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

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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.