region has previously been implicated in two BPAD linkage studies, and, interestingly, it harbors a known potassium dependant sodium/calcium exchanger gene, SLC24A3, that plays a critical role in neuronal calcium homeostasis. Our findings replicate a previously identified migraine linkage locus on chromosome 4 (not co-segregating with BPAD) in a sample of BPAD families with co-morbid migraine, and suggest a susceptibility locus on chromosome 20, harboring a gene for the migraine/BPAD phenotype. Together these data suggest that some genes may predispose to both bipolar disorder and migraine.

Genome-wide association study of bipolar disorder in European American and African American individuals.

To identify bipolar disorder (BD) genetic susceptibility factors, we conducted two genome-wide association (GWA) studies: one involving a sample of individuals of European ancestry (EA; n=1001 cases; n=1033 controls), and one involving a sample of individuals of African ancestry (AA; n=345 cases; n=670 controls). For the EA sample, single-nucleotide polymorphisms (SNPs) with the strongest statistical evidence for association included rs5907577 in an intergenic region at Xq27.1 (P=1.6 x 10(-6)) and rs10193871 in NAP5 at 2q21.2 (P=9.8 x 10(-6)). For the AA sample, SNPs with the strongest statistical evidence for association included rs2111504 in DPY19L3 at 19q13.11 (P=1.5 x 10(-6)) and rs2769605 in NTRK2 at 9q21.33 (P=4.5 x 10(-5)). We also investigated whether we could provide support for three regions previously associated with BD, and we showed that the ANK3 region replicates in our sample, along with some support for C15Orf53; other evidence implicates BD candidate genes such as SLITRK2. We also tested the hypothesis that BD susceptibility variants exhibit genetic background-dependent effects. SNPs with the strongest statistical evidence for genetic background effects included rs11208285 in ROR1 at 1p31.3 (P=1.4 x 10(-6)), rs4657247 in RGS5 at 1q23.3 (P=4.1 x 10(-6)), and rs7078071 in BTBD16 at 10q26.13 (P=4.5 x 10(-6)). This study is the first to conduct GWA of BD in individuals of AA and suggests that genetic variations that contribute to BD may vary as a function of ancestry.

Singleton deletions throughout the genome increase risk of bipolar disorder.

An overall burden of rare structural genomic variants has not been reported in bipolar disorder (BD), although there have been reports of cases with microduplication and microdeletion. Here, we present a genome-wide copy number variant (CNV) survey of 1001 cases and 1034 controls using the Affymetrix single nucleotide polymorphism (SNP) 6.0 SNP and CNV platform. Singleton deletions (deletions that appear only once in the dataset) more than 100 kb in length are present in 16.2% of BD cases in contrast to 12.3% of controls (permutation P=0.007). This effect was more pronounced for age at onset of mania

Impaired postnatal development of hippocampal dentate gyrus in Sp4 null mutant mice.

Sp4, a member of the Sp1 family of transcription factors, is expressed restrictively in the developing nervous system and abundantly in the hippocampus. Previously, we demonstrated that hypomorphic Sp4 mice display hippocampal vacuolization and concomitant deficits in memory and sensorimotor gating. Here, we report further analyses of Sp4 functions during postnatal development of the dentate gyrus in Sp4 null mutant mice. A reduced cell proliferation restrictively in hippocampus, but not cerebellum, was observed in the first week of postnatal development of Sp4 null mutant mice. The dendritic growth and arborization of dentate granule cells was decreased in hippocampal cultures in vitro from mutant neonatal mice. The adult Sp4 null mutant mice displayed decreased dentate granule cell density with reduced width of both dentate gyrus and the molecular layer. The abnormality of the molecular layer was indicated by a reduced level of synaptophysin expression in the mutant mice. The Sp4 transcription factor therefore appears to predominantly regulate the development of dentate granule cells.

Identification of additional variants within the human dopamine transporter gene provides further evidence for an association with bipolar disorder in two independent samples.

The dopamine transporter (DAT) is the site of action of stimulants, and variations in the human DAT gene (DAT1) have been associated with susceptibility to several psychiatric disorders including attention deficit hyperactivity disorder (ADHD) and bipolar disorder. We have previously reported the association of bipolar disorder to novel SNPs in the 3' end of DAT1. We now report the identification of 20 additional SNPs in DAT1 for a total of 63 variants. We also report evidence for association to bipolar disorder in a second independent sample of families. Eight newly identified SNPs and 14 previously identified SNPs were analyzed in two independent samples of 50 and 70 families each using the transmission disequilibrium test. Two of the eight new SNPs, one in intron 8 and one in intron 13, were found to be moderately associated with bipolar disorder, each in one of the two independent samples. Analysis of haplotypes comprised of all 22 SNPs in sliding windows of five adjacent SNPs revealed an association to the region near introns 7 and 8 in both samples (empirical P-values 0.002 and 0.001, respectively, for the same window). The haplotype block structure observed in the gene in our previous study was confirmed in this sample with greater resolution allowing for discrimination of a third haplotype block in the middle of the gene. Together, these data are consistent with the presence of multiple variants in DAT1 that convey susceptibility to bipolar disorder.

Reduced expression of the Sp4 gene in mice causes deficits in sensorimotor gating and memory associated with hippocampal vacuolization.

HF-1B/SP4:, a member of the Sp1 family of transcription factors, is expressed restrictively in the developing nervous system and most abundantly in adult hippocampus in mice. Here, we report the generation of hypomorphic Sp4 allele mice, in which the Sp4 deficiency can be rescued by the expression of Cre recombinase. Vacuolization was detected in the hippocampal gray matter of the mutant Sp4-deficient mice. Expression analysis of Sp4 mutant hippocampi revealed an age-dependent decrease in neurotrophin-3 expression in the dentate granule cells. Hypomorphic Sp4 mutant mice displayed robust deficits in both sensorimotor gating and contextual memory. The restoration of Sp4 expression, via a Cre-dependent rescue strategy, completely rescued all the observed molecular, histological and behavioral abnormalities. Our studies thus reveal a novel Sp4 pathway that is essential for hippocampal integrity and modulates behavioral processes relevant to psychiatric disorders.

Evidence that a single nucleotide polymorphism in the promoter of the G protein receptor kinase 3 gene is associated with bipolar disorder.

In a genome-wide linkage survey, we have previously shown evidence suggesting that the chromosome 22q12 region contains a susceptibility locus for bipolar disorder (BPD). Two independent family sets yielded lod scores suggestive of linkage at markers in this region near the gene G protein receptor kinase 3 (GRK3). GRK3 is an excellent candidate risk gene for BPD since GRK3 is expressed widely in the brain, and since GRKs play key roles in the homologous desensitization of G protein-coupled receptor signaling. We have also previously shown GRK3 expression to be induced by amphetamine in an animal model of mania using microarray-based expression profiling. To identify possible functional mutations in GRK3, we sequenced the putative promoter region, all 21 exons, and intronic sequence flanking each exon, in 14-22 individuals with BPD. We found six sequence variants in the 5'-UTR/promoter region, but no coding or obvious splice variants. Transmission disequilibrium analyses of one set of 153 families indicated that two of the 5'-UTR/promoter variants are associated with BPD in families of northern European Caucasian ancestry. A supportive trend towards association to one of these two variants (P-5) was then subsequently obtained in an independent sample of 237 families. In the combined sample, the P-5 variant had an estimated allele frequency of 3% in bipolar subjects, and displayed a transmission to non-transmission ratio of 26 : 7.7 (chi(2)=9.6, one-sided P value=0.0019). Altogether, these data support the hypothesis that a dysregulation in GRK3 expression alters signaling desensitization, and thereby predisposes to the development of BPD.

Linkage of a bipolar disorder susceptibility locus to human chromosome 13q32 in a new pedigree series.

Bipolar (BP) disorder or manic depressive illness is a major psychiatric disorder for which numerous family, twin and adoption studies support a substantial genetic contribution. Recently, we reported the results of a genome-wide search for BP disorder susceptibility loci in 20 pedigrees. Suggestive evidence for linkage was found in this study at three markers on 13q, representing possibly two peaks separated by 18 cM. We have now collected a second set of 32 pedigrees segregating BP disorder and have tested for evidence of linkage to markers on human chromosome 13q. In this sample, we have replicated the linkage result in 13q32 at D13S154 (lod=2.29), the more proximal of the two original peaks. When all 52 pedigrees were combined, the multipoint maximum lod score peaked approximately 7 cM proximal to D13S154 (lod=3.40), with a second peak occurring between D13S225 and D13S796 (lod=2.58). There have been several other reports of significant linkage to both BP disorder and schizophrenia in this region of chromosome 13. These pedigrees provide additional evidence for at least one locus for BP disorder in 13q32, and are consistent with other reports of a possible genetic overlap between these disorders.

Segmental linkage disequilibrium within the dopamine transporter gene.

The dopamine transporter gene (DAT) has been implicated in a variety of disorders, including bipolar disorder, attention-deficit hyperactivity disorder, cocaine-induced paranoia, Tourette's syndrome, and Parkinson's disease. As no clear functional polymorphism has been identified to date, studies rely on linkage disequilibrium (LD) to assess the possible genetic contribution of DAT to the various disorders. A better understanding of the complex structure of LD across the gene is thus critical for an accurate interpretation of the results of such studies, and may facilitate the mapping of the actual functional variants. In the process of characterizing the extent of variation within the DAT gene, we have identified a number of single nucleotide polymorphisms (SNPs) suitable for LD studies, 14 of which have been analyzed, along with a 3' repeat polymorphism, in a sample of 120 parent-proband triads. Calculations of pairwise LD between the SNPs in the parental haplotypes revealed a high degree of LD (P < 0.00001) in the 5' (distal promoter through intron 6) and 3' (exon 9 through exon 15) regions of DAT. This segmental LD pattern is maintained over approximately 27 kb and 20 kb in these two regions, respectively, with very little significant LD between them, possibly due to the presence of a recombination hotspot located near the middle of the gene. These analyses of the DAT gene thus reveal a complex structure resulting from both recombination and mutation, knowledge of which may be invaluable to the design of future studies.