Astrocyte-Derived Exosomes in an iPSC Model of Bipolar Disorder.

Bipolar I Disorder (BP) is a serious, recurrent mood disorder that is characterized by alternating episodes of mania and depression. To begin to identify novel approaches and pathways involved in BP, we have obtained skin samples from BP patients and undiagnosed control (C) individuals, reprogrammed them to form induced pluripotent stem cells (iPSC), and then differentiated the stem cells into astrocytes. RNAs from BP and C astrocytes were extracted and RNAseq analysis carried out. 501 differentially expressed genes were identified, including genes for cytoskeletal elements, extracellular matrix, signaling pathways, neurodegeneration, and notably transcripts that identify exosomes. When we compared highly expressed genes using hierarchial cluster analysis, "Exosome" was the first and most highly significant cluster identified, p < 5 × 10-13, Benjamini correction. Exosomes are membrane-bound vesicles that package and remove toxic proteins from cells and also enable cell to cell communication. They carry genetic material, including DNA, mRNA and microRNAs, proteins, and lipids to target cells throughout the body. Exosomes are released by cortical neurons and astrocytes in culture and are present in BP vs C postmortem brain tissue. Little is known about what transcripts and proteins are targeted to neurons, how they regulate biological functions of the acceptor cell, or how that may be altered in mood disorders. Since astrocyte-derived exosomes have been suggested to promote neuronal plasticity, as well as to remove toxic proteins in the brain, alterations in their function or content may be involved in neurodevelopmental, neuropathological, and neuropsychiatric conditions. To examine exosome cargos and interactions with neural precursor cells, astrocytes were differentiated from four bipolar disorder (BP) and four control (C) iPSC lines. Culture supernatants from these astrocytes were collected, and exosomes isolated by ultra-centrifugation. Western blot analysis demonstrated the presence of the exosome markers CD9, CD81, and Hsp70. Nanosight technology was used to characterize exosomes from each astrocyte cell line, suggesting that exosomes were slightly more concentrated in culture supernatants derived from BP compared with C astrocytes but there was no difference in the mean sizes of the exosomes. Analysis of their function in neuronal differentiation is being carried out by labeling exosomes derived from bipolar patient and control astrocytes and adding them to control neural progenitor cells. Given the current interest in clearing toxic proteins from brains of patients with neurodegenerative disorders, exosomes may present similar opportunities in BP.

Correction: Ankyrin-G regulates forebrain connectivity and network synchronization via interaction with GABARAP.

In the original version of this article, affiliation 3 was given as: "Division of Life Sciences, State Key Laboratory of Molecular Neuroscience, Hong Kong, University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China". This has now been corrected to: "Division of Life Sciences, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China".Additionally in the 'Data availability' section an incorrect accession code was given. The accession code has now been changed from 'PDB A9X (AnkG:GABARAPL)' to 'PDB 6A9X (AnkG:GABARAP)'.These errors have been corrected in both the PDF and HTML versions of the Article.

Ankyrin-G regulates forebrain connectivity and network synchronization via interaction with GABARAP.

GABAergic circuits are critical for the synchronization and higher order function of brain networks. Defects in this circuitry are linked to neuropsychiatric diseases, including bipolar disorder, schizophrenia, and autism. Work in cultured neurons has shown that ankyrin-G plays a key role in the regulation of GABAergic synapses on the axon initial segment and somatodendritic domain of pyramidal neurons, where it interacts directly with the GABAA receptor-associated protein (GABARAP) to stabilize cell surface GABAA receptors. Here, we generated a knock-in mouse model expressing a mutation that abolishes the ankyrin-G/GABARAP interaction (Ank3 W1989R) to understand how ankyrin-G and GABARAP regulate GABAergic circuitry in vivo. We found that Ank3 W1989R mice exhibit a striking reduction in forebrain GABAergic synapses resulting in pyramidal cell hyperexcitability and disruptions in network synchronization. In addition, we identified changes in pyramidal cell dendritic spines and axon initial segments consistent with compensation for hyperexcitability. Finally, we identified the ANK3 W1989R variant in a family with bipolar disorder, suggesting a potential role of this variant in disease. Our results highlight the importance of ankyrin-G in regulating forebrain circuitry and provide novel insights into how ANK3 loss-of-function variants may contribute to human disease.

Data-driven classification of bipolar I disorder from longitudinal course of mood.

The Diagnostic and Statistical Manual of Mental Disorder (DSM) classification of bipolar disorder defines categories to reflect common understanding of mood symptoms rather than scientific evidence. This work aimed to determine whether bipolar I can be objectively classified from longitudinal mood data and whether resulting classes have clinical associations. Bayesian nonparametric hierarchical models with latent classes and patient-specific models of mood are fit to data from Longitudinal Interval Follow-up Evaluations (LIFE) of bipolar I patients (N=209). Classes are tested for clinical associations. No classes are justified using the time course of DSM-IV mood states. Three classes are justified using the course of subsyndromal mood symptoms. Classes differed in attempted suicides (P=0.017), disability status (P=0.012) and chronicity of affective symptoms (P=0.009). Thus, bipolar I disorder can be objectively classified from mood course, and individuals in the resulting classes share clinical features. Data-driven classification from mood course could be used to enrich sample populations for pharmacological and etiological studies.

Obesity, but not metabolic syndrome, negatively affects outcome in bipolar disorder.

OBJECTIVE: Examine the effects of obesity and metabolic syndrome on outcome in bipolar disorder. METHOD: The Comparative Effectiveness of a Second Generation Antipsychotic Mood Stabilizer and a Classic Mood Stabilizer for Bipolar Disorder (Bipolar CHOICE) study randomized 482 participants with bipolar disorder in a 6-month trial comparing lithium- and quetiapine-based treatment. Baseline variables were compared between groups with and without obesity, with and without abdominal obesity, and with and without metabolic syndrome respectively. The effects of baseline obesity, abdominal obesity, and metabolic syndrome on outcomes were examined using mixed effects linear regression models. RESULTS: At baseline, 44.4% of participants had obesity, 48.0% had abdominal obesity, and 27.3% had metabolic syndrome; neither obesity, nor abdominal obesity, nor metabolic syndrome were associated with increased global severity, mood symptoms, or suicidality, or with poorer functioning or life satisfaction. Treatment groups did not differ on prevalence of obesity, abdominal obesity, or metabolic syndrome. By contrast, among the entire cohort, obesity was associated with less global improvement and less improvement in total mood and depressive symptoms, suicidality, functioning, and life satisfaction after 6 months of treatment. Abdominal obesity was associated with similar findings. Metabolic syndrome had no effect on outcome. CONCLUSION: Obesity and abdominal obesity, but not metabolic syndrome, were associated with less improvement after 6 months of lithium- or quetiapine-based treatment.

Cytomegalovirus Antibody Elevation in Bipolar Disorder: Relation to Elevated Mood States.

The neurobiology of mood states is complicated by exposure to everyday stressors (e.g., psychosocial, ubiquitous environmental infections like CMV), each fluctuating between latency and reactivation. CMV reactivation induces proinflammatory cytokines (e.g., TNF-α) associated with induction of neurotoxic metabolites and the presence of mood states in bipolar disorder (BD). Whether CMV reactivation is associated with bipolar diagnoses (trait) or specific mood states is unclear. We investigated 139 BD type I and 99 healthy controls to determine if concentrations of IgG antibodies to Herpesviridae (e.g., CMV, HSV-1, and HSV-2) were associated with BD-I diagnosis and specific mood states. We found higher CMV antibody concentration in BD-I than in healthy controls (T234 = 3.1, P uncorr = 0.002; P corr = 0.006) but no difference in HSV-1 (P > 0.10) or HSV-2 (P > 0.10). Compared to euthymic BD-I volunteers, CMV IgG was higher in BD-I volunteers with elevated moods (P < 0.03) but not different in depressed moods (P > 0.10). While relationships presented between BD-I diagnosis, mood states, and CMV antibodies are encouraging, they are limited by the study's cross sectional nature. Nevertheless, further testing is warranted to replicate findings and determine whether reactivation of CMV infection exacerbates elevated mood states in BD-I.

Transcripts involved in calcium signaling and telencephalic neuronal fate are altered in induced pluripotent stem cells from bipolar disorder patients.

Bipolar disorder (BP) is a chronic psychiatric condition characterized by dynamic, pathological mood fluctuations from mania to depression. To date, a major challenge in studying human neuropsychiatric conditions such as BP has been limited access to viable central nervous system tissue to examine disease progression. Patient-derived induced pluripotent stem cells (iPSCs) now offer an opportunity to analyze the full compliment of neural tissues and the prospect of identifying novel disease mechanisms. We have examined changes in gene expression as iPSC derived from well-characterized patients differentiate into neurons; there was little difference in the transcriptome of iPSC, but BP neurons were significantly different than controls in their transcriptional profile. Expression of transcripts for membrane bound receptors and ion channels was significantly increased in BP-derived neurons compared with controls, and we found that lithium pretreatment of BP neurons significantly altered their calcium transient and wave amplitude. The expression of transcription factors involved in the specification of telencephalic neuronal identity was also altered. Control neurons expressed transcripts that confer dorsal telencephalic fate, whereas BP neurons expressed genes involved in the differentiation of ventral (medial ganglionic eminence) regions. Cells were responsive to dorsal/ventral patterning cues, as addition of the Hedgehog (ventral) pathway activator purmorphamine or a dorsalizing agent (lithium) stimulated expression of NKX2-1 (ventral identity) or EMX2 (dorsal) in both groups. Cell-based models should have a significant impact on our understanding of the genesis and therefore treatment of BP; the iPSC cell lines themselves provide an important resource for comparison with other neurodevelopmental disorders.

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.

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

Genome-wide linkage scan of 98 bipolar pedigrees and analysis of clinical covariates.

Despite compelling evidence that genetic factors contribute to bipolar disorder (BP), attempts to identify susceptibility genes have met with limited success. This may be due to the genetic heterogeneity of the disorder. We sought to identify susceptibility loci for BP in a genome-wide linkage scan with and without clinical covariates that might reflect the underlying heterogeneity of the disorder. We genotyped 428 subjects in 98 BP families at the Center for Inherited Disease Research with 402 microsatellite markers. We first carried out a non-parametric linkage analysis with MERLIN, and then reanalyzed the data with LODPAL to incorporate clinical covariates for age at onset (AAO), psychosis and comorbid anxiety. We sought to further examine the top findings in the covariate analysis in an independent sample of 64 previously collected BP families. In the non-parametric linkage analysis, three loci were nominally significant under a narrow diagnostic model and seven other loci were nominally significant under a broader model. The top findings were on chromosomes 2q24 and 3q28. The covariate analyses yielded additional evidence for linkage on 3q28 with AAO in the primary and independent samples. Although none of the linked loci were genome-wide significant, their congruence with prior results and, for the covariate analyses, their identification in two separate samples increases the likelihood that they are true positives and deserve further investigation. These findings further demonstrate the value of considering clinical features that may reflect the underlying heterogeneity of disease in order to facilitate gene mapping.

Linkage of bipolar affective disorder on chromosome 8q24: follow-up and parametric analysis.

Our group first reported a linkage finding for bipolar (BP) disorder on chromosome 8q24 in a study of 50 multiplex pedigrees, with an HLOD score reaching 2.39. Recently, Cichon et al reported an LOD score of 3.62 in the same region using two-point parametric analysis. Subsequently, we published the results of a genome scan for linkage to BP disorder using a sample extended to 65 pedigrees in which chromosome 8q24 provided the best finding, an NPL score of 3.13, approaching the accepted score for suggestive linkage. We have now fine mapped this region of chromosome 8 in our 65 pedigrees by the addition of 19 microsatellite markers reaching a marker density of 0.8 cM and an information content of 0.84. After the addition of the new data, the original NPL score slightly increased to 3.25. Two-point parametric analysis using the model employed by Cichon et al obtained an LOD score of 3.32 for marker D8S256 at theta=0.14 exceeding the proposed threshold for genomewide significance. After adjusting the parameters in accordance with the 'common disease-common variant' hypothesis, multipoint parametric analysis resulted in an HLOD of 2.49 (alpha=0.78) between D8S529 and D8S256, and defined a 1-LOD interval corresponding to a 2.3 Mb region. No allelic association with the disease was observed for our set of microsatellite markers. Biologically, plausible candidate genes in this region include thyroglobulin, KCNQ3 coding for a voltage-gated potassium channel and the gene for brain adenyl-cyclase (ADCY8).

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.

Novel CAG/CTG repeat expansion mutations do not contribute to the genetic risk for most cases of bipolar disorder or schizophrenia.

The possible presence of anticipation in bipolar affective disorder and schizophrenia has led to the hypothesis that repeat expansion mutations could contribute to the genetic etiology of these diseases. Using the repeat expansion detection (RED) assay, we have systematically examined genomic DNA from 100 unrelated probands with schizophrenia and 68 unrelated probands with bipolar affective disorder for the presence of CAG/CTG repeat expansions. Our results show that 28% of the probands with schizophrenia and 30% of probands with bipolar disorder have a CAG/CTG repeat in the expanded range, but that each expansion could be explained by one of three nonpathogenic repeat expansions known to exist in the general population. We conclude that novel CAG/CTG repeat expansions are not a common genetic risk factor for bipolar disorder or schizophrenia.

Trapping and sequence analysis of 1138 putative exons from human chromosome 18.

In a search for novel genes on chromosome 18 (HC18), on which several regions have been linked to bipolar disorder, we applied exon trapping to HC18-specific cosmids. Among the 1138 exons trapped, 1052 of them have been mapped to HC18, and the remaining 86 have not been localized. No exons were localized to genomic regions other than HC18. BLAST database search revealed that 190 exons were identical to 98 Unigenes on HC18; 98 identical to additional 82 clusters of ESTs not present in the HC18 Unigene set; 39 homologous to genes from human and other species (e<10(-3)); and the remaining 811 exons had no significant homology to transcripts in public databases. The mapped exons were compared to the 867 annotated genes on HC18 in the Celera databases; 216 exons were identical to 104 Celera 'genes' and the remaining 836 exons were not found in the Celera databases. On average, there were two exons for a matched transcript (known genes and ESTs). Therefore, the 850 novel exons may represent hundreds of novel genes on chromosome 18.

Genome-wide scan of bipolar disorder in 65 pedigrees: supportive evidence for linkage at 8q24, 18q22, 4q32, 2p12, and 13q12.

The purpose of this study was to assess 65 pedigrees ascertained through a Bipolar I (BPI) proband for evidence of linkage, using nonparametric methods in a genome-wide scan and for possible parent of origin effect using several analytical methods. We identified 15 loci with nominally significant evidence for increased allele sharing among affected relative pairs. Eight of these regions, at 8q24, 18q22, 4q32, 13q12, 4q35, 10q26, 2p12, and 12q24, directly overlap with previously reported evidence of linkage to bipolar disorder. Five regions at 20p13, 2p22, 14q23, 9p13, and 1q41 are within several Mb of previously reported regions. We report our findings in rank order and the top five markers had an NPL>2.5. The peak finding in these regions were D8S256 at 8q24, NPL 3.13; D18S878 at 18q22, NPL 2.90; D4S1629 at 4q32, NPL 2.80; D2S99 at 2p12, NPL 2.54; and D13S1493 at 13q12, NPL 2.53. No locus produced statistically significant evidence for linkage at the genome-wide level. The parent of origin effect was studied and consistent with our previous findings, evidence for a locus on 18q22 was predominantly from families wherein the father or paternal lineage was affected. There was evidence consistent with paternal imprinting at the loci on 13q12 and 1q41.

Family-based association study of 76 candidate genes in bipolar disorder: BDNF is a potential risk locus. Brain-derived neutrophic factor.

Identification of the genetic bases for bipolar disorder remains a challenge for the understanding of this disease. Association between 76 candidate genes and bipolar disorder was tested by genotyping 90 single-nucleotide polymorphisms (SNPs) in these genes in 136 parent-proband trios. In this preliminary analysis, SNPs in two genes, brain-derived neurotrophic factor (BDNF) and the alpha subunit of the voltage-dependent calcium channel were associated with bipolar disorder at the P<0.05 level. In view of the large number of hypotheses tested, the two nominally positive associations were then tested in independent populations of bipolar patients and only BDNF remains a potential risk gene. In the replication samples, excess transmission of the valine allele of amino acid 66 of BDNF was observed in the direction of the original result in an additional sample of 334 parent-proband trios (T/U=108/87, P=0.066). Resequencing of 29 kb surrounding the BDNF gene identified 44 additional SNPs. Genotyping eight common SNPs identified three additional markers transmitted to bipolar probands at the P < 0.05 level. Strong LD was observed across this region and all adjacent pairwise haplotypes showed excess transmission to the bipolar proband. Analysis of these haplotypes using TRANSMIT revealed a global P value of 0.03. A single haplotype was identified that is shared by both the original dataset and the replication sample that is uniquely marked by both the rare A allele of the original SNP and a novel allele 11.5 kb 3'. Therefore, this study of 76 candidate genes has identified BDNF as a potential risk allele that will require additional study to confirm.

Genetic analysis of the (CTG)n NOTCH4 polymorphism in 65 multiplex bipolar pedigrees.

A strong genetic association between the NOTCH4 locus on chromosome 6 and schizophrenia was recently reported. Based on the data suggesting overlapping susceptibility for schizophrenia and bipolar disorder, we genotyped the polymorphic (CTG)n encoding polyleucine repeat in exon 1 of NOTCH4 in 65 pedigrees ascertained for a genetic linkage study of bipolar disorder. In addition, we analyzed a subset of our pedigrees with psychotic features at this locus. We failed to find any association between the (CTG)n NOTCH4 polymorphism and either the bipolar or the psychotic bipolar phenotype in our 65 pedigrees.

Linkage of bipolar disorder to chromosome 18q and the validity of bipolar II disorder.

BACKGROUND: An analysis of the relationship between clinical features and allele sharing could clarify the issue of genetic linkage between bipolar affective disorder (BPAD) and chromosome 18q, contributing to the definition of genetically valid clinical subtypes. METHODS: Relatives ascertained through a proband who had bipolar I disorder (BPI) were interviewed by a psychiatrist, assigned an all-sources diagnosis, and genotyped with 32 markers on 18q21-23. Exploratory findings from the first 28 families (n = 247) were tested prospectively in an additional 30 families (n = 259), and the effect of confirmed findings on the linkage evidence was assessed. RESULTS: In exploratory analyses, paternal allele sharing on 18q21 was significantly (P =.03) associated with a diagnostic subtype, and was greatest in pairs where both siblings had bipolar II disorder (BPII). Prospective analysis confirmed the finding that BPII-BPII sibling pairs showed significantly (P =.016) greater paternal allele sharing. Paternal allele sharing across 18q21-23 was also significantly greater in families with at least one BPII-BPII sibling pair. In these families, multipoint affected sibling-pair linkage analysis produced a peak paternal lod score of 4.67 (1-lod confidence interval, 12 centimorgans [cM]) vs 1.53 (1-lod confidence interval, 44 cM) in all families. CONCLUSIONS: Affected sibling pairs with BPII discriminated between families who showed evidence of linkage to 18q, and families who did not. Families with a BPII sibling pair produced an increased lod score and improved linkage resolution. These findings, limited by the small number of BPII-BPII sibling pairs, strengthen the evidence of genetic linkage between BPAD and chromosome 18q, and provide preliminary support for BPII as a genetically valid subtype of BPAD.

The familial aggregation of psychotic symptoms in bipolar disorder pedigrees.

OBJECTIVE: Symptomatic overlap between affective disorders and schizophrenia has long been noted. More recently, family and linkage studies have provided some evidence for overlapping genetic susceptibility between bipolar disorder and schizophrenia. If shared genes are responsible for the psychotic manifestations of both disorders, these genes may result in clustering of psychotic symptoms in some bipolar disorder pedigrees. The authors tested this hypothesis in families ascertained for a genetic study of bipolar disorder. METHOD: Rates of psychotic symptoms-defined as hallucinations or delusions-during affective episodes were compared in families of 47 psychotic and 18 nonpsychotic probands with bipolar I disorder. The analysis included 202 first-degree relatives with major affective disorder. RESULTS: Significantly more families of psychotic probands than families of nonpsychotic probands (64% versus 28%) contained at least one relative who had affective disorder with psychotic symptoms. Significantly more affectively ill relatives of psychotic probands than of nonpsychotic probands (34% versus 11%) had psychotic symptoms. An analysis of clustering of psychotic subjects across all families revealed significant familial aggregation. Clustering of psychosis was also apparent when only bipolar I disorder was considered the affected phenotype. CONCLUSIONS: Psychotic bipolar disorder may delineate a subtype of value for genetic and biological investigations. Families with this subtype should be used to search for linkage in chromosomal regions 10p12-13, 13q32, 18p11.2, and 22q11-13, where susceptibility genes common to bipolar disorder and schizophrenia may reside. Putative schizophrenia-associated biological markers, such as abnormal evoked response, oculomotor, and neuroimaging measures, could similarly be explored in such families.