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.

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.

Genome-wide linkage analysis of 972 bipolar pedigrees using single-nucleotide polymorphisms.

Because of the high costs associated with ascertainment of families, most linkage studies of Bipolar I disorder (BPI) have used relatively small samples. Moreover, the genetic information content reported in most studies has been less than 0.6. Although microsatellite markers spaced every 10 cM typically extract most of the genetic information content for larger multiplex families, they can be less informative for smaller pedigrees especially for affected sib pair kindreds. For these reasons we collaborated to pool family resources and carried out higher density genotyping. Approximately 1100 pedigrees of European ancestry were initially selected for study and were genotyped by the Center for Inherited Disease Research using the Illumina Linkage Panel 12 set of 6090 single-nucleotide polymorphisms. Of the ~1100 families, 972 were informative for further analyses, and mean information content was 0.86 after pruning for linkage disequilibrium. The 972 kindreds include 2284 cases of BPI disorder, 498 individuals with bipolar II disorder (BPII) and 702 subjects with recurrent major depression. Three affection status models (ASMs) were considered: ASM1 (BPI and schizoaffective disorder, BP cases (SABP) only), ASM2 (ASM1 cases plus BPII) and ASM3 (ASM2 cases plus recurrent major depression). Both parametric and non-parametric linkage methods were carried out. The strongest findings occurred at 6q21 (non-parametric pairs LOD 3.4 for rs1046943 at 119 cM) and 9q21 (non-parametric pairs logarithm of odds (LOD) 3.4 for rs722642 at 78 cM) using only BPI and schizoaffective (SA), BP cases. Both results met genome-wide significant criteria, although neither was significant after correction for multiple analyses. We also inspected parametric scores for the larger multiplex families to identify possible rare susceptibility loci. In this analysis, we observed 59 parametric LODs of 2 or greater, many of which are likely to be close to maximum possible scores. Although some linkage findings may be false positives, the results could help prioritize the search for rare variants using whole exome or genome sequencing.

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.

Quantitative neuroanatomy in schizophrenia. A controlled magnetic resonance imaging study.

Twenty-four patients with schizophrenia and 14 normal control subjects underwent magnetic resonance imaging scans using a 0.5-tesla scanner and 600-ms inversion recovery technique. A midsagittal section and twelve 1-cm coronal sections beginning at the frontal pole were obtained, and linear, area, and signal intensity measurements were made on nine brain regions. Volume estimates were made by summing consecutive sections for four of the following regions: the precallosal frontal lobes, temporal lobes, lateral ventricles, amygdala-hippocampal complexes, and cerebral hemispheres. The area of the third ventricle in its most anterior coronal slice was increased by 73% in schizophrenic subjects (0.83 +/- 0.08 cm2) in comparison with controls (0.48 +/- 0.04 cm2). Lateral ventricular volume was increased by 62% in schizophrenic subjects (24.7 +/- 2.6 mL) in comparison with controls (15.2 +/- 1.4 mL). The lateral ventricular enlargement in schizophrenic subjects was more pronounced posteriorly than anteriorly, especially at the level of the anterior thalamus and the colliculi. There were no other significant differences between schizophrenic and control groups in any other spatial or signal intensity measures. There was no brain region the size of which correlated with ventricular size. These data corroborate third and lateral ventriculomegaly in schizophrenia using magnetic resonance imaging but fail to further localize the structural abnormality.

Early parental loss and development of adult psychopathology.

We assessed the effect of parental loss during childhood on the development of psychopathology in 90 adults. The subjects with a history of adult psychopathology (PATH group), in comparison with subjects with no history of a psychiatric disorder (NO PATH group), had poorer quality of childhood home life and personal adaptation subsequent to parental loss as assessed by the Home Life and Personal Adaptation (HAPA) scale developed by us. Total HAPA scale scores were the single most powerful predictor of adult psychopathology, accounting for correct prediction of adult psychopathology in 80% (72/90) of the subjects. The PATH subjects had significantly increased plasma levels of cortisol and beta-endorphin immunoreactivity. Moreover, cortisol and adrenocorticotropic hormone levels significantly correlated with total HAPA scores. First-degree family history of psychiatric disorders, age at loss, and parental vs maternal loss were not significantly different between PATH and NO PATH subjects. We conclude that the quality of home life subsequent to early parental loss is critically related to the development of adult psychopathology. The hypothesis that early trauma results in enduring neuroendocrine alterations in hypothalamic-pituitary-adrenal axis function is examined.

Alprazolam augmentation of the antipsychotic effects of fluphenazine in schizophrenic patients. Preliminary results.

Alprazolam was added, under double-blind conditions, to stable fluphenazine hydrochloride regimens in 12 symptomatic, chronically ill inpatients with schizophrenia. The addition of alprazolam was associated with significant, albeit modest, reductions in global psychosis, thought disorder, and paranoia ratings, with a return to pretreatment levels on discontinuation of alprazolam treatment. Improvement in "negative symptoms" such as emotional withdrawal paralleled the changes in "positive symptoms" but did not, in itself, reach statistical significance. There were no significant changes in group mean plasma levels of homovanillic acid or 3-methoxy-4-hydroxyphenylglycol during alprazolam treatment, although group mean serum cortisol levels were significantly decreased by alprazolam treatment. Patients who responded favorably to alprazolam treatment were significantly more psychotic or anxious before treatment, were older, showed significant alprazolam-associated reductions in plasma levels of homovanillic acid, and had significantly more prominent prefrontal cortex atrophy on computed tomographic scans than patients in whom alprazolam was without therapeutic effect. These preliminary data, based on a small sample, suggest that some patients with schizophrenia who are only partially responsive to standard neuroleptic treatment may benefit from the addition of triazolobenzodiazepines, such as alprazolam.

The cholinergic rapid eye movement induction test with arecoline in depression.

The cholinergic rapid eye movement (REM) induction test using arecoline hydrobromide, a cholinergic muscarinic receptor agonist, was studied in patients with affective disorder and in normal controls to determine whether or not depression is associated with enhanced induction of REM sleep by muscarinic agonists. Arecoline induced REM sleep in a dose-dependent fashion in both patients and controls compared with placebo infusions. Compared with normal controls, patients entered REM sleep significantly more rapidly following intravenous administration of 1.0 mg of arecoline hydrobromide than they did following administration of 0.5 mg of arecoline hydrobromide or placebo. These results, as well as those of previous studies, support the hypothesis that patients with affective disorder show a functional supersensitive induction of REM sleep in response to muscarinic receptor agonists and may be consistent with the hypothesis that functional muscarinic receptor "up regulation" is associated with depression.

Sleep and sleep electroencephalogram in depressed patients treated with phenelzine.

BACKGROUND: The beneficial effect of antidepressant interventions has been proposed to depend on suppression of rapid eye movement (REM) sleep or inhibition of electroencephalographic (EEG) slow-wave activity (SWA) in non-REM sleep. Use of the monoamine oxidase inhibitor phenelzine sulfate can eliminate REM sleep. We studied the relation between REM sleep suppression and antidepressant response and the effect of phenelzine therapy on sleep EEG power spectra. METHODS: Open-labeled prescriptions of 30 to 90 mg of phenelzine were given to 11 patients with major depressive disorder (6 men and 5 women; mean age, 41.4 years); all were physically healthy. Mood, dream recall, sleep, sleep EEG, and ocular and muscular activity during sleep were studied before treatment and during the third and fifth weeks of pharmacotherapy. RESULTS: Six patients remitted from depression, 2 responded partially, and 3 showed no antidepressant response. Independent from clinical response, REM sleep was dramatically suppressed. On average, only 4.9 minutes of REM sleep was observed in treatment week 5, and it was completely absent in 6 patients. This effect was compensated for by increased stage 2 sleep. In non-REM sleep, EEG power was higher than at baseline between 16.25 and 25 Hz. Slow-wave activity (power within 0.75-4.5 Hz) and the exponential decline of SWA during sleep were not affected. CONCLUSIONS: Antidepressant response to phenelzine treatment does not depend on elimination of REM sleep or inhibition of SWA in non-REM sleep. In depressed patients, REM sleep is regulated independently from non-REM sleep and can be manipulated without altering the dynamics of SWA.

Rapid tryptophan depletion, sleep electroencephalogram, and mood in men with remitted depression on serotonin reuptake inhibitors.

BACKGROUND: In previous studies, depletion of brain serotonin by administration of a tryptophan-free amino acid drink (TFD) (1) temporarily reversed the antidepressant effects of selective serotonin reuptake inhibitors (SSRIs) in euthymic patients who had a history of major depression, and (2) enhanced rapid eye movement (REM) sleep in normal volunteers. In this study, we hypothesized that the TFD would not only increase depressive symptoms but also the propensity for REM sleep in euthymic patients treated with SSRIs. METHODS: Ten fully remitted, medicated male patients who had a history of major depressive episode ingested a 100-g TFD (the experimental dose) or a 25-g TFD (designed to be the control drink) in double-blind, random order on separate days. The effects were assessed with mood ratings, plasma tryptophan concentrations, and an all-night sleep electroencephalogram. RESULTS: The TFDs produced a dose-dependent reduction in plasma tryptophan concentrations, sleep latency, and REM latency, as well as increased REM percentage, REM minutes, REM density, and total sleep time. Neither strength of TFD altered mood to a clinically significant degree. CONCLUSIONS: Although the TFD affected plasma tryptophan concentrations and various sleep measures, our study did not confirm previous reports that TFD temporarily reversed the antidepressant effects of SSRIs in euthymic patients. Our patients, however, had been treated for a longer period with SSRIs and were more fully remitted at the time of the study. Our results suggest that TFD-induced relapse in SSRI-treated patients in remission decreases as a function of treatment duration, degree of remission, or both.

Identifying a series of candidate genes for mania and psychosis: a convergent functional genomics approach.

We have used methamphetamine treatment of rats as an animal model for psychotic mania. Specific brain regions were analyzed comprehensively for changes in gene expression using oligonucleotide GeneChip microarrays. The data was cross-matched against human genomic loci associated with either bipolar disorder or schizophrenia. Using this convergent approach, we have identified several novel candidate genes (e.g., signal transduction molecules, transcription factors, metabolic enzymes) that may be involved in the pathogenesis of mood disorders and psychosis. Furthermore, for one of these genes, G protein-coupled receptor kinase 3 (GRK3), we found by Western blot analysis evidence for decreased protein levels in a subset of patient lymphoblastoid cell lines that correlated with disease severity. Finally, the classification of these candidate genes into two prototypical categories, psychogenes and psychosis-suppressor genes, is described.

Sleep and dreams in Vietnam PTSD and depression.

The present study compares the sleep and dreams of three groups of subjects: 1) Vietnam veterans with posttraumatic stress disorder (PTSD) and major depression, 2) veterans with depression alone, and 3) veterans with neither PTSD nor depression (i.e., normal controls). Sleep recordings indicate only one significant difference between the PTSD/depressed and depressed alone groups: sleep latency was prolonged in the depressed alone patients compared with the other two groups. The two patient groups differed from controls in the manner already reported for depressed patients (decreased REM latency, increased REM density, reduced total sleep time, reduced sleep efficiency), with some of the differences significant only at the trend level. Dreams were obtained from REM awakenings. Dream recall rate and report length did not differ between groups. Mean anxiety level in dreams was less than 1 (mild) for all three groups, with major depression patients scoring significantly higher than controls. Dreams of PTSD/depressed patients were significantly less likely to be set in the present than dreams of the other two groups.

Effects of the acute administration of caffeine in patients with schizophrenia.

Caffeine, 10 mg/kg, was administered to 13 schizophrenic patients in a double-blind placebo-controlled study of its behavioral effects. Some measures of psychopathology were significantly increased: Brief Psychiatric Rating Scale (BPRS) total, BPRS subscales thought disorder, unusual thought content, and euphoria-activation, and several individual BPRS items. Nurses' Bunney-Hamberg ratings of psychosis and mania, comparing the day before with the day after pharmacological challenge, increased significantly. Compared to placebo, caffeine also produced significant increases of diastolic blood pressure and cortisol. Thus, these findings indicate that caffeine increases arousal and has a psychotogenic effect when administered to schizophrenic patients. The possible roles of various neurotransmitters is discussed with special emphasis on caffeine's actions on dopaminergic and adenosinergic systems.

Lack of association between an RFLP near the D2 dopamine receptor gene and severe alcoholism.

Blum et al (1990) have recently examined a restriction fragment length polymorphism (RFLP) detected by TaqI RFLP to the dopamine D2 receptor gene (DRD2) in deceased alcoholics and nonalcoholics, and reported an association between alcoholism and the A1 allele. Subsequent studies, however, by other investigators have failed to confirm this. We have examined the DRD2 TaqI RFLP in 47 living Caucasian males with severe alcoholism. All alcoholic subjects were thoroughly characterized by a structured interview, and met DSM-III-R criteria for alcohol dependence. Only 9/47 (19%) (1990) of these alcoholics had the AI allele compared to 14/22 (64%) reported by Blum et al. This rate was not significantly different from the rates reported in control populations by Blum et al (1990), CEPH, or Bolos et al (1990), and differed only slightly from those reported by Grandy et al (1990). Alcoholics selected for severe medical complications also displayed a similar rate. Our data do not support an association between alcoholism and the D2 dopamine receptor gene in this population.

Sleep electroencephalographic response to muscarinic and serotonin1A receptor probes in patients with major depression and in normal controls.

BACKGROUND: To test the hypothesis that depression is associated with an increased ratio of cholinergic to serotonergic neurotransmission, we compared the effects of pilocarpine, a muscarinic agonist, and ipsapirone, a serotonin (5-HT)1A agonist, on electroencephalographic (EEG) sleep in depressed and healthy subjects. We hypothesized, adopting the reciprocal interaction model, that the effects on REM sleep of these probes within the same individuals are negatively correlated and unmask neurobiological changes in depression. METHODS: Polysomnographic recordings were obtained in 12 unmedicated patients with a current major depression and 12 normal controls. They received placebo, pilocarpine 25 mg, or ipsapirone 10 mg (orally, 15 min before bedtime, after premedication with the peripheral anticholinergic probanthine 30 mg, double blind, counterbalanced) on three occasions. RESULTS: Pilocarpine shortened and ipsapirone prolonged REM latency equally in both groups. These effects were not correlated. Pilocarpine decreased slow-wave sleep and EEG delta power during the first nonREM episode more in controls than in patients, and enhanced EEG sigma power equally in both groups. Ipsapirone had no significant effects on EEG power. CONCLUSION: These data do not support the postulate of muscarinic receptor up-regulation and 5-HT1A receptor down-regulation in depression. The significance of blunted delta power suppression in patients following pilocarpine warrants further investigations.