Epigenetic studies in rhabdomyosarcoma

该项目的直接目标是研究融合阳性和融合阴性横纹肌肉瘤（RMS）肿瘤之间DNA甲基化的差异。在准备该分析时，编辑Infinium HumanMethylation 27 BeadChip数据库，以删除定位于X和Y染色体或重复区域的探针、含有多态性位点的探针、具有模糊基因组位置的探针或没有可检测信号的探针。将无监督分析算法应用于该编辑的数据库证明了两个不同的聚类，一个包含融合阳性RMS病例，另一个包含融合阴性RMS病例。这种差异甲基化的进一步特征在于与融合阴性肿瘤相比，融合阳性肿瘤中低甲基化位点的发生率增加。在两种RMS亚型之间差异甲基化的探针中，融合阳性亚组中低甲基化位点比高甲基化位点多3倍。这种差异甲基化在定位于启动子区域和/或CpG岛的探针中特别突出。由于最初的分析确定甲基化状态足以对RMS肿瘤的融合状态进行分类，我们接下来研究了该分类所需的探针数量。找到最小数目的区分性CpG位点的训练算法确定11个探针的甲基化状态足以将融合阳性RMS肿瘤与融合阴性RMS肿瘤分类。在这组11个探针中，与融合阴性病例相比，融合阳性病例中有7个低甲基化，4个高甲基化。8个相关基因的焦磷酸测序研究独立地证实了在阵列上查询的CpG位点以及其他附近的CpG位点是差异甲基化的。为了验证这些发现，我们评估了10个RMS细胞系（5个融合阳性和5个融合阴性）的甲基化状态。无监督分析显示，这些细胞系根据融合状态聚类，并且11个探针特征足以正确分类这10个细胞系中的融合状态。在另一组分析中，我们研究了这些RMS肿瘤中甲基化和基因表达之间的关系。在具有甲基化（HumanMethylation 27）和表达（U133 Plus 2.0）数据的RMS病例中，mRNA表达和启动子甲基化总体呈负相关。然而，在145个同时存在差异启动子甲基化和差异表达的基因中，57%的基因甲基化与表达呈负相关，而43%的基因甲基化与表达呈正相关。为了确定融合转录因子（PAX 3-FOX 01和PAX 7-FOXO 1）是否影响这些问题，我们分析了PAX 3-FOXO 1结合位点在这些差异甲基化基因中的分布。尽管在具有和不具有差异甲基化的基因之间PAX 3-FOXO 1结合位点的分布没有显著差异，但PAX 3-FOXO 1结合位点在也差异表达的差异甲基化基因中富集。

The immediate goal of this project was to investigate differences in DNA methylation between fusion-positive and fusion-negative rhabdomyosarcoma (RMS) tumors. In preparation for this analysis, the Infinium HumanMethylation27 BeadChip database was edited to remove probes that localized to the X and Y chromosomes or repetitive regions, probes containing polymorphic sites, probes with ambiguous genomic locations or probes without detectable signal. Application of unsupervised analysis algorithms to this edited database demonstrated two distinct clusters, one containing the fusion-positive RMS cases and one containing the fusion-negative RMS cases. This differential methylation was further characterized by an increased incidence of hypomethylated sites in the fusion-positive compared to the fusion-negative tumors. Among probes that are differentially methylated between the two RMS subtypes, there were 3-fold more hypomethylated than hypermethylated sites in the fusion-positive subset. This differential methylation was particularly prominent in probes mapping to promoter regions and/or CpG islands. Since the initial analysis determined that methylation status was sufficient to classify the fusion status of an RMS tumor, we next investigated the number of probes that were needed for this classification. A training algorithm to find the minimum number of discriminating CpG sites determined that the methylation status of 11 probes is sufficient to classify the fusion-positive from the fusion-negative RMS tumors. In this group of 11 probes, seven were hypomethylated and four were hypermethylated in the fusion-positive compared to the fusion-negative cases. Pyrosequencing studies of eight of the associated genes independently confirmed that the CpG site queried on the array as well as other nearby CpG sites were differential methylated. To validate these findings, we assessed the methylation status in a collection of ten RMS cell lines (five fusion-positive and five fusion-negative). Unsupervised analysis revealed that these cell lines clustered according to fusion status and that the eleven probe signature was sufficient to correctly classify the fusion status in these 10 lines. In another set of analyses, we investigated the relationship between methylation and gene expression in these RMS tumors. In RMS cases with both methylation (HumanMethylation27) and expression (U133 Plus 2.0) data, there was an overall inverse relationship of mRNA expression and promoter methylation. However, among the 145 genes with both differential promoter methylation and differential expression, 57% showed an inverse correlation between methylation and expression whereas 43% showed a direct correlation. To determine whether the fusion transcription factors (PAX3-FOX01 and PAX7-FOXO1) impact on these issues, we analyzed the distribution of PAX3-FOXO1 binding sites among these differentially methylated genes. Though there was no significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation, PAX3-FOXO1 binding sites are enriched among differentially methylated genes that are also differentially expressed.