Analysis of bipolar and schizophrenia candidate alleles

描述(由研究人员提供)：由于遗传连锁和关联研究，已经确定了几个精神分裂症(SZ)的候选基因，包括NRG1和DTNBP1等。然而，尽管对外显子、内含子-外显子边界和转录起始点上游的启动子区域进行了深入的分析，但特定的致病等位基因尚未被鉴定。一种可能性是这些候选基因的表达受到位于调控元件中的变异等位基因的影响，这些变异等位基因位于尚未确定的调控元件中，这些变异等位基因可以在远离转录起始点的几千个碱基处发挥作用，例如增强子和基因座控制区。该建议的主要目的是确定几个SZ候选基因的末端调控元件，并确定是否存在潜在的功能等位基因，这些等位基因可能是SZ易感性的候选基因。我们将通过实时定量PCR定位NRG1和DTNB1基因座上的DNase1高敏感位点，并使用pGL2-荧光素酶报告系统检测这些位点附近DNA中的启动子和增强子功能，从而筛选远端调控元件。不同的等位基因将在同一系统中进行测试，以确定启动子/增强子功能是否受损。还将使用电迁移凝胶位移分析(EMSA)测试它们的蛋白质结合亲和力，以确定DNA-蛋白质复合体中是否存在等位基因特异性差异。功能突变将在病例对照关联研究中进行分析，以确定SZ对照组和患者之间的等位基因和基因型分布是否不同。对于最有希望的变体，我们将通过使用针对最有可能的候选抗体的EMSA超移动来鉴定DNA结合蛋白，或者通过使用肝素琼脂糖层析和DNA琼脂糖层析使用DNA结合蛋白来纯化它们。通过识别与基因调控区结合的蛋白质，我们将对SZ的发病机制有新的认识，特别是在发育过程中可能在某些情况下发挥作用的基因-环境相互作用。正是通过基因表达的改变，环境应激源与大脑相互作用。

DESCRIPTION (provided by investigator): 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.