Functional Analysis of Rare Variants in Genes Associated with Autism

这个职业发展奖的目标是整合自闭症谱系障碍（ASD）的遗传学，发育神经科学和罕见变异的功能分析，以促进我们对ASD基本生物学机制的理解。霍夫曼博士是一位经过委员会认证的执业儿童精神病学家，目前正在耶鲁儿童研究中心的马修州立大学医学博士实验室攻读调查医学博士学位。 她的职业目标是成为一名独立的研究者，在儿童精神疾病的遗传学和脊椎动物系统的神经生物学方面拥有双重专业知识。在这 她建议利用斑马鱼作为一种新的翻译工具，将利用人类遗传研究的结果作为推进我们对ASD病理生理学知识的一种手段。霍夫曼博士将通过她的主要导师，ASD遗传学的领导者State博士和共同导师，斑马鱼发育专家Antonio Giraldez博士的联合指导来获得这种专业知识，这将促进建立这种创新方法来调查ASD易感基因在神经回路形成中的作用。 本研究的目的是通过研究ASD风险基因Contactin Associated Protein-2（CNTNAP 2）在神经发育中的功能来阐述ASD的基本机制，并确定ASD个体中鉴定的该基因的序列变体如何破坏其功能。使用锌指核酸酶的新兴技术，它具有上级准确性超过吗啉代，霍夫曼博士诱导两个斑马鱼CNTNAP 2基因，CNTNAP 2a和2b的靶向种系突变。据我们所知，这是第一个通过这种方法产生的ASD风险基因的斑马鱼敲除。我们的假设是CNTNAP 2a/2b双敲除将显示可再现的形态和/或行为表型，这将产生对CNTNAP 2在神经发育中的功能的重要见解。该假设将通过追求以下目标进行测试：1）通过杂交携带种系突变的鱼来产生两种斑马鱼CNTNAP 2基因的双敲除; 2）鉴定CNTNAP 2双敲除中的可定量形态学和/或行为表型;和3）表征具有ASD个体中发现的罕见序列变体的人CNTNAP 2基因逆转表型的能力。我们采用这种方法的理由是，开发一种体内系统来快速评估罕见遗传变异的功能后果是理解ASD生物学的关键下一步。 博士霍夫曼组建了一个杰出的导师和合作者团队，包括斑马鱼神经回路分析和行为表型分析的先驱，因为该系统有望为CNTNAP 2在简单行为背后的神经回路中的作用提供独特的见解。耶鲁儿童研究中心的广泛资源，以及她在神经科学和发育生物学教学的正式计划，将进一步支持霍夫曼博士阐明ASD的分子和细胞机制的目标。

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.