2012 — 2016 |
Hoffman, Ellen J |
K08Activity Code Description: To provide the opportunity for promising medical scientists with demonstrated aptitude to develop into independent investigators, or for faculty members to pursue research aspects of categorical areas applicable to the awarding unit, and aid in filling the academic faculty gap in these shortage areas within health profession's institutions of the country. |
Functional Analysis of Rare Variants in Genes Associated With Autism
The goal of this career development award is to integrate the genetics of autism spectrum disorders (ASD), developmental neuroscience, and the functional analysis of rare variants, in order to advance our understanding of the basic biological mechanisms underlying ASD. Dr. Hoffman is a board certified, practicing child psychiatrist, who is currently pursuing her PhD in Investigative Medicine in the laboratory of Matthew State, MD, PhD, at the Yale Child Study Center. Her career goal is to become an independent investigator with dual expertise in the genetics of child psychiatric disorders and the neurobiology of vertebrate systems. In this application, she proposes to utilize zebrafish as a novel translational tool that will leverage findings from human genetic studies as a means of advancing our knowledge of the pathophysiology of ASD. Dr. Hoffman will gain this expertise through the combined guidance of her primary mentor, Dr. State, a leader in ASD genetics, and co-mentor, Antonio Giraldez, PhD, an expert in zebrafish development, which will promote the establishment of this innovative approach to investigating the role of ASD susceptibility genes in neural circuit formation. The objective of this research is to elaborate basic mechanisms of ASD by investigating the function of the ASD risk gene, Contactin Associated Protein-2 (CNTNAP2) in neural development, and to determine how sequence variants in this gene identified in individuals with ASD disrupt its function. Using the emerging technology of zinc finger nucleases, which have superior accuracy over morpholinos, Dr. Hoffman induced targeted germline mutations in the two zebrafish CNTNAP2 genes, CNTNAP2a and 2b. To our knowledge, this is the first zebrafish knockout of an ASD risk gene generated by this method. Our hypothesis is that CNTNAP2a/2b double knockouts will display reproducible morphological and/or behavioral phenotypes that will yield important insights into the function of CNTNAP2 in neural development. This hypothesis will be tested by pursuing these aims: 1) Generate double knockouts of the two zebrafish CNTNAP2 genes by crossing fish carrying germline mutations; 2) Identify quantifiable morphological and/or behavioral phenotypes in CNTNAP2 double knockouts; and 3) Characterize the ability of the human CNTNAP2 gene with rare sequence variants found in individuals with ASD to reverse the phenotypes. Our rationale for this approach is that the development of an in vivo system to rapidly assess the functional consequences of rare genetic variants is the crucial next step in understanding the biology of ASD. Dr. Hoffman has assembled an outstanding team of mentors and collaborators, including pioneers in zebrafish neural circuit analysis and behavioral phenotyping, as this system is anticipated to provide unique insights into the role of CNTNAP2 in the neural circuitry underlying simple behaviors. The extensive resources of the Yale Child Study Center, together with her formal plan for didactics in neuroscience and developmental biology, will further support Dr. Hoffman¿s goal of elucidating the molecular and cellular mechanisms of ASD.
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2018 — 2021 |
Hoffman, Ellen J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High-Throughput Functional Analysis of Autism Risk Genes
The central objective of this research is to elucidate the function of genes that are strongly associated with autism spectrum disorders (ASD) in fundamental processes of vertebrate brain development. While whole-exome sequencing has led to a growing list of ?high confidence? ASD (hcASD) risk genes, which are beginning to converge on common pathways, our understanding of the mechanisms by which hcASD gene disruption alters the development of specific cell types and neural circuits, resulting in behavioral dysfunction, remains limited. The long-term goal of this research is to illuminate the basic biological mechanisms underlying ASD, which will provide a much-needed avenue for the discovery of targeted pharmacological treatments. Here, we will capitalize on the unique advantages of zebrafish as an in vivo, biologically relevant system for the rapid functional analysis of multiple hcASD genes in parallel, including: (i) the ability to directly visualize basic neurodevelopmental processes and neural activity in a whole vertebrate brain through transparent embryos; (ii) large progenies, which are ideal for conducting high-throughput in vivo screens to identify small molecule suppressors of behavioral phenotypes; and (iii) ease of genetic manipulation, such that we have already generated zebrafish mutants in 10 top hcASD genes, which we will analyze in the present study. Our central hypothesis is that zebrafish mutants of hcASD genes will display quantifiable morphological, simple behavioral, and circuit-level phenotypes that will converge on common pathways, providing new insights into the roles of these genes in the developing vertebrate brain. This hypothesis is based on evidence from our study of zebrafish mutants of the ASD risk gene, CNTNAP2, which revealed dysregulation of GABAergic and glutamatergic signaling and identified a novel pharmacological suppressor of an ASD gene-associated mutant behavioral phenotype. To test this hypothesis, we will pursue the following aims: 1) Identify quantifiable brain phenotypes across 10 hcASD mutants using light-sheet imaging and automated deep phenotyping of CNS- specific markers; 2) Conduct high-throughput pharmaco-behavioral profiling of hcASD mutants using a novel, automated visual-startle assay and perform small molecule screens to identify suppressors of mutant startle phenotypes; and 3) Characterize neural circuit deficits underlying altered sensory processing behaviors in hcASD mutants and identify the circuit-level mechanisms of pharmacological suppressors using whole-brain in vivo two-photon imaging. This approach is highly innovative and is the first to analyze the function of multiple hcASD genes in parallel at the structural, behavioral, and circuit levels, allowing us to progress rapidly from risk gene discovery to the elucidation of convergent pathways with relevance to ASD. Therefore, we expect that this research will lead to critical advances in our understanding of the basic biology of ASD and provide a path forward in the discovery of mechanism-based pharmacological treatments.
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