Julie Korenberg, MD PhD - US grants

The long-range goals are to elucidate the molecular basis for the meiotic abnormalities that result in trisomy 21, and for the clinical phenotype that results from trisomy 21. The specific aims are to: 1) Examine features of the organization of DNA sequences about the centromere of chromosome 21, and their relation to meiotic abnormalities involving chromosome 21. 2) Isolate genes on chromosome 21 that encode surface proteins responsible for the increased adhesiveness of trisomy 21 fetal fibroblasts; these genes are potentially responsible for the congenital malformations associated with Down syndrome. The DNA studies will begin with a family of DNA sequences, the "724" family, that we localized to the pericentromeric region of all the human acrocentric chromosomes, including chromosome 21. An efficient recombination-based assay, which we continue to refine, will be used to "walk" to adjacent DNA sequences in chromosome 21-specific recombinant libraries. These studies will be conducted analogously to previous studies in which 724 and ribosomal DNA families were shown to be apposed within several thousand base pairs of each other in the human genome. Single copy DNA probes for the centromere of chromosome 21 will be isolated to study the parental origin of nondisjunction events, and to study the assortment of individual acrocentric centromeres in interphase and meiosis. The evolutionary basis for chromosome rearrangements involving the acrocentric chromosomes will be studied by localizing the 724 family in higher primates, as the apposition of 724 and ribosomal DNA sequences postdates the divergence of man from the New world owl monkey 35 million years ago. Our finding that trisomy-21 fetal lung and endocardial-cushion fibroblasts aggregate more rapidly in vitro than do normal control cells suggests that cell surface markers encoded by chromosome 21 may mediate this effect and thereby interfere with normal pulmonary and cardiac development in Down syndrome. Monoclonal antibodies that react with chromosome 21-encoded cell surface markers will be used to isolate genes on chromosome 21 that encode such markers. Particular attention will be focused on one such antibody that appears to inhibit the rapid aggregation of trisomy 21 fetal fibroblasts in vitro. Isolation of a gene that is responsible for the increased aggregation of trisomy 21 fetal fibroblasts could ultimately be used to study the genesis of congenital heart defects in trisomy 16 in mouse, and in trisomy 21 in man.

We will utilize a novel recombination-based assay (RBA) to select genes and thereby perform a time and tissue analysis of transcription for expressed sequences on chromosome 19. Reversal of this selection, counterselection, will be performed to yield the gene of interest and allow rapid DNA sequencing of the gene. Rapid performance of these steps will permit us to append a "genic" initiative onto the "genomic" initiative. We have elaborated novel bacterial strains, plasmids and schemes for the RBA. The bacterial strains include DM21,, a dnaB amber line that enables us to select for phages that share homology with a defined sequence; pMAD3, a supF plasmid without homology to ColE1 that allows us to select without background for phages - that share homology to a sequence cloned in pMAD3; PCR counterselection, that permits us to select efficiently against supF, thereby allowing us to isolate rapidly a desired sequence selected by recombination. This methodology will be applied in tandem with efforts at Lawrence Livermore to isolate and catalogue expressed sequences on chromosome 19. Regardless of whether this or other technology is utilized to identify transcribed sequences, the RBA will be employed to identify the time and tissue of transcription in an efficient manner.

DESCRIPTION: (Applicant's Description) Using a sensitive fluorescent quantitative-PCR (QPCR) energy-transfer assay, we can determine sequence copy number directly and efficiently. Application of this technology to cancer documents a surprisingly frequent assortment of copy number changes that are not seen in the stable normal genome. In preliminary studies, this technique was compared with standard loss of heterozygosity (LOH) techniques and found to be as accurate and more efficient in determining anomalies of bladder tumors. More recently, we have shown that this technology can be used to diagnose anomalies associated with bladder cancer in the abnormal genomes of tumor cells found in urine sediment and serum. Further, we have found that the absolute amount of DNA found assayed by QPCR in urine sediment is a marker for bladder abnormality. Putting all these techniques together, we propose an effort focused on bladder tumors to determine how QPCR can be used to diagnose and assist the therapy of bladder tumors. Since we have seen these genomic changes in a wide variety of tumors, the technology can ultimately be applied to all cancers. Questions to be addressed are: 1. Can a simplified variant of QPCR be used to screen efficiently for bladder tumors in urine sediment of populations at increased risk? 2. Can QPCR be used to diagnose and stage bladder tumors? 3. Can QPCR be used to monitor the therapy of bladder tumors when the bladder is left by monitoring urine sediment? 4. When the bladder is removed because of extensive disease, can QPCR be used to monitor the therapy of bladder tumors by monitoring serum? 5. Can QPCR be used to monitor the occurrence of metastatic disease by monitoring serum? Answering these questions will determine the roles that are appropriate for QPCR in the diagnosis and treatment of bladder tumors.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area: (06) Enabling Technologies and specific Challenge Topic: 06-MH-103 New technologies for neuroscience research. Develop technologies for neuroscience research that are software-based, (e.g., informatics tools, implementation of data analytic algorithms), hardware-based (e.g., instrumentation or devices), or biology-based (e.g., driven by conditional gene expression or bioactive agents). Contact: Michael F. Huerta, Ph.D., 301-443-1815, mhuert1@mail.nih.gov Establishing a network diagram of the brain is one of the major challenges of modern neurobiology and medicine, particularly a diagram of genetic connectivity. Nowhere is this need more clear than for the social-emotional system, where dysregulation of circuitry has been implicated in the most devastating mental illnesses, depression, schizophrenia and autism. This proposal seeks to deploy the next generation neurobiologic technologies and novel software tools needed to generate gene-specific 3-dimensional reconstructions from serial sections of long range axon projections or wiring diagrams of the limbic system, at the axon level and on the size scale of primate brain. This software will fill two striking gaps that have seriously hindered the generation of maps of primate brain circuitry. Blocked by lack of existing software for both aligning sequential sections and for handling large datasets, current circuit reconstruction methods cannot handle axon projections that travel through more than a single, typical ~30[unreadable]m section. Therefore, although there are elegant emerging approaches to tract tracing, current technology permits only visualization of cells in a single section of a mouse hippocampus but prevents providing answers to the critical questions at the systems level. The long range links to other cortical or subcortical structures involved in regulation of emotion or cognition, particularly at the scale needed for primate systems cannot yet be visualized. Moreover, there are no programs for making the leap between axon projections and larger tracts defined by DTI or MRI. The current proposal takes advantage of a unique multidimensional collaboration among the Scientific Computing and Imaging Institute (SCI) experts in image reconstruction and three-dimensional visualization (Tasdizen, Jones) who have recently (Anderson, 2009) developed a computational framework for 3-dimensional neurocircuitry of the retina and in fluorescence image reconstruction (Roysam), experts in non-human primate brain circuitry and neuroanatomy (Angelucci, Hof), experts in animal and human DTI/ MRI (Hsu), and experts in multicolor fluorescence imaging of axon projections (Korenberg, Angelucci). This unique collaborative project will help to maintain two junior investigators and three early career investigators, and will create seven new research jobs in the fields of neurobiology and computation. The four aims will: 1) Generate high signal- to-noise multicolor fluorescence images of neuropeptide projections in the macaque limbic system (hypothalamus- a subset of limbic targets) at axon resolution, 2) Acquire and store images of more than 400 serial sections in mosaic 2 [unreadable]m stacks equal to158 terabytes, 3) Reconstruct the hypothalamic- projections found with arginine-vasopressin and a Williams syndrome gene product to a subset of limbic targets from serial images into a single continuous dataset, and 4) Automatically reconstruct axon pathways to their targets. The goal is to create and integrate this novel set of software and neurobiologic technologies that will accelerate wiring the brain of mammals. These technologies will provide the critical missing framework necessary for image acquisition, manipulation of terabyte datasets, serial section reconstruction, automatic and manual tract tracing, and gene specific wiring diagrams or connectomes at the scale of primate brains and for bridging the gap between axon projections and high resolution tracts obtained by MRI and DTI of non-human primates and humans. of this research to public health: Establishing a network diagram of the brain is one of the major challenges of modern neurobiology and medicine, particularly a diagram of genetic connectivity. Nowhere is this need clearer than for the brain system controlling social behavior and emotion, where dysregulation of circuitry has been implicated in the most devastating mental illnesses, depression, schizophrenia and autism that together affect more than 13 million Americans. Creating solutions to understand these brain systems will broadly accelerate unraveling the connectivity of many circuits that are disturbed in other neurologic diseases, including those involved in Parkinson's, Alzheimer's, and addiction. In summary, the tools we are creating will transform the field of mental illness by providing genetic links to the underlying brain circuitry. This knowledge will provide insights into new drug targets to prevent, treat, and ultimately cure mental illness.

DESCRIPTION (provided by applicant): Down syndrome (DS) or trisomy for chromosome 21, is a major cause of mental retardation and congenital heart disease, that affects more than 400,000 individuals in the USA. In addition, people with DS have defects in memory, language and neuroanatomy. The ultimate goal of this proposal is to elucidate the genes, neuroanatomy and neurocircuitry linked to the neurocognitive defects of DS. Recent advances in human genome sequencing provide a complete list of chromosome 21 genes, but until recently (Korbel, 2009), there were no genes linked to any specific feature and no successful treatment for the cognitive deficits. Treatment of DS has been hindered by lack of human models for narrowing the genes responsible for cognitive features, the need for high resolution neuroimaging of the brain defects in DS and the need for cognitive tests focused on DS deficits. To address these gaps, we propose an innovative multidisciplinary approach integrating neuroimaging, cognitive testing and genetics. In DS as in genetics, rare events can illuminate common processes. Rare individuals have DS caused by duplication of only parts of chromosome 21 and provide a unique opportunity to link the defects of brain development and function with the genes responsible. The PI of this proposal has generated the largest cohort in existence, consisting of 45 persons, of whom 30 are alive, in the USA, 27 aneuploid only for 21. Recently, these rare cases were used to map specific gene subsets in DS for congenital malformations but little was known about the brain or cognition. Further, a unique and rare set of identical twins discordant for DS, has been identified and studied. To characterize these cohorts, a unique team of scientists has been created, with expertise in partial trisomy 21 (Korenberg), neurocognitive testing targeted to DS (Nadel, Yurgelun-Todd), Magnetic Resonance Imaging and Diffusion tensor imaging (Yurgelun- Todd, Gerig), NMR Spectroscopy (Renshaw) and genome organization (Korenberg). This team will integrate high resolution imaging of full and rare partial trisomy 21 to establish the regional neuroanatomy and neurocircuitry of DS and to link these to subsets of chromosome 21 genes. We will test three levels of hypotheses, first to define in DS vs normals, distinct volumetric and circuit brain structures and defects of cognitive function, including language and memory; second, to link within DS, neural substrates with cognitive features and third, by using each rare partial trisomy, to begin to parse gene subsets for DS neural and cognitive features. Further, we will test in DS, whether the glutamatergic or, as in mouse, the GABAergic neurotransmitter systems are disturbed in vivo. We propose two aims: 1) to characterize 60 normal and 60 full trisomy 21 individuals with cognitive testing, high resolution MRI, DTI and MRS, 2) to characterize the rare DS cohort with partial trisomy 21 and the unique set of identical twins discordant for DS as in aim 1. The results of these studies will provide unprecedented knowledge of the defects of brain development and function in DS, GABAergic and glutamatergic pathways, and the relationship of both to genes duplicated. These results will help to elucidate the neurobiology of DS and to develop novel treatments for DS and other intellectual disabilities.

The PPG is committed to a tightly integrated, cross-disciplinary design, bringing multiple levels of analysis and techniques to bear on shared specific themes in linking social phenotype to brain to gene. An overarching goal for the PPG is to create a framework to link each Project with the others. Core B is at the heart of this function, ensuring that, to the extent possible, participants are investigated by each of the disciplines represented by the different Projects. Core B, based at The Salk Institute's Laboratory for Cognitive Neuroscience (LCN), with Dr. Ursula Bellugi serving as Principal Investigator, is an efficient model for achieving this goal through its provision of a central infrastructure for the following services: (i)Planning and executing outreach activities that establish and maintain connections with the target participant communities (WS, DD) through referral sources as well as local, national, and international organizations; (ii)ldentifying and recruiting potential participants; (iii) Performing the induction screening, administering and scoring the Core Diagnostic Battery, and gathering medical and background information necessary for the application of inclusionary/exclusionary criteria; (iv) Administering and scoring a Core Cognitive Battery; (v) Coordinating an efficient schedule of participation across Projects; (vi) Tracking and ensuring the participation of all subjects across Projects; (vii) Coordinating the acquisition, transport, and collection of human tissue samples (e.g., blood samples for Project I; dental pulp for Project 11; brain tissue for Project 111). Overall, Core B centralizes the participant recruitment efforts and oversees participant tracking and participation inappropriate Projects.

The overarching goal of Core A is to provide the basic Adnninistrative, Data Centralization and Management, and Statistical Services/Data Integration required by all projects to: (i) Provide unified administrative services; (ii) Facilitate scientific and administrative communication; (iii) Manage the central data archives and facilitate exchange of verified data; (iv) Coordinate statistical services consultation for the participating investigators; and (v) Apply bioinformatics / neurocomputational approaches to integrate our multimodal data, from the molecular genetic level to that of social behavior. To this end, the Objectives are: (a) Administrative Services Fostering Interactivity and Integration, to coordinate daily functioning of a complex interdisciplinary research enterprise by coordinating communications and scientific activity among the Projects and Cores, providing budgetary planning and control, and facilitating and promoting collaboration and interactivity among investigators and consultants; (b) Data Centralization and Management, Data Quality Control, Core Statistical Services, and Multidimensional Data Integration, to maintain a secure, centralized, and immediately sharable database in order to facilitate collaboration and integration, to provide basic statistical consulting services, and to coordinate data integration by applying bioinformatics / neurocomputational approaches to our large database to reveal cross-level associations. The vitally important functions of Core A thus help to manage and integrate data from different domains with the ultimate goal of characterizing the system of human social behavior against the backdrop ofthe WS social phenotype. The testimony of the enormous success of Core A function as an Administrative and Data Centralization and Integration Core is our highly impressive list of interdisciplinary publications stemming from the decade of this Program Project. RELEVANCE (See instructions): Our goal is to integrate the gene, neural systems and behavioral project findings to forward our understanding of Williams syndrome. This study may help identify educational, social and medical-health support approaches appropriate for WS. The new bioinformatics and computer modeling techniques developed for this study will also be applicable to other multi-level brain research studies.

DESCRIPTION [Provided by Applicant): The overarching goal of this application is to link social-affective functions to their underlying neurobiological and molecular genetic bases, using Williams syndrome (WS) as a model. The theme leads from the investigators' past studies to the hypothesis that genetic and neuropeptide dysregulation underlies the social-affective features of WS. They continue to employ a multi-pronged approach incorporating new techniques and approaches to understanding the neurogenetic systems involved in the WS social phenotype. To this end, Project I: Gene Networks for Social Cognition, will determine the role of WS genes in social-emotional behavior using individuals with both full and atypical deletions; the role of genetic variations for neurotransmitters and neuropeptides in WS social-emotional behavior; the expression of WS and neuropeptide receptor genes in WS brain; and the genetic transcriptional networks altered in WS. Project II: Modeling WS using Human Neurons, will model typical and affected human nervous system development in WS using pluripotent stem cells. Studies will target genes implicated in social behavior within the WS region. Project III: Cellular Architectonics and Local Circuits, will examine the structures implicated in the social brain in WS on a unique collection of WS brains. Project IV: Neuroimaging of Social Circuitry, will utilize new multimodal, integrated structural and functional neuroimaging techniques to test hypotheses about the relationships among brain anatomy, physiology, cognition, and genetics in WS. Project V: Characterization of the Social Phenotype of WS, will characterize three independent key dimensions of altered social-affective behavior WS, including the pathways of several dissociations, incorporating new technologies and probes for behavior, psychophysiology, and electrophysiology. Genetic concepts have been used to map out the program project, and will be integrated at the level of the organism (Projects IV and V), brain/gene expression (Project III), and cell (Project II), to parse genes and transcriptional networks underlying social behavior. Results have the potential to move our understanding forward in unprecedented ways, providing a critical contribution to Social Neuroscience. RELEVANCE: A mission of NICHD includes research that leads to increased understanding and treatment of social behavior and emotional disorders. The investigators propose research that targets the study of genes, neural circuits, and social behavior in new and innovative ways, the results of the studies will provide unprecedented integration of the genetic and brain processes responsible for human social behavior, and key to novel treatments.

DESCRIPTION (provided by applicant): Establishing a genetic network diagram of the primate brain linked to social disorders is one of the major challenges of modern neurobiology and medicine. Nowhere is this need clearer than in social-emotional systems, where dysregulation of circuitry is implicated in mental illnesses: autism, depression and anxiety. We will generate multiple-gene-specific 3D reconstructions from serial sections of long range axon projections of social circuitry in primate, including hypothalamus, bed nucleus and stria terminalis, basal forebrain nuclei, amygdala and basal ganglia, and integrate with MRI. This will address two barriers to understanding primate brain and human social behavior; lack of knowledge of endogenous circuitry, and lack of computational systems for large scale multicolor axon projections in serial registration and for handling the terabyte datasets. We have generated a novel pipeline of multicolor confocal image acquisition integrated with a computational suite of algorithms for reconstructing and visualizing TB image sets. We have applied these to generate a 3D reconstruction of 45,295,200 images (1,620mm) at the axon level for social circuitry involving oxytocin (OT) and vasopressin (AVP) and the Williams syndrome gene, GTF2IRD1, all implicated in social behavior. We have identified novel OT and AVP structures and sparse tracts that may fill critical gaps as substrates and biomarkers for human behavior. Moreover, we have developed approaches for hierarchical 3D integration of axon-level images to MRI images. The proposal emerges from a unique multidimensional team and advisors*; experts in segmentation, large scale image reconstruction and 3D visualization (Tasdizen, Joshi, Pascucci, Roysam*), in primate brain circuitry and neuroanatomy (Angelucci, Hof*, Dong*, Korenberg), in animal and human MRI (Hsu, Joshi), and in genetics and multicolor fluorescence imaging of axon projections (Korenberg, Angelucci). We will: 1) Establish a genetic wiring diagram of axonal projections for OT, AVP and GTF2IRD1 in macaque. We will generate, validate and image more than 667 serial coronal sections (21,100mm) of macaque brains multicolor fluorescence immunohistochemistry of ligands and their receptors, using confocal microscopy and tract tracing. Pre-/post-mortem MRI(150-200mm resolution) and block-faces images will be acquired. Novel circuitry will be validated in cognate regions of human brain. 2) Build a Neural Information System that integrates and visualizes a multiscale volumetric TB dataset of primate genetic connectivity for social neuropeptides, aligned using automated slice-to-slice image registration at the resolution of axonal projections to MRI, and annotated neuroanatomy. The results will identify novel OT/AVP related social circuitry and establish a pipeline of integrated technologies for bridging (macro)connectome with (micro)axome and both with genetics of mental illness. These will provide novel biomarkers for disease features and receptor targets, and accelerate translating the dissonant orchestration of social behavior by neuropeptides to therapeutic harmony in humans.

ABSTRACT Down syndrome (DS) or Trisomy 21, is the major genetic cause of intellectual disabilities (ID) affecting millions worldwide. Even more striking, DS is a major risk for autistic spectrum disorder (ASD), Alzheimer?s disease (AD), congenital heart disease, and deficits of the immune, endocrine and hematopoetic systems. There are no preventatives or treatments of these deficits in DS, due in part to the need for deeply annotated and deeply phenotyped DS study and discovery cohorts. To fill this gap, the goal of this Administrative Supplement to the Utah Clinical and Translational Science Award (CTSA), under the leadership of Julie R. Korenberg, is to harmonize with and expand the NIH INCLUDE (INvestigation of Co-occurring conditions across the Lifespan to Understand Down syndromE) consortium, DS-ConnectTM registry, the Data Management and Portal for INCLUDE (DAPI) Project, and the Data Coordinating Center (DCC) by completing and integrating two novel DS cohorts each with existing deep annotation, and one with deep brain phenotyping and pan-omics that will overlap the Crnic Institute?s Human Trisome Project (HTP). Enabled by this supplement, we will deliver: ? Recruitment of DS-UPDB, a large cohort of 300 participants (200 DS families, 100 age and gender-matched controls) covering the entire lifespan, derived from the unique multi-generational Utah Population Database (UPDB) that includes >4000 confirmed DS diagnoses, with family data and the electronic medical record (EMR). The next phase will establish the biobank and pan-omics for this unique cohort. ? Deeply annotated, portal-ready demographics, clinical and family datasets for 300 participants in DS-UPDB. ? Establishment of an INCLUDE cohort and Public Gateway using the pre-existing DS Brain Discovery Cohort, a unique live cohort with multidimensional linked datasets: deeply annotated, deeply phenotyped and biobanked, with extensive pre-existing datasets (cognition, behavior, MRI, DTI, fMRI, karyotypic, DNA array, methylome, labs). ? Completion of Pan-omics datasets (Transcriptomics, Proteomics, Cytokines and Metabolomics) of the DS Brain Discovery Cohort (30 DS, 37 parents, 14 controls) embedded within the larger cohort. ? The first inter-cohort collaboration integrating the Immune tests with brain imaging using the Brain Discovery Cohort biobank. The results will add a future dimension to DS research collaboration by establishing a deeply annotated DS cohort enriched for co-occurring conditions, within the multigenerational UPDB, and the first DS Brain Discovery Cohort (also UPDB) deeply phenotyped for brain imaging, genes and pan-omics, as an unparalleled resource for collaborative data mining by the INCLUDE consortia, HTP, for the DS-ConnectTM registry, DAPI, and DCC. This proposal is responsive to NOT-OD-20-024, maintains the scope of the Utah parent CTSA, attracts and trains junior DS investigators, and will accelerate the speed of translation to therapeutics for DS.