A tool for analysis of gene-specific DNA methylation in clinical samples

描述（由申请人提供）：在许多情况下，早期发现疾病可以改善结果；因此，寻找分子生物标志物成为生物医学研究的一个重要方向。理想情况下，疾病特异性生物标志物应该是敏感的、特异性的、廉价的、通用的、快速的、独立于观察者的，以便对无症状人群进行客观筛查。此外，拥有一个统一的平台来开发多种疾病的生物标志物将是有利的。DNA甲基化的变化可在不同疾病中发现，并可在血液中循环的无细胞DNA中检测到；然而，每个特定位点只有一定的甲基化概率。为了解决这一问题并提高检测的准确性，可以使用具有多个信息元素的复合生物标志物。虽然大量的甲基化位点表明每个生物标志物存在多个潜在成分，但信息成分的选择是困难的，并且需要一个平台来同时测量每个临床样本的多个位点的甲基化。本项目将开发这样一个工具（目标1），并通过选择乳腺癌生物标志物的信息元素来测试其性能（目标2）。所提出的工具包括用于在已知在不同疾病中甲基化的基因启动子中进行甲基化检测的定制阵列；从少量血浆中分离无细胞DNA的方法；以及一种检测DNA甲基化的技术。该方法用56个基因原型进行了测试；对不同疾病的检测准确率为70-90%。所提出的阵列将包含大多数异常甲基化基因，可以通过该技术进行分析，以选择额外的高信息量基因并提高准确性。为了确认该工具的实用性，将开发用于检测浸润性乳腺癌和导管原位癌的生物标志物。因此，女性最常见的癌症之一的生物标志物成分也将被识别出来。一旦找到信息元素的问题得到解决，只有它们将在专门的乳腺癌筛查试验中进行测量。该工具很可能会在不同的临床领域（如肿瘤学、精神病学、产前诊断和药理学）以及生物学的其他领域（如胚胎学和干细胞研究，其中甲基化涉及印迹和胚胎编程）的生物标志物研究中找到应用。

DESCRIPTION (provided by applicant): In many cases, early detection of disease improves outcomes; as a result, search for molecular biomarkers became an important direction in biomedical research. Ideally, disease-specific biomarkers should be sensitive, specific, inexpensive, versatile, rapid, and observer-independent to allow objective screening of asymptomatic people. In addition, it would be advantageous to have a uniform platform for development of biomarkers for multiple diseases. Changes in DNA methylation are found in different diseases and can be detected in circulating cell-free DNA from blood; however, there is only a certain probability of methylation for each specific site. To solve this problem and increase the accuracy of detection, a composite biomarker with multiple informative elements can be used. While the multitude of methylation sites indicates that multiple potential components exit for each biomarker, selection of informative components is difficult and requires a platform for simultaneous measurements of methylation in multiple sites of each clinical sample. Such a tool will be developed in this project (Aim 1) and its performance will be tested by selecting informative elements for a breast cancer biomarker (Aim 2). The proposed tool consists of a customized array for methylation detection in promoters of genes that are known to be methylated in different diseases; a procedure for isolating cell-free DNA from small amounts of plasma; and a technique for methylation detection in this DNA. The approach was tested with a 56 gene prototype; accuracy of detection was 70-90% for different diseases. The proposed array will contain the majority of abnormally methylated genes that can be analyzed by this technique in order to select additional highly informative genes and increase the accuracy. To confirm the utility of the tool, biomarkers for detection of invasive breast cancer and ductal carcinoma in situ will be developed. As a result, biomarker components for one of the most frequent cancers in females will be identified as well. Once the problem of finding informative elements is solved, only they will be measured in a dedicated screening assay for breast cancer. It is likely that the tool will find application for biomarker research in different clinical areas - e.g. in oncology, psychiatry, prenatal diagnostics, and pharmacology - and in other areas of biology, e.g. in embryology and in stem cell research, where methylation is involved in imprinting and embryonic programming. Public Health Relevance Statement: In this project a platform for testing DNA methylation in multiple sites will be developed and validated with DNA from blood of healthy women and breast cancer patients. The developed platform will allow selection of blood-based biomarkers for different diseases, which will translate into a rapid, inexpensive, and very accurate test for their detection.