Chromatin Immunoprecipitation (ChIP) Assay Development for FFPE human brain secti

描述(由申请人提供)：抑郁症是15-44岁之间的个人残疾的主要原因，影响到大约2100万美国人，也是自杀的主要原因。目前可供医疗专业人员使用的诊断工具仅限于问卷和其他固有的主观方法。提高诊断的准确性被认为是该领域的迫切需要。最近的进展揭示了精神病理疾病的遗传基础，这不仅包括基因表达的变化，也包括表观基因组的变化。表观组学机制在不改变DNA密码的情况下调节基因的活性，主要包括胞嘧啶环上第五位CpG-二核苷酸的DNA甲基化，以及通过组蛋白的翻译后修饰来调节染色质结构。在这个第一阶段的提案中，我们打算开发一种临床染色质免疫沉淀分析(C-CHIP)，用于甲醛固定的石蜡包埋(FFPE)人脑样本，这将使分析正常和自杀脑样本中组蛋白翻译后修饰的变化成为可能。染色质免疫沉淀是一种捕获DNA结合蛋白的强大技术，并能够相对于输入定量特定的免疫沉淀DNA序列。C-CHIP分析的开发将通过首先建立一个大鼠组织模型系统来实现，并结合分离一组与转录活跃或抑制基因座相关的组蛋白修饰的高度特征化和特异性的单抗，将用于系统地调整为体外培养细胞开发的现有芯片方案，使其成为与FFPE切片兼容的高度敏感的分析方法。临床样本的检测验证将在大鼠母体护理模型上合作进行，随后将在FFPE对照和自杀脑样本上进行。C-芯片检测的成功开发将产生两个影响。首先是将C-芯片检测商业化，使其面向广阔的研究市场，首次提供一种能够对存档的临床样本进行功能性基因组分析的检测方法。这一第一阶段应用的第二个结果是，C-芯片检测将使随后的第二阶段研究成为可能，在这一阶段，对大量自杀大脑样本的基因组调查将检查表观遗传变化，这些变化可能作为抑郁症的生物标记。有了这些信息，就有可能随后确定与大脑标记相关的外围标记，从而开发出一种用于评估抑郁和自杀风险的诊断方法。 公共卫生相关性：1-3自杀和抑郁症是主要的公共卫生问题。最近的研究已经确定了一些与自杀和抑郁有关的分子机制，这些7-15机制包括大脑中基因表达调控的变化。这一阶段的应用描述了一种测试的开发，该测试将能够识别整个基因组中的基因表达变化，这可能随后导致开发出急需的抑郁症和自杀风险诊断方法。

DESCRIPTION (provided by applicant): Depression is the leading cause of disability among individuals between the ages of 15-44, affecting approximately 21 million Americans and is the leading cause of suicide. Current diagnostic tools available for medical professionals are limited to questionnaires and other inherently subjective approaches. Improving accuracy of diagnosis has been cited as a pressing need in the field. Recent advances have shed light on the genetic basis for psychopathological conditions, which not only includes changes in gene expression, but also epigenomic changes as well. Epigenomic mechanisms, which regulate gene activity without altering the DNA code, consist fundamentally of DNA methylation of CpG-dinucleotides, which occurs at the fifth position of the cytosine pyrimidine ring, and regulation of chromatin structure through post-translational modification of histones. In this Phase I proposal , we intend to develop a Clinical Chromatin ImmunoPrecipitation assay (C- ChIP), for use with formaldehyde-fixed paraffin embedded (FFPE) human brain specimens, which will enable analysis of changes in histone post-translational modifications in normal and suicide brains samples. Chromatin immunoprecipitation is a powerful technique that captures DNA bound proteins, and enables quantification of the specific immunoprecipitated DNA sequences relative to input. Development of the C- ChIP assay will be achieved by first establishing a rat tissue model system, and in conjunction with the isolation of a panel of highly characterized and specific monoclonal antibodies to histone modifications associated with either transcriptionally active or repressed loci, will be used to systematically adapt existing ChIP protocols developed for in vitro cultured cells, into a highly sensitive assay compatible for use with FFPE sections. Assay validation with clinical samples will be performed collaboratively on a rat maternal care model and subsequently on FFPE control and suicide brain samples. The successful development of the C-ChIP assay will have two effects. First, would be the commercialization of the C-ChIP assay for the broad research market, providing for the first time, an assay which enables the functional genomic analysis of archived clinical samples. The second outcome of this Phase I application is that the C-ChIP assay will enable subsequent Phase II studies in which genomic-wide survey of a large cohort of suicide brains specimens will be examined for epigenetic alternations which could possibly serve as biomarkers for depression. With this information, it may be possible to subsequently identify peripheral markers that correlate with the brain markers, leading to the development of a diagnostic assay for assessing depression and suicide risk. PUBLIC HEALTH RELEVANCE: 1-3 Suicide and depression are major public health concerns . Recent studies have identified some of the molecular mechanisms involved in suicide and depression and these 7-15 mechanisms include changes in the regulation of gene expression in the brain . This Phase 1 application describes the development of an assay that will enable identification of gene expression changes through the whole genome, which could subsequently lead to the development of a much needed diagnostic for depression and suicide risk.