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

抑郁症是15-44岁人群残疾的主要原因，影响了大约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调查和其他固有的主观方法。提高诊断的准确性已被认为是该领域的迫切需要。最近的进展揭示了精神病理学条件的遗传基础，不仅包括基因表达的变化，还包括表观基因组的变化。表观基因组机制，其调节基因活性而不改变DNA密码，基本上包括CpG-二核苷酸的DNA甲基化，其发生在胞嘧啶嘧啶环的第五位，以及通过组蛋白的翻译后修饰调节染色质结构。在该I期提案中，我们打算开发一种临床染色质免疫沉淀分析（C-ChIP），用于甲醛固定石蜡包埋（FFPE）人脑标本，这将能够分析正常和自杀脑样本中组蛋白翻译后修饰的变化。染色质免疫沉淀是捕获DNA结合蛋白的强大技术，并且能够相对于输入定量特异性免疫沉淀的DNA序列。C-ChIP测定的开发将通过首先建立大鼠组织模型系统来实现，并结合一组高度表征的和特异性的单克隆抗体的分离，以针对与转录活性或抑制基因座相关的组蛋白修饰，将用于系统地调整为体外培养细胞开发的现有ChIP方案，与FFPE切片兼容的高灵敏度测定。将在大鼠孕产妇护理模型上以及随后在FFPE对照和自杀脑样本上协作进行临床样本的试验验证。C-ChIP检测的成功开发将产生两个影响。首先，将C-ChIP测定商业化用于广泛的研究市场，首次提供能够对存档的临床样品进行功能基因组分析的测定。第一阶段申请的第二个结果是，C-ChIP检测将使后续的第二阶段研究成为可能，在该研究中，将对大量自杀脑样本的全基因组调查进行检查，以确定可能作为抑郁症生物标志物的表观遗传改变。有了这些信息，就有可能随后确定与大脑标记物相关的外周标记物，从而开发出一种用于评估抑郁症和自杀风险的诊断测定法。 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涉及自杀和抑郁症的分子机制，这些7-15机制包括大脑中基因表达调节的变化。该第一阶段申请描述了一种检测方法的开发，该方法将能够通过整个基因组识别基因表达变化，这可能随后导致开发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.