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

项目总结 对精神疾病的全基因组关联研究一再将精神分裂症和 双相情感障碍到突触组织相关基因内或附近的非编码区，包括 CACNA1C基因第三内含子的间隔，它编码泛神经元的成孔亚基 突触后电压门控钙通道。因为这些变体位于非编码的DNA中，所以它们不会改变 蛋白质的后续氨基酸序列。相反，许多风险变种预计将是 表达突触基因的数量性状基因座(EQTL)，极有可能是包含风险的 序列是调节靶向突触基因转录的增强子。这增加了广泛的 已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， 尽管有强有力的证据将风险eQTL与突触组织联系起来，但这种关系的性质 目前还不清楚。在这个项目中，我的目标是检验以下假设：(1)非编码精神障碍风险eQTL 突触基因是青少年或青少年皮质突触发育的增强剂，(2)风险- CACNA1C中高优先级增强子的相关序列变异破坏了细胞类型特异性能力 在皮质突触发生过程中起增强剂作用的序列。我将通过以下方式验证第一个假设： 包含eQTL的候选基因组增强子的大规模平行报告分析(MPRA) 在大脑皮质突触发生期间对双相情感障碍和精神分裂症的体内跨诊断风险 老鼠。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 发育过程。这些实验将使我能够确定顶级精神病患者的功能角色 哺乳动物皮质突触发生过程中发育过程中的紊乱相关风险区域。我也会测试一下 对于等位基因效应，这可能使我能够识别其风险等位基因对转录有强烈影响的eQTL 监管。我将通过对已确定的增强剂进行降低复杂性的MPRA来测试第二个假设 在小鼠皮质突触发生的活体CACNA1C中，包括包含危险等位基因的序列， 参考等位基因，以及以风险相关SNP为中心的缺失。我还将测试细胞类型或区域 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，我的敏感性 检测活动中的等位基因差异将得到极大的改进，我预计能够确定 与风险相关的序列变异相关的活动的潜在差异。这些实验的结果 将阐明与风险相关的非编码基因组变异与疾病病因机制之间的联系 哺乳动物的皮质突触发生。这项研究的未来方向包括确定如何表达 与风险变异相关的变化影响突触生物学和跨诊断基因组学研究 这项研究为未来的治疗开发确定了分子靶点。

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.