NCT00001174 In order to better interpret the impact of genetic variation on the brain biology of bipolar disorder, we are pursuing a variety of functional genomics studies, including brain imaging, microarray gene expression and RNA-seq in post-mortem brain tissue, and cellular phenotyping of neurons derived from induced pluripotent stem cells. Current neuroimaging genetics work is focused on structural MRI. We contribute data to the Enhancing NeuroImaging Genetics through Meta-Analysis (ENIGMA) brain imaging consortium, which is using genome-wide association methods in large samples to detect genetic markers associated with the volumes of various cortical and subcortical brain regions. These important endophenotypes may shed light on the mechanisms whereby common genetic variants influence risk for a variety of psychiatric disorders. Following upon published pilot studies, this year we are doing RNA-sequencing in an additional 200 postmortem brain samples obtained from people with and without major psychiatric disorders. This study focusses on the subgenual anterior cingulate cortex because neuroimaging and brain stimulation studies have implicated this region in people with mood disorders. We also seek to model the functional genomics of disease-related genes in cells derived from induced pluripotent stem cell (iPSC) lines. This project aims to explore the 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 NIH Center for Regenerative Medicine, the National Institute of Neurological Disorders and Stroke (NINDS) and the National Heart, Lung and Blood Institute (NHLBI) stem cell cores we have so far successfully reprogrammed fibroblasts into iPSCs from 15 individuals ascertained in our ongoing family studies of bipolar disorder. We are differentiating the cells into neurons and glia, and characterizing their morphology, neuronal action potentials, gene expression profiles, and response to medications and toxins. Careful analysis of these phenotypes could reveal differences between control and patient-derived cells. We are also studying multiple functions of those lines in collaborating labs (JHU). Our preliminary data suggest that chronic valproic acid treatment induces growth inhibition and promotes neuronal differentiation of neural progenitor cells, and chronic lithium treatment reduces calcium response to glutamate stimulation on iPSC derived neurons. In the coming year, we plan to expand our iPSC lines from bipolar families. We are also exploring ways to measure the functional impact of genetic mutations at the cellular level and to use genome editing tools such as CRISPR-Cas9 to rescue cellular phenotypes and establish a causal role for specific genetic mutations. Our current collection of fibroblast lines were generated from >40 skin biopsies from family members in our bipolar disorder study. Each line has >20 vials stored frozen. Of these, seven were reprogrammed into iPSCs this year and for each fibroblast line. At least 2 iPSC clones, previously tested for pluripotency, were grown, passaged, karyotyped and aliquots at various passage numbers are stored frozen. Clones that carry a normal karyotype are used in succeeding work on neuronal differentiation, phenotype characterization, toxicological and drug discovery research. iPSC-derived neural progenitor cells (NPCs) provide a renewable cellular template to conduct various investigations into disease-associated features and mechanisms at the early stage of neuronal development. In collaboration with Joseph Steiner (NINDS), we are developing a morphologic assay to measure the effect of stressors on dendritic number and length following initiation of differentiation NPCs into neurons with or without pretreatment with mood stabilizers. The above approaches may illuminate important features that contribute to disruption of biological mechanisms during the disease process and provide reproducible assays for high throughput screening for novel therapeutics. We are also studying Smith-Magenis syndrome (SMS), a neurodevelopmental disorder characterized by various presentations including behavioral abnormalities and a profound disruption of the circadian rhythm. The majority of SMS cases are caused by a deletion on 17p11.2. We are focusing our studies on non-deletion patients that carry a mutation in RAI1, a gene located in this deleted region. Fibroblast cells from cases obtained through a collaboration with Ann Smith (NHGRI), the co-discoverer of SMS, were reprogrammed into iPSCs. Study samples that carry an established single gene defect permits gene editing for comparative studies of phenotypes generated by 2 different cells that carry the identical genetic background. Findings from this study may have relevance to other neuropsychiatric disorders with circadian rhythm disturbances, such as depression and bipolar disorder.
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