Molecular analysis of bipolar and schizophrenia candidate genes

描述（由申请人提供）：尽管使用全基因组关联研究（GWAS）在理解精神分裂症（SZ）和双相情感障碍（BD）的遗传基础方面取得了重大进展，但在患者中识别致病功能变异仍然是一个问题。一个例外是结构改变的发现，如患者特异性拷贝数变异（CNV），这已经在相当多的少数SZ和自闭症谱系障碍（ASD）患者中发现，特别是新发病例。在GWAS中鉴定的疾病相关SNP通常不是其自身的功能变体，而是可能与真正的致病功能突变处于连锁不平衡（LD）中。然而，识别这样的变体是困难的，特别是当相关的SNP被发现在非编码区-深入内含子内或远离最近的注释基因的基因间区域，这一直是GWAS中的一个共同主题。考虑到显著的LD仅维持约10-20 kb，一个合理的假设是许多致病突变发生在远离候选基因编码区的调控元件中，例如非编码RNA的远端增强子、抑制子和启动子。许多调节元件的特征是序列保守性。然而，单独的序列保守性不足以建立生物学功能，并且已经观察到人特异性增强子元件。哪些调控因素控制着与精神疾病相关的基因的调节？这些区域的遗传变异是否会导致疾病风险？在GWAS中确定的正相关信号是否是由于LD具有这些特征？我们在最初的资助期间成功实施的一个策略是使用基于染色质免疫沉淀的方法-ChIP芯片-作为筛选工具来识别SZ和BD候选基因中的新调控元件。基本的ChIP芯片策略涉及使用针对蛋白质的抗体（Ab）对染色质进行免疫沉淀，所述蛋白质例如共价修饰的组蛋白，所述组蛋白与DNA结合并且在调节结构域中富集。然后可以测定这些区域的生物活性，并且如果可以证明这种活性，则重新测序以鉴定与对照相比在患者中具有更高等位基因频率的罕见患者特异性变体或SNP。也可以对这种潜在的致病变体进行功能分析以确定生物活性。然而，ChIP芯片受到可以询问的基因数量和DNA量的限制。一种更全面的调控域筛选分析是ChIP-Seq，其主要优点是它提供了无偏见的全基因组覆盖。因此，ChIP-Seq有可能识别所有先前表征的候选基因中的调控元件，以及未来识别的那些，为研究人员提供可以有效筛选以识别患者特异性突变的生物活性位点。ChIP-Seq方法还将用于鉴定与SZ和BD病理生理学相关的转录因子在整个人类基因组中的结合位点。

DESCRIPTION (provided by applicant): Although there have been major advances in understanding the genetic basis of schizophrenia (SZ) and bipolar disorder (BD) using genome-wide association studies (GWAS), identifying disease causing functional variants in patients remains a problem. An exception is the finding of structural alterations, such as patient-specific copy number variants (CNVs), which have been found a sizeable minority of patients with SZ and autism spectrum disorders (ASD), especially do novo cases. Disease-associated SNPs identified in GWAS are usually not functional variants on their own, but are presumably in linkage disequilibrium (LD) with bona fide disease-causing functional mutations. However, identifying such variants is difficult, especially when associated SNPs are found in non-coding regions - deep within introns or in intergenic regions far removed from the nearest annotated genes, which has been a common theme in GWAS. A reasonable assumption, considering that significant LD is maintained for only ~10-20 kb, is that many disease-causing mutations are occurring in regulatory elements that are far removed from a candidate gene's coding region, such as distal enhancers, suppressors and promoters of non-coding RNAs. A feature of many regulatory elements is sequence conservation. However, sequence conservation alone is not sufficient to establish biological function and human-specific enhancer elements have been observed. What are the regulatory elements that control the regulation of genes involved in psychiatric disorders? Does genetic variation in these regions contribute to disease risk? Are the positive association signals identified in GWAS due to LD with these features? One strategy we successfully implemented in the initial grant period was to use a chromatin immunoprecipitation based method - ChIP-chip - as a screening tool to identify novel regulatory elements in SZ and BD candidate genes. The basic ChIP-chip strategy involves immunoprecipitation of chromatin using antibodies (Ab) to proteins, such as covalently modified histones, that bind to DNA and are enriched in regulatory domains. These regions can then be assayed for biological activity and, if such activity can be demonstrated, resequenced to identify rare patient-specific variants or SNPs that have higher allele frequencies in patients compared with controls. Such potential disease- causing variants can also be subjected to functional analysis to establish biological activity. However, ChIP-chip is limiting by the number of genes and amount of DNA that can be interrogated. A much more comprehensive analysis for regulatory domain screening is ChIP-Seq, the primary advantage of which is that it provides unbiased, genomewide coverage. Thus, ChIP-Seq has the potential to identify regulatory elements in all previously characterized candidate genes, as well as those identified in the future, providing researchers with biologically active sites that can be efficiently screened to identify patient-specific mutations. The ChIP-Seq approach will also be used to identify binding sites, throughout the entire human genome, of transcription factors relevant to SZ and BD pathophysiology.