Validation studies and multi-omics analysis of Zhx2 as a candidate quantitative trait gene underlying brain oxycodone metabolite (oxymorphone) levels and behavior

Sensitivity to the subjective reinforcing properties of opioids has a genetic component and can predict addiction liability of opioid compounds. We previously identified Zhx2 as a candidate gene underlying increased brain concentration of the oxycodone (OXY) metabolite oxymorphone (OMOR) in BALB/cJ (J) versus BALB/cByJ (By) females that could increase OXY state-dependent reward. A large structural intronic variant is associated with a robust reduction of Zhx2 expression in J mice, which we hypothesized enhances OMOR levels and OXY addiction-like behaviors. We tested this hypothesis by modeling the loss-of-function variant through knocking out the Zhx2 coding exon (E3KO). Integrative transcriptomic and proteomic analysis of E3KO mice identified astrocyte function, cell adhesion, extracellular matrix properties, and endothelial cell functions as pathways influencing brain OXY metabolite concentration and behavior. These results support Zhx2 as a quantitative trait gene underlying brain OMOR concentration that is associated with changes in OXY behavior and implicate potential quantitative trait mechanisms that together inform our overall understanding of Zhx2 in brain function. Overall design: To investigate potential mechanisms for how Zhx2 influences OXY metabolism and state-dependent reward learning, we established a CRISPR-mouse line where Zhx2 Exon3 (the only coding exon) has been constitutively knocked out in all tissues in BALB/cByJ mice. Brains were harvested from drug-naïve CRISPR offspring and the University of Chicago Genomics Facility performed RNA-sequencing on right brain hemisphere for gene expression profilling. Pathway enrihment anaysis using Gene-Set Enrichment Analysis was performed on transcriptomic data as well as combined transcriptomic and proteomic data of shared genes.