Epigenetic studies in rhabdomyosarcoma

该项目的近期目标是研究融合阳性和融合阴性横纹肌肉瘤(RMS)之间DNA甲基化的差异。在2013财年，一名工作人员科学家开始分析这个DNA甲基化项目，直到该人于2012年11月离开实验室。从2013年3月到现在，该实验室的一名研究遗传学家继续进行了这些研究。不同的生物信息学策略被应用于现有的Infinium人类甲基化27珠芯片数据，以识别融合阳性和融合阴性RMS肿瘤之间的差异甲基化基因。用监督分析比较融合阴性和融合阳性肿瘤中每个基因的平均甲基化水平，然后确定两组肿瘤中平均甲基化水平显著不同的基因。发现融合阳性RMS高甲基化、融合阴性RMS低甲基化的基因，以及融合阳性RMS高甲基化、融合阴性RMS高甲基化的基因。由于每个甲基化测量都适用于一个CpG位点，当在同一基因中发现多个CpG的关系时，信心和兴趣增加。我们的下一个目标是通过确定融合阴性和阳性RMS肿瘤中较大的包含CpG的调节区的甲基化状态来验证选定基因的差异甲基化。我们选择焦磷酸测序是因为它能够定量评估选定的CpG区域内多个CpG位点的甲基化程度。特别是，这项技术测量了亚硫酸氢盐处理的DNA样本中CpG二核苷酸中多个胞嘧啶的C：T比率，从而确定了这些位点在CpG-包含区域内的甲基化状态。由于这一程序需要用不含CpG二核苷酸的侧翼引物扩增选定的CpG富集区(因此没有依赖于甲基化状态的可变序列)，我们使用了现有的PyroMark CpG分析(QIGEN)，或者使用了PyroMark分析设计软件2.0软件来设计定制的引物。基于这些不同的要求，我们选择了四个在融合阳性的RMS肿瘤中可能高甲基化的基因，以及四个在融合阴性肿瘤中可能高甲基化的基因。我们最初使用RMS肿瘤细胞系来研究这一方法学。我们的细胞系结果表明，融合阴性和融合阳性的RMS肿瘤之间的甲基化差异，如Infinium人类甲基化27珠芯片所确定的，在RMS细胞系中没有完全保持。接下来，我们将使用RMS肿瘤样本的DNA，特别是，我们将从27珠芯片上检测的样本DNA开始。在一组单独的研究中，我们分析了四个基因的表达，其中至少有一个CpG位点显示融合阳性和融合阴性RMS肿瘤的甲基化存在显著差异。对于一个基因，其中两个CpG位点中的一个在融合阳性肿瘤中显示出显著高于融合阴性肿瘤的甲基化，其在融合阴性肿瘤中的表达显著高于融合阴性肿瘤。对于融合阳性肿瘤中多个CpG位点的甲基化高于融合阴性肿瘤的两个基因，融合阳性肿瘤中的表达水平高得令人惊讶。最后，对于一个基因，其中两个CpG位点中的一个在融合阴性肿瘤中表现出比融合阳性肿瘤明显更高的甲基化，两个融合亚群之间的表达没有显著差异。

The immediate goal of this project is to investigate difference in DNA methylation between fusion-positive and fusion-negative rhabdomyosarcoma (RMS) tumors. During FY 2013, a staff scientist commenced analysis of this DNA methylation project until that individual left the laboratory in November 2012. A research geneticist in the laboratory then continued these studies from March 2013 to the present. Various bioinformatic strategies were applied to the existing Infinium Human Methylation 27 BeadChip data to identify genes that are differentially methylated between fusion-positive and fusion-negative RMS tumors. Supervised analysis was used to compare mean methylation levels for each gene in the fusion-negative and fusion-positive tumors, and then to determine those genes whose mean methylation levels were significantly different between the two groups of tumors. Genes were found for which there was both evidence of hypermethylation in fusion-positive RMS and hypomethylation in fusion-negative RMS, and other genes were found for which there was evidence of hypomethylation in fusion-positive RMS and hypermethylation in fusion-negative RMS. As each methylation measurement applies to a single CpG site, confidence and interest increased when the relationship was found for more than one CpG in the same gene. Our next goal was to validate the differential methylation of selected genes by determining the methylation status of the larger CpG-containing regulatory region in fusion-negative and positive RMS tumors. We selected pyrosequencing because of its ability to quantitatively assess the extent of methylation of multiple CpG sites within a selected CpG-containing region. In particular, this technology measures the C:T ratio at multiple cytosines within CpG dinucleotides in a bisulfite-treated DNA sample and thereby determines the methylation status of these sites within the CpG-containing region. As this procedure requires amplification of the selected CpG-rich regions with flanking primers that do not contain CpG dinucleotides (and thus do not have a variable sequence dependent on methylation status), we used existing PyroMark CpG Assays (Qiagen) when available or used the PyroMark Assay Design Software 2.0 software to design custom primers. Based on these various requirements, we selected four genes that are potentially hypermethylated in fusion-positive RMS tumors and four genes that are potentially hypermethylated in fusion-negative tumors. We initially used RMS tumor cell lines to work out this methodology. Our cell line results indicate that the methylation differences between fusion-negative and fusion-positive RMS tumors, as determined on the Infinium Human Methylation 27 BeadChip, are not fully maintained in RMS cell lines. We will next use DNA from RMS tumor samples, and in particular, we will start with DNA from the samples assayed on the 27 BeadChip. In a separate set of studies, we assayed expression of four genes for which there was at least one CpG site that showed a substantial difference in methylation between fusion-positive and fusion-negative RMS tumors. For one gene in which one of two CpG sites showed substantially higher methylation in fusion-positive than fusion negative tumors, the expression was significantly higher in the fusion-negative tumors. For two genes in which multiple CpG sites demonstrated higher methylation in fusion-positive than fusion-negative tumors, the expression was surprisingly higher in the fusion-positive tumors. Finally, for one gene in which one of two CpG sites showed substantially higher methylation in fusion-negative than fusion-positive tumors, there was no significant difference in expression between the two fusion subsets.