Bisulphite-seq to investigate the proto CpG island methylator phenotype of sessile serrated adenoma/polyps, dataset 2

BACKGROUND & AIM: Sessile serrated adenoma/polyps (SSA/Ps) are the likely culprit of ~20% colon cancers but they are molecularly poorly understood. We investigated their epigenetic phenotype using high-throughput analysis of DNA methylation and gene expression. METHODS: 17 SSA/Ps and, as a comparative group, 15 conventional adenomas (all with matched samples of normal mucosa) were prospectively collected during colonoscopy. DNA from the 64 tissues was analyzed, via bisulfite next generation sequencing, for methylation at ~2.7 million CpG sites located prevalently in gene regulatory regions spanning 80.5Mb (~2.5% of the genome). The transcriptome of these samples was also investigated using RNA sequencing. An independent series of 61 archival lesions was used for targeted verification of DNA methylation. RESULTS: Both SSA/Ps and conventional adenomas showed a profound remodelling of their methylome. Cytosine hypermethylation was more pervasive in SSA/Ps than adenomas, in terms of number of hypermethylated regions (22,147 vs 15,965, respectively) and genes (4938 vs 3443, respectively). In addition, the extent of hypermethylation in a given region was higher in SSA/Ps than adenomas. This methylation pattern of SSA/Ps was reminiscent of the CpG island methylator phenotype (CIMP) of their descendant cancers. We have called this phenotype proto-CIMP since most of the hypermethylation occurred in CpG islands and shores. SSA/Ps were also protected from a wave of demethylation that instead occurred in adenomas outside of CpG islands/shores (4288 vs 18,417 hypomethylated regions in SSAPs vs adenomas). Verification studies of six hypermethylated regions demonstrated the high potential of DNA methylation markers for predicting the diagnosis of SSA/Ps and/or adenomas. Proto-CIMP of SSA/Ps was surprisingly associated with a higher number of up- (618) than downregulated (349) genes, while adenomas showed the opposite trend (516 up- vs 712 downregulated genes). CONCLUSIONS: The epigenetic landscape of SSA/Ps differs substantially from that of conventional adenomas. This huge amount of epigenetic variation represents a rich source of promising diagnostic tools, such as novel DNA markers or histologic stainings, for the tailored management of the two most frequent colon cancer precursors.

背景与研究目的：无蒂锯齿状腺瘤/息肉（Sessile serrated adenoma/polyps，SSA/Ps）是约20%结肠癌的潜在致病元凶，但目前学界对其分子层面的特征仍知之甚少。本研究通过DNA甲基化与基因表达的高通量分析，探究了SSA/Ps的表观遗传表型。
研究方法：本研究在结肠镜检查中前瞻性采集了17例SSA/Ps样本，以及作为对照队列的15例常规腺瘤样本，所有样本均配有配对的正常黏膜组织。研究人员对64份组织样本的DNA进行亚硫酸氢盐高通量测序（bisulfite next generation sequencing）分析，检测了广泛分布于80.5Mb（约占基因组的2.5%）基因调控区域内的约270万个CpG位点的甲基化水平；同时通过RNA测序分析了所有样本的转录组特征。另外纳入61例存档病灶作为独立队列，用于DNA甲基化的靶向验证。
研究结果：SSA/Ps与常规腺瘤均呈现出甲基化组的显著重塑。从高甲基化区域数量（分别为22147个与15965个）及高甲基化基因数量（分别为4938个与3443个）来看，SSA/Ps的胞嘧啶高甲基化程度较常规腺瘤更为广泛；此外，单个区域内的高甲基化程度在SSA/Ps中也高于常规腺瘤。SSA/Ps的这种甲基化模式与其衍生癌症的CpG岛甲基化表型（CpG island methylator phenotype，CIMP）极为相似，由于该表型的大部分高甲基化事件均发生在CpG岛及其侧翼岸区域，我们将其命名为前CpG岛甲基化表型（proto-CIMP）。同时，SSA/Ps未出现常规腺瘤中出现的全基因组范围去甲基化浪潮——常规腺瘤在CpG岛及侧翼岸区域外存在18417个低甲基化区域，而SSA/Ps仅存在4288个。针对6个高甲基化区域的验证研究表明，DNA甲基化标志物在预测SSA/Ps或常规腺瘤的诊断方面具有极高应用潜力。令人意外的是，SSA/Ps的前CpG岛甲基化表型与上调基因数量（618个）多于下调基因数量（349个）的特征相关，而常规腺瘤则呈现相反趋势（上调基因516个，下调基因712个）。
研究结论：SSA/Ps的表观遗传调控网络与常规腺瘤存在显著差异。这种丰富的表观遗传变异为开发新型DNA标志物或组织化学染色等诊断工具提供了充足的资源，可用于两种最常见结肠癌前病变的个体化诊疗。