DNA methylation in the development of pediatric leukemias

描述（由申请人提供）： 项目总结/摘要：众所周知，遗传畸变（如染色体易位）与继发性畸变（可能的表观遗传）互补，后者导致白血病克隆。然而，互补的表观遗传事件及其在儿童白血病常见亚型发展中的作用还没有得到很好的表征。我们的目标是了解儿童白血病全基因组表观遗传改变的范围，然后开发表观遗传筛查标记物来测试存档的新生儿血点（古特里卡），以早期检测白血病。我们已经成功地使用GoldenGate甲基化癌症组I微阵列（Illumina，Inc），使用白血病骨髓和儿童白血病患者的相应存档古特里卡进行了初步实验，以测试一大组癌症相关基因在白血病发展中的贡献.我们还用白血病细胞系进行了实验，以测试对去甲基化剂的基因表达反应。该技术涉及表观遗传修饰的药理学抑制，再加上基因表达微阵列（Affytek外显子1.0），并产生了一些候选基因，基因启动子甲基化可能在白血病中发挥功能性作用。我们目前的重点是在原发性白血病样本中使用验证分析集中于一组初步候选基因，然后进一步集中于我们选择的靶点的古特里卡。在目前的应用中，我们将使用甲基化特异性PCR和亚硫酸氢盐测序来减小大小，并集中我们目前的90个基因靶标的大面板。我们将选择在TEL-AML 1白血病的最高部分中甲基化的30个，并且其在正常CD 34细胞和正常古特里卡片DNA中不甲基化。然后，我们将在古特里卡中测试这组表观遗传标记，这些卡来自长大后患TEL-AML 1白血病的儿童，并与500名未患白血病的儿童进行比较，以评估预测价值。我们专门关注最常见的细胞遗传学亚型，即占所有ALL 22%的TEL-AML 1易位患者。本研究的最终目的是表征白血病发展中的早期表观遗传突变和印迹异常，并使用这些标记物进行儿童白血病的早期检测。 公共卫生相关性： 项目叙述：通过这个项目，我们正在探索出生时白血病的早期标志物的存在。这将通过增加我们对儿童白血病病因学机制的认识，并提供出生时早期发现白血病或易患白血病的标志物来影响公共卫生。

DESCRIPTION (provided by applicant): PROJECT SUMMARY / ABSTRACT: It is well recognized that genetic aberrations, such as chromosomal translocations, are complemented by secondary aberrations (possible epigenetic) that give rise to leukemic clones. However, complementary epigenetic event(s) and their roles in development of common subtypes of childhood leukemias are not well characterized. Our goals are to understand the scope of genome-wide epigenetic alterations in childhood leukemia, and then develop epigenetic screening markers to test archived neonatal blood spots (Guthrie cards) for early detection of leukemia. We have successfully performed preliminary experiments using GoldenGate Methylation Cancer Panel I microarrays (Illumina, Inc), using leukemic bone marrows, and corresponding archived Guthrie cards of childhood leukemias patients, to test the contribution of a large panel of cancer- related genes in the development of leukemia. We have also performed experiments with leukemia cell lines to test the gene expression response to a demethylating agent. This technique involved pharmacologic inhibition of epigenetic modifications, coupled with gene expression microarrays (Affymetrix Exon 1.0), and has yielded a number of candidate genes for which gene promoter methylation may play a functional role in leukemia. Our current focus is to focus on a panel of preliminary candidate genes using validation assays in primary leukemia samples, and then focusing further in Guthrie cards for our chosen targets. In the current application we will use methylation-specific PCR and bisulfite sequencing to reduce the size, and to focus our current large panel of 90 gene targets. We will choose the 30 which are methylated in the highest fraction of TEL-AML1 leukemia and which are not methylated in normal CD34 cells and normal Guthrie card DNA. We will then test this panel of epigenetic markers in Guthrie cards from children who grew up to get TEL-AML1 leuekmia and compare to a group of 500 children who did not, to assess predictive value. We focus exclusively on the most common cytogenetic subltype, those with TEL-AML1 translocations who are 22% of all ALL. This research aims ultimately at the characterization of early epigenetic mutations and imprinting abnormalities in the development of leukemia, and the use these markers for the early detection of pediatric leukemia. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE: With this project we are exploring the existence of early markers of leukemia at birth. This will impact public health by increasing our knowledge in the mechanisms of the etiology of childhood leukemia and providing markers for the early detection of, or predisposition to, leukemia at birth.