Functional Genomics of Bipolar Disorder

National Institute Of Mental Health

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Abstract

NCT00001174 Despite strong evidence of heritability and discovery of genetic markers for major mental illness, little is known about how gene expression in the brain differs across psychiatric diagnoses, or how inherited genetic risk factors shape these differences. We have studied expression of genes and gene transcripts in postmortem subgenual anterior cingulate cortex (sgACC), a key component of limbic circuits linked to mental illness. Deep sequencing was carried out in RNA obtained postmortem from 200 donors diagnosed with bipolar disorder, schizophrenia, major depression, or no psychiatric disorder. Case-control comparisons detected modest expression differences that were similar across disorders, although transcript-level differences were more pronounced. The 250 rare transcripts that were differentially expressed were enriched for genes involved in synapse formation, cell junctions, and heterotrimeric G-protein complexes. Relative abundances of alternatively spliced transcripts were associated with common genetic variants that accounted for disproportionate fractions of diagnosis-specific heritability. Inherited genetic risk factors shape the brain transcriptome and contribute to diagnostic differences between broad classes of mental illness. We seek to model the impact of disease-related genes in cells derived from induced pluripotent stem cell (iPSC) lines. This project aims to explore ways in which we can use iPSC technology to study the biological impact of genes and genetic mutations that we identify in our other ongoing studies. Working with the National Heart, Lung and Blood Institute (NHLBI) stem cell core we have so far successfully reprogrammed fibroblasts into iPSCs from 89 study participants. We are developing a large iPSC-based resource and associated work-flows that constitute a living catalog of psychiatric risk alleles. iPSC-derived cells are studied with high-resolution microscopic imaging, electrophysiology, and gene expression methods. These data could reveal differences between control and patient-derived cells and the impact of known and novel therapeutic agents. In collaboration with scientists at the New York Stem Cell Foundation Research Institute, we are also exploring ways to measure the functional impact of genetic mutations at the cellular level and use genome editing tools to establish a causal role for specific genetic mutations. A previous proof-of-concept study demonstrated the value of iPSC-based assays for translating common, low-risk alleles identified by GWAS into novel genetic, neurobiological, and pharmacological insights. Currently we are working on methods to map regulatory chromatin contacts in neural progenitor cells. This will enable the identification of developmental stage and treatment specific influences on chromatin structure and gene expression. Our ongoing copy number variant (CNV) studies examine known pathogenic CNVs in iPSC-derived neural cells and post-mortem brain. Over the past year, we carried out extensive morphological and transcriptomic characterization of neural cells carrying reciprocal CNVs (duplication or deletion) on chromosome 16p11.2. These CNVS have previously been associated with BD and other psychiatric disorders. Transcriptomic analyses indicated that most genes in the CNV region show expression changes in neurons that were consistent with copy number: increased in duplication carriers and decreased in deletion carriers. Many other genes were also dysregulated in carriers. Genes with concordant expression changes in carriers were enriched for several pathways, including neuronal growth and proliferation, synapse development, and cell migration. Consistent with these transcriptomic results, in vitro comparisons between carriers and matched non-carriers revealed major differences in cellular differentiation and growth and reduced synaptic structures. While non-carrier NPCs easily differentiated into mature astrocytes using standard protocols, NPCs carrying the 16p11.2 duplication did not. Astrocytes play a critical role in the development and maintenance of healthy neurons and synapses. Exogenous astrocytes from mouse rescued most of the neurodevelopmental and synaptic deficits in 16p11.2 duplication carriers. These results demonstrate that CNVs on 16p11.2 confer convergent effects on gene expression, leading to neurodevelopmental deficits that may play a causal role in psychiatric disorders. In the past year, we have also screened a variety of established and novel psychotropic medications to identify drugs that may rescue or ameliorate the impact of CNVs in neurons. Multigenic disorders such as BD pose special challenges for experimental studies, since a single causative mutation is usually not identifiable. Thus we are also studying rare, single-gene disorders whose symptoms overlap with those seen in common mental illnesses. Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by behavioral abnormalities and disruptions in circadian rhythm. Cells from people living with SMS obtained in collaboration with Ann Smith (NHGRI) have been reprogrammed into iPSCs and differentiated into neurons and other brain cells. We have used these cells to explore gene-expression, molecular, and morphological traits associated with SMS mutations in cultured brain cells. The results suggest that SMS mutations cause increases in proliferation and neurite outgrowth especially among excitatory neurons, consistent with proposed excitation/inhibition imbalance models of pathogenesis. Findings from this study may have relevance to other neuropsychiatric disorders such as depression, autism, and BD. A new set of studies started over this year aims to leverage novel, single-cell RNA sequencing methods to characterize gene expression changes in neurons at cellular resolution. These methods also allow us to detect changes in the proportions of neuronal cell types that may contribute to psychiatric illness. Recent postmortem transcriptomic studies of human brain have shown hundreds of genes differentially expressed in donors with psychiatric illnesses. However, it is unclear which of these gene expression changes are the result of exposure to psychotropic medications rather than the illness itself. We have now explored this question using antipsychotic drug exposure in schizophrenia (SCZ) as a proof of concept. We compared differential gene expression in the prefrontal cortex from donors with SCZ who tested positive or negative for antipsychotics at the time of death. APD exposure was associated with numerous changes in the brain transcriptome, especially among donors exposed to atypical APDs. Brain transcriptome data from macaques chronically treated with APDs showed that APDs affect the expression of many functionally relevant genes, some of which show expression changes in the same directions as those observed in SCZ. In contrast, major cell type shifts inferred in SCZ were primarily unaffected by APD use. These results suggest that APD use confounds gene expression changes in postmortem brain tissue. Disentangling these effects will help identify causal genes and improve our neurobiological understanding of psychiatric disorders. In the coming year, we will continue studies in neural cells and post-mortem brain tissue derived from people with psychiatric disorders, carriers of high-risk CNVs, and rare damaging mutations within GWAS or CNV loci that run together with BD and related conditions in families. If successful, these projects will help unpack the biology behind genetic risk for mental illness, identify high-risk alleles, and shed new light on how risk alleles exert biological changes in the brain. The findings may ultimately identify new targets that lead to better methods of diagnosis and treatment for neuropsychiatric disorders.