Several candidate genes for schizophrenia (SZ) have been identified as a result of genetic linkage and association studies, including NRG1 and DTNBP1, among others. However, despite intensive analysis of exons, intron-exon borders, and promoter regions immediately upstream from transcription start sites, specific disease-causing alleles have not yet been identified. One possibility is that the expression of these candidate genes is being affected by variant alleles located in regulatory elements that have not yet been identified, that can function many kilobases away from transcription start sites, such as enhancers and locus control regions. The primary aims of this proposal are to identify distal regulatory elements of several SZ candidate genes and determine if potential functional alleles, which would be candidates for SZ susceptibility, exist within. We will screen for distal regulatory elements by mapping DNAse 1 hypersensitive sites in the NRG1 and DTNB1 loci by quantitative real-time PCR, and testing for promoter and enhancer function in the DNA near these sites using the pGL2-luciferase reporter system. Variant alleles will be tested in the same system to determine if promoter/enhancer function is impaired. They will also be tested for protein binding affinity using electromobility gel shift assays (EMSA) to determine whether or not allele-specific differences in DNA-protein complexes exist. Functional mutations will be analyzed in a case control association study to determine if the allele and genotype distributions differ between controls and patients with SZ. For the most promising variants, we will identify the DNA-binding protein by EMSA supershift using antibodies to the most likely candidates, or by purifying them using DNA-binding proteins using heparin Sepharose and DNA Sepharose chromatography. By identifying the proteins that bind to a gene's regulatory regions, we will gain new insight into the pathogenesis of SZ, in particular, gene-environment interactions during development that may play a role in some cases. It is through alterations in gene expression that environmental stressors interact with the brain.
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