Analysis of bipolar and schizophrenia candidate alleles

描述（由研究者提供）：作为遗传连锁和关联研究的结果，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，尽管对转录起始位点上游的外显子、内含子-外显子边界和启动子区域进行了深入分析，但尚未确定特定的致病等位基因。一种可能性是，这些候选基因的表达受到位于尚未确定的调控元件中的变异等位基因的影响，这些等位基因可以在转录起始位点（如增强子和基因座控制区）以外的数千个碱基处发挥作用。本研究的主要目的是确定几个SZ候选基因的远端调控元件，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。我们将通过实时荧光定量PCR绘制NRG1和DTNB1位点上的DNAse 1超敏感位点来筛选远端调控元件，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。变异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。他们还将使用电迁移凝胶位移法（EMSA）测试蛋白质结合亲和力，以确定dna -蛋白质复合物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，以确定对照组和SZ患者之间的等位基因和基因型分布是否存在差异。对于最有希望的变体，我们将使用抗体对最有可能的候选物进行EMSA超移鉴定DNA结合蛋白，或者使用肝素Sepharose和DNA Sepharose色谱法使用DNA结合蛋白纯化它们。通过鉴定与基因调控区域结合的蛋白，我们将对SZ的发病机制有新的认识，特别是在某些情况下，发育过程中基因与环境的相互作用可能起作用。环境压力源通过基因表达的改变与大脑相互作用。

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.