Joachim Hallmayer - US grants

DESCRIPTION (provided by applicant): Of all multifactorial child psychiatric disorders, autism is the most strongly genetically influenced. The inherited liability is not restricted to the full clinical syndrome of autism but encompasses a range of behavioral and cognitive characteristics. Family studies have failed to distinguish whether the genetic liability to autism falls on a continuous spectrum of severity or whether it may be better subdivided into distinct categorical subtypes. From a genetic standpoint this raises the question, whether different aspects of the autism phenotype are influenced by different loci. The proposed study will be the largest, population-based study of twins with autism. With this unique sample we will be able to unravel the relationship between genes and environment as it pertains to the cognitive impairments and the clinical symptoms seen in children with autism spectrum disorders. We will assess twin pairs using (a) clinical measures of autistic-like behaviors (b) more specific measures for repetitive and stereotyped behaviors, (c) measures of general cognitive ability d) measures of more specific neurocognitive abilities. This will allow us to address several fundamental questions: (1) what is the heritability of autism (2) what is the contribution of genetic factors to variation in symptom dimensions? (3) is there a continuum between the quantitative neurocognitive traits and clinical disorder? (4) what proportion of the variance in the neurocognitive traits is accounted for by genetic and non-genetic factors?

DESCRIPTION (provided by applicant): Autism is most likely inherited by an oligogenic mechanism with epistasis. Thus, large samples will be required to locate the contributing genes, and a number of complementary methods will be needed. Genome screens of sib pair families have identified a few consistent chromosomal regions of interest, but there are numerous other suggestive signals that need to be verified in much larger samples. The purpose of this application is to facilitate the discovery of the genes that contribute to autism, by developing and maintaining an infrastructure by which research groups studying the genetics of autism can work collaboratively. This will be accomplished through workshops, a Virtual Private Network (VPN), and access to a database that includes phenotype and genotype data from all participating groups. Other goals are to develop and continually update information on autism genetics for families and practitioners. There will be substantial quality control to assure the accuracy of the data. The six groups participating in this application have been working together for 18 months toward the goal of establishing an effective means to share data. Any new group may join at any time, as long as their data meet the volume and quality standards described in the application. The current groups are the Collaborative Linkage Study of Autism (Tufts/New England Medical Center, University of North Carolina at Chapel Hill, Vanderbilt University and the University of Iowa); Duke University and the University of South Carolina; Mt. Sinai Medical Center in New York and Trinity University in Dublin, Canadian Autism Genetics (McMaster University, and the University of Toronto/Hospital for Sick Children); Stanford; the Paris Autism Research International Sib-pair Study (INSERM in Paris and Creteil. Goteborg University in Sweden, Ben-Gurion University in Israel and Hebrew University of Jerusalem in Israel). Currently, our collective sample numbers nearly 800 sib pair families and also includes cousin pairs and connecting relatives, uncle/nephew and father/offspring pairs, trios (one proband and both parents). We expect that having a forum (both virtual - the VPN and database, and actual - the workshops) where information can be shared openly and easily will facilitate the discovery of genes contributing to the cause of autism. The database will contain large numbers of families and a wealth of phenotype data which will allow the drawing of sub-samples based on a common phenotype, such as macrocephaly, absence of functional language, absence of any structural language abnormality, or prominence of resistance to change.

DESCRIPTION (provided by applicant): During the last 18 years of funding of this program project, the only NIH supported program project focused on narcolepsy, we have made significant strides in understanding the pathophysiology of narcolepsy, as well as the physiology of normal sleep. Our discovery during the last funding period that most cases of human narcolepsy cataplexy are caused by hypocretin (also called orexin) deficiency significantly changed the sleep field. In this revised competitive renewal proposal, based on our recent discoveries, we have refocused our research efforts and recruited new members to expand the expertise of the Center. In this revised proposal, we have brought together a unique group of independent investigators working across disciplines toward a common goal. Based on the reviewers' critiques, we have removed two projects from our original proposal, leaving four projects and a core (Project A). The core (Project A) provides the necessary core resources to support research projects at the Stanford Center for Narcolepsy, most notably biological samples. The goal of Project B, directed by Dr. Terry Young at the University of Wisconsin, Madison is to determine the prevalence of narcolepsy without cataplexy using an epidemiological approach and to study its association with HLA and lypocretin deficiency. Project D, directed by Dr. Juliette Faraco, will use a zebrafish model to isolate novel genes regulating hypocretin and histamine neurotransmission. In Project F, directed by Dr. Luis de Lecea, the discoverer of hypocretins, is seeking to identify novel genes with preferential expression in hypocretin-containing cells; an accessory goal of this project will be to study the neuropathology of narcolepsy without cataplexy. Project E, directed by Dr. Joachim Hallmayer, will use a human genetic approach to identify novel narcolepsy susceptibility genes. Narcolepsy is a frequent and disabling neurological disorder affecting more than 1 in 2,000 Americans. Our recent findings have led to new diagnostic procedures but have not yet changed therapeutic options. Our aims are improved diagnosis, a better understanding of the narcolepsy pathophysiology and the discovery of new treatments, if not a cure for narcoleptic patients. PROJECT A PI: Emmanuel Mignot Title: Research Administration Core, Human DNA Samples Bank, Animal Models, & Databases Description (provided by applicant): A seasoned and well-trained research administration staff is essential for the effective management of a complex multi-disciplinary program project. During the last consecutive 18 years of funding of this program project the Principal Investigator and the Assistant Director of the Sleep Research Center have worked together and built a team of research administrators and research support staff that are responsible for the day-to-day activities of managing the Center for Narcolepsy. This includes budgetary and fiscal reporting planning, oversight of policy and guidelines, animal and human database management, subject recruitment and the integration of this project into the Sleep Research Center and Sleep Clinic, and in general ensuring that the program runs smoothly. To support this revised research proposal, five main core activities are recognized: 1) A research administrative and coordinating function to facilitate the research & interactions among investigators and projects 2) Management of the Stanford University Center for Narcolepsy animal colonies used in this proposal; this includes zebrafish, mice, and rats. 3) Recruitment of human narcolepsy patients for research projects: this includes the banking of DNA, serum, cerebrospinal fluid and brain specimens from narcolepsy and control subjects. 4) Maintenance of computerized databases containing human, mouse/rat, canine narcolepsy data and tissue sample data; maintenance of the project's website and data-sharing on-line. 5) Maintenance of all documentation supporting adherence to all federal guidelines and policies for conducting research. It is the responsibility of the Principal Investigator to assure that the Center for Narcolepsy scientists work as an integrated team. His direction and scientific leadership, is essential to the pursuit of the overall goals of the program. A weekly research meeting is held to facilitate communication among the investigators and between the research staff and research administration staff.

DESCRIPTION (provided by applicant): Autism spectrum disorders (ASD) are highly heritable complex neurodevelopmental disorders of the brain, which cannot be explained by mutation or mutations in any single gene. In the last couple of years linkage and association studies have led to the identification of several mutations that confer susceptibility to ASDs. Studying the functional effects of these mutations offers a unique window to a better understanding of the underlying neurobiology. One of the major obstacles is the difficulty in obtaining neurons and glial cells from patients with an ASD. The goal of this project is to develop the methods to convert skin cells from patients with ASDs into neurons and to characterize these neurons using high content screens. To achieve this goal we will convert fibroblasts into pluripotent progenitor (iPS) cells. In the next step we will differentiate these iPS cells into neurons in vitro. Finally we will study the specific cell- intrinsic aspects of neuronal function that are likely to be disrupted in ASDs including synapse formation, axonal and dendritic morphology and calcium signaling. We have already established all of these techniques in our laboratory. Before we can apply these techniques on a larger scale we need to first address some of their limitations. The focus of R21 phase of the proposal is to improve and standardize the methodology. We will first generate and characterize iPS cells from human fibroblasts harvested from healthy controls and ASD patients with mutations in the CACNA1C and SHANK3 gene, mutations known to affect neuronal development, and optimize and characterize the differentiation of iPS cells (Specific Aim 1). We will develop standardized protocols for differentiating iPS cells into mixed populations of cortical, dopaminergic, and inhibitory neurons (Specific Aim 2). We will then characterize the cellular phenotypes of neurons from ASD and from controls, focusing on calcium signaling, dendritic arborization, and cell survival (Specific Aim 3). In the R33 phase of the project we will target a larger number of individuals with ASD a known to have a mutation in others gene/s affecting neuronal development (Specific Aim 4 and 5). PUBLIC HEALTH RELEVANCE: Autism is considered to be among the most common of the serious developmental disabilities, second only to mental retardation. The lifetime per capita incremental societal cost is estimated at $3.2 million. Because the deficits in autism affect human- specific social behaviors, the mechanisms underlying autism will need to be studied in human patients and in cells. The goal of this project is to develop the methods to convert skin cells from patients with autism into neurons and to characterize these neurons using high content screens. These experiments will allow researchers to study the neurons of individuals diagnosed with autism and will lead to a better understanding of the development and differentiation of neurons.

3' Untranslated Regions; Adult; Affect; Aging; Alleles; Autoimmune Diseases; Autoimmune Process; base; burden of illness; case control; Cataplexy; Caucasians; cell type; Chronic; cost; Custom; Cytotoxic T-Lymphocytes; design; Development; Disease; Early Diagnosis; Early treatment; Etiology; Excessive Daytime Sleepiness; Functional disorder; Gene Frequency; Genes; genetic epidemiology; Genetic Polymorphism; Genome; genome wide association study; genome-wide; Genotype; Health; Healthcare; Hereditary Disease; Human; Human Genetics; Human Genome; hypocretin; Individual; infancy; Knowledge; Large-Scale Sequencing; Minor; Mutation; Narcolepsy; nervous system disorder; North America; novel; Odds Ratio; Pathogenesis; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Predisposition; Productivity; Proteins; Psyche structure; Purinoceptor; receptor; Recruitment Activity; Reporting; Research; Research Design; Resources; Sampling; Serum; Single Nucleotide Polymorphism; Sleep Disorders; social; Societies; socioeconomics; Streptococcal Infections; streptolysin O; T-Cell Receptor; T-Cell Receptors alpha-Chain; T-Lymphocyte and Natural Killer Cell; TNFRSF5 gene; TRA@ gene cluster; Variant; Variation (Genetics);

DESCRIPTION (provided by applicant): 22q11.2 microdeletion syndrome (Velocardiofacial Syndrome; 22q11DS) occurs in about 1/3000 live births, and is the most frequent known recurrent genetic cause of schizophrenia, accounting for 1-2 % of schizophrenia cases in the general population The overall goal of this project is to study neural progrenitors and neurons derived from human induced pluripotent stem cells (iPSCs), in order to identify the cellular and molecular mechanisms underlying the neuropsychiatric phenotype in patients with 22q11DS. In the last five years we have developed highly reproducible methods for studying the differentiation of iPSCs into neurons, and for characterizing these cells using well-validated genetic and cell biological assays. Using this approach, we found evidence of reproducible changes in gene expression that implicate calcium (CA) signaling and developmental dysregulation in 22q11DS neurons. We experimentally validated the presence of aberrant CA signaling and dopaminergic D2 receptor dysfunction, as well as defects in dendritic branching. We now have the technology in place to significantly increase the throughput, and in this study we will expand our investigations to a much larger sample of patients with 22q11DS, in order to connect the cellular defects with patient characteristics. Specifically, we will: 1) generate an iPSC patient resource by obtaining skin fibroblasts from 40 well-characterized patients with 22q11DS - 20 with a diagnosis of psychotic disorder and 20 without - and 20 demographically comparable controls. 2) Using these resources, we will first validate our preliminary findings of defects in dopaminergic signaling, and then determine which aspects of calcium signaling are impacted by the deletion. By rescuing the phenotype through selectively expressing each of the genes deleted within the 22q11.2 region we will determine which gene(s) are causally implicated in the specific defects. 3) In parallel, we will comprehensively analyze the transcriptome in order to identify key hubs and pathways that are dysregulated in neurons from 22q11DS patients, and compare the co-expression modules in 22q11DS patients with and without psychosis, to explore potential pathways that may be specifically relevant to the development of schizophrenia in 22q11DS. Finally we will: 4) connect cellular, gene expression and behavioral phenotypes, by comparing cellular phenotypes derived from 22q11DS patients with and without psychosis, which will be integrated with gene expression data, in order to connect molecular pathways to morphological or physiological phenotypes, and actual clinical presentations in 22q11DS patients.

PROJECT SUMMARY ? PROJECT 3 Autism spectrum disorder (ASD) is a complex condition characterized by important changes to the brain and behavior. 15% of boys with ASD have disproportionate megalencephaly (ASD-DM), or enlarged brain relative to body size. An increase in brain size often precedes the first clinical signs of the disorder, suggesting that understanding the mechanisms leading to brain overgrowth could provide a window of opportunity to intervene and possibly prevent disease onset. Here, the research team will use human induced pluripotent stem cell (hiPSC) technology to model ASD-DM and investigate the underlying cellular and molecular mechanisms involved. They will obtain skin fibroblasts from 40 individuals in Project 2 and derive human iPSCs from: A) 10 ASD subjects with megalencephaly, ASD-DM; B) 10 ASD subjects with normal sized brains, ASD-N C) 10 Typically developing (TD) subjects with megalencephaly, TD-DM, and D) 10 TD subjects with normal sized brains, TD-N. Following iPSC generation, they will differentiate each of the iPSC lines into neural progenitor cells (NPCs), oligodendrocyte progenitor cells (OPCs), and microglia (the primary immune cells in the brain that maintain homeostasis). The overarching goals of their project are two-fold: 1) to investigate whether ASD-DM is due to an increase in cell proliferation, increase in cell survival, improper elimination of damaged cells, and/or a combination of all; and 2) to identify therapeutic targets by understanding the underlying cellular and signaling mechanisms involved. In Specific Aim 1, they will identify the cellular mechanisms underlying ASD-DM by investigating changes in the cell cycle, cell proliferation, and apoptosis of iPSC-derived NPCs, OPCs, and microglial cells. In Specific Aim 2, they will investigate the functional activity of microglia in ASD-DM by directly differentiating each of the iPSC lines into microglia and assessing their phagocytic capacity by co-culturing them with mixed neuroglial cultures derived from the same lines. This will test their hypothesis that microglia are compromised in ASD-DM, failing to eliminate damaged cells and synapses and contributing to brain overgrowth. In Specific Aim 3, they will identify the underlying regulatory signaling mechanisms that lead to the changes at the cellular level. They will differentiate the iPSCs into NPCs, OPCs, and microglia, sort them by flow cytometry using antibodies specific for each cell type, and perform RNA-sequencing to identify gene networks and signaling mechanisms that are significantly regulated in each condition. Using these mechanistic insights, they will identify therapeutic targets to directly test in the in vitro models. Their overall goal across the projects is to collect imaging, behavioral, and mechanistic data on the same cohort of subjects. In Specific Aim 4, they will correlate the cellular and mechanistic data obtained in Project 3 with the imaging and behavioral data from Project 2 to identify broader trends and characteristics specific to ASD-DM. This comprehensive body of data will be a valuable resource for the broader research and medical communities in identifying predictive biomarkers of ASD and/or ASD-DM and potentially more tailored therapies.

Autism Spectrum Disorder (ASD) is a genetically and phenotypically heterogeneous neurodevelopmental disorder. Hundreds of genes contribute to the risk to develop ASD with no individual genetic locus accounting for more than 1% of cases. This raises the issue of whether, and how such diverse mechanisms converge on a smaller number of biological pathways that ultimately result in one phenotype, namely ASD. The inability to study neurons and brains from living subjects has blocked progress toward understanding the cellular and molecular mechanisms underlying ASD and other neurodevelopmental disorders. Neurons derived from human induced pluripotent stem cell (hiPSC) recapitulate multiple stages of in vivo neural development in vitro. Because they retain the genetic makeup of the patient they enable in vitro studies of neurons that harbor the complex genetic background associated with ASD. Deriving neurons from hiPSCs is labor intensive and costly, and to date studies have examined very small numbers of patients. hiPSC studies of the scope required to capture idiopathic ASD have not been possible, entirely limiting our ability to determine the mechanistic underpinnings of this form of the disorder. We propose to conduct an investigation of hiPSC derived neurons on an unprecedented scale by leveraging our California Institute for Regenerative Medicine (CIRM) funded existing collection of over 300 hiPSCs, to examine 100 individuals with idiopathic ASD and 100 age- and sex-matched controls. We will use commercially derived neurons (iCell Neurons) which are a >95% pure population of glutamatergic (excitatory) and GABAergic (inhibitory) neurons. To identify dysregulated molecular pathways in these neurons we will sequence the human transcriptome at three time points during maturation of the neurons. We will also conduct assays of cell viability, morphology, neurite outgrowth using live cell imaging, and function through calcium imaging. We aim to identify dysregulated molecular pathways in these hiPSC-derived neurons from individuals with ASD, by identifying genes that are differentially expressed from controls at each time point and perform a factorial analysis to study interactive effects between time point and disease variable. This will identify genes showing differentiation- induced expression changes across neuronal differentiation in individuals with ASD. We will identify key drivers of biological pathway changes using Weighted Gene Co-expression Network Analysis (WGCNA). Finally, we will relate gene expression profiles to cellular and behavioral phenotypes.

Project Summary/Abstract After a century of debate about the fundamental nature of neuropsychiatric disorders, we know that genetics lie at their core, yet do not fully understand the critical underlying mechanisms of their disease-causing pathology. The overall goal of our proposal is the creation of comprehensive and integrated maps of chromatin accessibility, chromosome folding and transcriptional patterns, delineating regulatory regions in the genomes of key disease relevant anatomical regions of adult and fetal brains, in brains from patients with Schizophrenia, Autism Spectrum Disorder, Bipolar Affective Disorder and matched controls, and those with known CNVs (Copy-Number Variants) that may unmask regional or long-range targets of epigenomic regulatory interactions that may also be of great relevance in patients with the same clinical phenotype. We will use comprehensive and highly-resolving epigenomics assays, that were recently developed by us, and novel ways to integrate the data for the first time in neuropsychiatrically relevant brain tissues. We will generate comprehensive maps of the spectrum of organization and function of regulatory regions by integrating complementary techniques: single-cell ATAC-seq (scATAC-seq) to characterize chromatin openness and HiChIP to characterize long- range folding interactions of sorted neuronal and non-neuronal cells, both of which are coupled to single-cell RNA-seq and long-range RNA-seq for expression information, further complimented by information about transcription factors through proteomic analysis of nuclear fractions. These maps will then be combined with coding or non-coding/regulatory variants in the genomic sequence in the candidate regions and integrated into the overall PsychENCODE database, which will allow us to create and validate reference maps for epigenomic marks and interactions, determine aberrations to the reference state in patient tissue, and connect such aberrations to genetic disease loci as well as assemble such loci into disease pathways. This project will not only greatly expand our understanding of regulatory information encoded in the human genome and its impact on human brain development and neuropsychiatric disorders, but also produce the bioinformatics tools necessary to analyze the complex data being generated in PsychENCODE.