Regulatory Role of CACNA1C Intronic DNA Variation Relevant to Psychiatric Disease

描述(申请人提供)：精神障碍全基因组关联研究中出现的最一致的发现之一是CACNA1C，这是一种编码电压依赖型L型钙通道α1亚单位的基因。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相关。然而，尽管强有力的遗传数据表明CACNA1C中的序列变异是一个危险因素，但位于该基因内的遗传变异如何改变风险尚不清楚。在CACNA1C中，所有Gwas鉴定的SNPs都位于单个大的内含子3中，不会直接导致编码蛋白序列的变化。指导本研究工作的中心假设得到了我们初步数据的支持，即CACNA1C内含子3中特定的遗传变异修改了调控功能，这可以通过生物信息预测和实验验证。我们的假设是基于我们对相关人类SNPs周围区域的生物信息学分析，以及我们对In Silico预测子集的体外功能验证的初步数据。这一建议提出了一种多方法战略，与实验室中已经进行的研究一致。在特定的目标#1中，我们将使用生物信息学方法来定义人类CACNA1C中可能含有调控元件的序列。我们预测人类CACNA1C基因含有可能含有等位基因的调控元件，这些元件与GWASSNPs存在连锁不平衡(LD)。具体目标#2建议测试报告载体系统中的人类候选区域，以评估调控活动。我们将把可能的调控元件克隆到报告载体中，以评估它们作为体外转录调节因子的功能，精细定位这些调控元件的位置，并使用共转染和TF特异性凝胶移位分析来评估这些元件可能与TF结合。我们预测，精神疾病相关的SNPs(或遗传变异)将通过顺式作用调控元件导致CACNA1C基因表达的等位基因特异性变化和/或功能改变，我们将识别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分析不能完全预测内源性活性，我们将绘制小鼠体内人类调节域的存在图(特定目标#3)。我们将在小鼠CACNA1C基因中定位相应的区域，在体外验证它们的活性，并在体内评估不同发育阶段的TF结合。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。总体而言，我们的研究将提高CACNA1C调控的基础知识，并显著提高对CACNA1C基因内含子变异可能改变发生精神障碍的风险的机制的理解。

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.