Identifying enhancers in schizophrenia and bipolar disorder-associated noncoding eQTLs during cortical synaptic organization

项目摘要 精神疾病的全基因组关联研究一再将精神分裂症的风险与 双相情感障碍的非编码区或附近的基因相关的突触组织，包括一个 CACNA1C基因的第三内含子中的间隔，其编码泛神经元的孔形成亚基。 突触后电压门控钙通道。由于这些变异体位于非编码DNA中， 蛋白质的后续氨基酸序列。相反，许多风险变量预计将 表达数量性状位点（eQTL）的突触基因，它是最有可能的风险， 序列是调节靶向突触基因转录的增强子。这增加了一个广泛的机构， 将精神分裂症和双相情感障碍确定为"突触障碍"的研究。然而，在这方面， 尽管有强有力的证据将风险eQTL和突触组织联系起来，但这种关系的性质 还不清楚在本项目中，我的目的是检验以下假设：（1）非编码精神病性障碍风险的eQTLs， 突触基因是幼年或青春期皮质突触发育的增强子，以及（2）风险- CACNA1C中高优先级增强子的相关序列变异破坏了CACNA1C的细胞类型特异性能力， 该序列在皮层突触发生过程中充当增强子。我将测试第一个假设， 候选基因组增强子的大规模平行报告基因测定（MPRA），所述候选基因组增强子含有eQTL， 在皮质突触发生过程中， 小鼠我将在代表青少年（P7）和青少年（P56）突触的两个时间点进行MPRA。 发展过程。这些实验将使我能够确定顶级精神病患者 在哺乳动物皮层突触发生过程中的发育障碍相关的风险区域。我还将测试 等位基因效应，这可能使我能够确定风险等位基因对转录水平有强烈影响的eQTL。 调控我将测试第二个假设进行简化的MPRA确定的增强子 在CACNA1C中，在小鼠皮质突触发生期间的体内，包括含有风险等位基因的序列， 参考等位基因，以及以风险相关SNP为中心的缺失。我还会检测细胞类型或区域 CACNA1C增强子表达的特异性。通过在我的第二个目标中测试更少的序列，我的灵敏度 检测活动中等位基因差异的能力将大大提高，我预计能够确定任何 与风险相关序列变异相关的活性的潜在差异。这些实验的结果 将阐明风险相关的非编码基因组变异和疾病病因机制之间的联系， 哺乳动物皮层突触发生这项研究的未来方向包括确定表达如何 与风险变异相关的变化影响突触生物学和转诊断基因组或 在这项研究中确定的分子靶点，用于未来的治疗开发。

PROJECT SUMMARY Genome-wide association studies of psychiatric disease have repeatedly linked risk for schizophrenia and bipolar disorder to noncoding regions that are in or near genes relevant in synaptic organization, including an interval in the third intron of the gene CACNA1C, which encodes the pore-forming subunit of a pan-neuronal postsynaptic voltage-gated calcium channel. As these variants are located in noncoding DNA, they do not alter the subsequent amino acid sequence of the protein. Rather, many of the risk variants are expected to be expression quantitative trait loci (eQTLs) of synaptic genes, and it is most likely that the risk-containing sequences are enhancers that regulate transcription of a targeted synaptic gene. This adds to a wide body of research that have identified schizophrenia and bipolar disorder as “disorders of the synapse.” However, though there is strong evidence linking the risk eQTLs and synaptic organization, the nature of this relationship is unclear. In this project, I aim to test the hypotheses that (1) non-coding psychotic disorder risk eQTLs of synaptic genes are enhancers during juvenile or adolescent cortical synaptic development and (2) risk- associated sequence variation in high-priority enhancers in CACNA1C disrupts the cell-type specific ability of the sequence to act as an enhancer during cortical synaptogenesis. I will test the first hypothesis by conducting a massively parallel reporter assay (MPRA) of candidate genomic enhancers that contain eQTLs that contribute transdiagnostic risk for bipolar disorder and schizophrenia in vivo during cortical synaptogenesis in mice. I will conduct this MPRA at two time points representing juvenile (P7) and adolescent (P56) synaptic developmental processes. These experiments will allow me to determine the functional role of top psychotic disorder-associated risk regions across development during mammalian cortical synaptogenesis. I will also test for allelic effects, which may allow me to identify eQTLs whose risk allele has a strong effect on transcriptional regulation. I will test the second hypothesis by conducting a reduced complexity MPRA of identified enhancers in CACNA1C in vivo during mouse cortical synaptogenesis, including sequences containing the risk allele, the reference allele, and a deletion centered at the risk-associated SNP. I will also test for cell-type or regional specificity of expression of CACNA1C enhancers. By testing fewer sequences in my second aim, my sensitivity to detect allelic differences in activity will be strongly improved and I anticipate being able to determine any potential differences in activity related to risk-associated sequence variation. The results of these experiments will clarify the link between risk-related noncoding genomic variation and mechanisms of disease etiology in mammalian cortical synaptogenesis. Future directions from this research include determining how expression changes associated with risk variants impact synaptic biology and the study of transdiagnostic genomic or molecular targets identified in this research for future therapeutic development.