Regulatory Role of CACNA1C Intronic DNA Variation Relevant to Psychiatric Disease

CACNA1C 内含子 DNA 变异与精神疾病相关的调节作用

• 批准号: 8684348
• 负责人: YEN PEI CHRISTY CHANG
• 金额: $ 23.03万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8684348
• 关键词: Adult; Affect; Alleles; Animal Model; Autopsy; Behavior; Binding; Binding Sites; Bioinformatics; Biological; Biological Assay; Biology; Bipolar Disorder; Brain; Cardiovascular system; Cells; Code; Complex; Computer Simulation; DNA; Data; Development; Diagnosis; Disease; Electrophoretic Mobility Shift Assay; Electroporation; Elements; Functional disorder; Future; Gene Expression; Gene-Modified; Genes; Genetic; Genetic Risk; Genetic Transcription; Genetic Variation; Genomics; Goals; Hereditary Disease; Human; Human Cell Line; In Vitro; Inbred Mouse; Inherited; Introns; Knowledge; L-Type Calcium Channels; Laboratories; Lead; Linkage Disequilibrium; Location; Maps; Mental Depression; Mental disorders; Messenger RNA; Methodology; Methods; Modeling; Molecular Genetics; Mood Disorders; Mus; Mutation; National Institute of Mental Health; Nucleic Acid Regulatory Sequences; Pathogenesis; Predisposition; Prevention; Proteins; Publishing; Regulation; Regulatory Element; Reporter; Research; Research Domain Criteria; Resources; Risk; Risk Factors; Role; Schizophrenia; Staging; System; Testing; Transfection; Transgenic Mice; Validation; Variant; base; brain tissue; chromatin immunoprecipitation; disorder risk; epigenetic marker; experience; genetic association; genetic variant; genome wide association study; improved; in vitro activity; in vivo; mouse model; neurobehavioral; neuropsychiatry; novel; novel strategies; programs; public health relevance; research study; risk variant; skills; transcription factor; vector

DESCRIPTION (provided by applicant): One of the most consistent findings to have emerged from psychiatric disorder genome wide association studies (GWAS) is with CACNA1C, a gene that codes for the alpha1 subunit of a voltage-dependent L-type calcium channel. Consistent with the NIMH Research Domain Criteria (RDOC) initiative, the biological implications of CACNA1C function are relevant to a diagnosis of bipolar disorder, depression, and schizophrenia. However, in spite of strong genetic data implicating sequence variations in CACNA1C as a risk factor, it is not known how genetic variants located within the gene modify risk. All GWAS-identified SNPs in CACNA1C are located within a single large intron 3 and do not lead directly to changes in the sequence of the coded protein. The central hypothesis guiding the present research effort, supported by our preliminary data, is that specific genetic variation in CACNA1C intron 3 modifies regulatory functions that can be bioinformatically predicted and experimentally validated. Our hypothesis is based on our bioinformatic analyses of the regions surrounding associated human SNPs and preliminary data from our in vitro functional validation of a subset of the in silico predictions. This proposal proposes a multiple methodology strategy, consistent with studies already underway in the laboratory. We will, in Specific Aim #1, use a bioinformatics approach to define sequences in human CACNA1C that are likely to harbor regulatory elements. We predict that the human CACNA1C gene harbors putative regulatory elements containing alleles in linkage disequilibrium (LD) with GWAS identified SNPs. Specific Aim #2 proposes to test human candidate regions in reporter vector systems to assess regulatory activity. We will clone putative regulatory elements into reporter vectors to assess their function as modifiers of transcription in vitro, fine map the location of these regulatory elements, and evaluate these elements for putative TF binding using co-transfection and TF-specific gel-shift assays. We predict that psychiatric condition-associated SNPs (or genetic variations inherited with them) will result in allele-specific changes in CACNA1C gene expression and/or altered function through cis-acting regulatory elements and that we will identify proteins that interact with such regions. As these in vitro and cell-based assays incompletely predict endogenous activity, we will map the presence of human regulatory domains in the mouse (Specific Aim #3). We will locate the corresponding regions in the mouse Cacna1c gene, validate their activity in vitro, and assess in vivo TF binding during different developmental stages. Our intent is to plan future in vivo studies with inbred or transgenic mice harboring similar genetic variations. Overall, our studies will improve basic knowledge of CACNA1C regulation, and significantly progress understanding of the mechanism by which CACNA1C gene intronic variation may modify risk for developing psychiatric disorders.

描述（由申请人提供）：精神疾病基因组全关联研究（GWAS）中出现的最一致的发现之一是与CACNA1C有关，CACNA1C是一种编码电压依赖性l型钙通道α 1亚基的基因。与NIMH研究领域标准（RDOC）倡议一致，CACNA1C功能的生物学意义与双相情感障碍、抑郁症和精神分裂症的诊断有关。然而，尽管强有力的遗传数据表明CACNA1C序列变异是一个危险因素，但尚不清楚位于基因内的遗传变异是如何改变风险的。在CACNA1C中，所有gwas鉴定的snp都位于单个大内含子3中，并且不会直接导致编码蛋白序列的变化。我们的初步数据支持了指导本研究工作的中心假设，即CACNA1C内含子3的特定遗传变异可以改变调节功能，这种功能可以通过生物信息学预测和实验验证。我们的假设是基于我们对相关人类snp周围区域的生物信息学分析，以及我们对计算机预测子集的体外功能验证的初步数据。该建议提出了一种与实验室中正在进行的研究相一致的多种方法策略。在Specific Aim #1中，我们将使用生物信息学方法来定义人类CACNA1C中可能包含调控元件的序列。我们预测，人类CACNA1C基因含有与GWAS鉴定的snp连锁不平衡（LD）等位基因的推定调控元件。具体目标2建议测试报告媒介系统中的人类候选区域，以评估调节活性。我们将把假设的调控元件克隆到报告载体中，以评估它们在体外作为转录修饰因子的功能，精细绘制这些调控元件的位置，并使用共转染和TF特异性凝胶转移试验评估这些元件是否与假定的TF结合。我们预测，精神疾病相关的snp（或遗传的遗传变异）将导致CACNA1C基因表达的等位基因特异性变化和/或通过顺式调控元件改变功能，我们将确定与这些区域相互作用的蛋白质。由于这些体外和基于细胞的测试不能完全预测内源性活性，我们将绘制小鼠中人类调节域的存在（Specific Aim #3）。我们将在小鼠Cacna1c基因中定位相应的区域，在体外验证其活性，并评估不同发育阶段的体内TF结合情况。我们的目的是计划未来对近亲繁殖或转基因小鼠进行体内研究，这些小鼠具有类似的遗传变异。总之，我们的研究将提高对CACNA1C调控的基础知识，并显著推进对CACNA1C基因内含子变异可能改变精神疾病发生风险的机制的理解。