Variants in NRG3 Associated with Delusion Have Regulatory Potential and Differentially Bind to Nuclear Proteins. M. Zeledon1,2,3, M. Taub5, N. Eckart1,2, M. Beer1,6, R. Wang3, M. Szymanski1,2, P. Chen7, A. E. Pulver3,4, J. A. McGrath3,4, P. Wolyniec3,4, D. Avramopoulos1,3, A. Sawa3, D. Valle1 1) McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD; 2) Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, MD; 3) Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD; 4) Epidemiology-Genetics Program, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; 5) Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; 6) Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, MD; 7) Department of Medical Genetics, National Taiwan University Hospital, Taipei City, Taiwan.

   Schizophrenia (SZ) is a severely disabling psychiatric disease that affects 1% of the worlds population. Symptoms include dysfunctions in cognition, thought/belief (delusions), perception/sensation (hallucinations) and affect. Previously, our group reported a linkage peak for SZ (NPL of 4.7) at 10q22 in the Ashkenazi Jewish (AJ) population and, in a follow-up fine mapping association study in the AJ, found strong evidence of association between a quantitative phenotypic trait delusion and three intronic SNPs in the 5 end of NRG3. Two other independent groups have replicated our findings, making NRG3 a strong candidate gene for a subtype of schizophrenia with delusions. To identify causative variants, we sequenced the 162 kb LD block covering the 5 end of NRG3 and containing the three associated SNPs in 47 AJ SZ patients at either extreme of the delusion quantitative trait. We identified 5 SNPs with minor alleles on the implicated haplotype and significantly overrepresented in high delusion patients. We tested these for regulatory potential and found that the delusion-associated alleles of rs10883866 and rs60827755 significantly and reproducibly decreased and increased (3-4 fold) expression of a reporter gene respectively, compared to the common allele in a variety of cell types including primary rat cortical neurons. To test whether these changes are due to differential binding of DNA regions surrounding the variants to nuclear protein(s), we performed electrophoretic mobility shift assays. We determined that the 21 bp surrounding the variants consistently bind to nuclear proteins and by competing with cold probe, found that binding of rs60827755 is influenced by the genotype of the SNP. In silico predictions of disrupted binding sites pointed to CNOT4 (CCR4-NOT transcription complex, subunit 4) as a candidate binding protein, and supershift data suggest that the shift is due in part to CNOT4 binding. In summary, we have identified cis-acting regulatory motifs that modify NRG3 expression and that account for the association with delusions in SZ have regulatory potential and bind to nuclear proteins, one of which may be CNOT4. Several proteins binding CNOT4 have been implicated in SZ by GWAS. We anticipate that these experiments will allow us to move from genetic observations to deciphering the mechanistic pathways that may lead to risk for SZ or modifications of the SZ phenotype.

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