肿瘤甲基化

Tumors are often characterized by an imbalance in cytosine methylation as manifested both by hypermethylation of CpG islands and by genome hypomethylation.[1]

1. CpG岛高甲基化 与 基因组低甲基化并无矛盾

CpG is shorthand for 5'—C—phosphate—G—3'
也就是我们常说的序列中的CG

CpG位点

CpG 岛可是说是连续密集出现的CG
Though objective definitions for CpG islands are limited, the usual formal definition is a region with at least 200 [bp], a GC percentage greater than 50%, and an observed-to-expected CpG ratio greater than 60%.
他们通常出现在基因启动子的位置，C5甲基化抑制基因的表达(m5C)
In humans, about 70% of promoters located near the transcription start site of a gene (proximal promoters) contain a CpG island.

DNA甲基化

黑色部分位甲基化，白色为非甲基化。可见甲基化沉默转座子
There was no relationship between the level of global hypomethylation and hypermethylation.Hypermethylation and hypomethylation contribute separately to the process of carcinogenesis[1].

甲基化检测方法
WGBS（Whole Genome Bisulfite Sequence）全基因组甲基化测序目前是金标准，利用重亚硫酸氢盐使DNA中未发生甲基化或者羟甲基化的胞嘧啶(C)脱第四位氨基转变成尿嘧啶(U)，而甲基化的胞嘧啶保持不变，然后通过PCR将U变为T，仅有甲基化的C可以成功保留，最后通过测序就可判断CpG位点是否发生甲基化。

概览

亚硫酸盐处理流程

PCR扩增

最后进行芯片杂交
Illumina的450K芯片采用两种策略：Infinium I和Infinium Ⅱ，前者有两种bead（微珠），分别是甲基化M和非甲基化U，后者则是一种bead（不区分甲基化和非甲基化）。
❶ 如下图：Infinium I，在未甲基化的GpC locus，U型bead尾部为A，与未甲基化CpG位点相匹配，能够成功进行单核苷酸延伸并被检测到（U型磁珠发光），而M型bead尾部为G，与未甲基化位点不能匹配，没有信号产生；在甲基化的GpC locus，M型bead能与甲基化CpG位点相匹配，单核苷酸延伸并产生信号（M型磁珠发光），而U型bead则不匹配，不产生信号。
❷ Infinium Ⅱ探针则不区分M和U，探针尾部为C，配对后只加入单个碱基（ddNTP-BioT， ddNTP-DNP），然后根据荧光颜色判断加入碱基的类型，进而确定该位点是否被甲基化
❸ 最后，通过计算甲基化和非甲基化位点的荧光信号比例，可确定某位点的甲基化水平（Beta值=M/(M+UM)）

Infinium

值得注意的是“肿瘤中全局甲基化降低，启动子区甲基化增高”也并非定律[7]肾透明细胞癌中就存在高甲基化，尤其是基因体部分。TET-mediated 5mC oxidation might be responsible for the 5hmC loss in kidney cancer. TET-mediated 5mC oxidation requires cosubstrates, such as 2-ketoglutarate (2-KG), which is mainly generated by IDHs during the tricarboxylic acid cycle.
相应的转座子高表达也并非是所有肿瘤中共有的特征
stomach, bladder (Supplementary Fig. 2f), liver, and head and neck tumors show predominantly overexpression of TEs, whereas thyroid, breast, kidney chromophobe, and lung adenocarcinoma tumors show predominantly reduced TE expression compared with normal (Fig. 2a).[8]

a.各种转座子高表达的癌症全局（尤其是转座元件附近）CpG位点的甲基化水平 b.甲基化水平与TEs表达水平有一致性

differentially methylated CpGs (DMCs)
对转座子表达上调的癌症类型进行后续分析
we examined in 10 TCGA cancer types DNA methylation changes from normal to tumor at both the genome-wide (global) and TE-proximal level using TCGA Illumina 450 K array data.
并且后续分析还发现：转座子区的去甲基化程度比全局更大。
这一切都印证了肿瘤中甲基化表观变化（主）调控转座子表达。

但是在转座子表达下调的癌症类型中，转座子与甲基化水平无明显相关性
We observed predominantly reduced levels of TE expression in tumor compared with normal tissue in a subset of cancer types (e.g., breast and lung adenocarcinoma). We examined DNA methylation status at six recurrently downregulated TEs but found no clear association between methylation and TE expression.
这些数据表明，肿瘤中许多TE的过表达与DNA甲基化的丧失有关，尤其是在TE近端CpG位点，这表明TE表达的主要机制可能是针对TE附近DNA甲基化变化。但其在下调类型中相关性不强，表明他表观修饰也起作用，例如H3F3A突变导致的基因间区域H3K27ac增加，H3K27me3减少[8.1]。

胶质瘤课题相关

异柠檬酸脱氢酶1突变型（IDH1mut）胶质瘤的侵袭性比IDH1野生型（IDH1wt）胶质瘤低，而全基因组甲基化水平高。IDH-1是一种参与细胞代谢的胞质NADP+依赖性酶，胶质瘤内的大多数突变是IDH-1突变。IDH-1突变后细胞内生成肿瘤代谢产物D2-HG，D2-HG对细胞内多种酶具有抑制效应，其中最关键的是DNA脱甲基酶（TET）。DNA去甲基酶的抑制结果是DNA甲基化增加，包括CpGTET岛的甲基化（G-CIMP表型）。CpG岛甲基化通过阻断CTCF绝缘子蛋白的结合位点而干扰DNA的三级结构的形成。CTCF结合受干扰，则破坏染色质环的形成，DNA物理分离遭受破坏，并与不同的基因聚集在一起。这一新发现的肿瘤发生机制，阐明了神经胶质瘤发病过程中IDH-1突变所起的影响，同时表明其与较好的预后和较大的放化疗敏感性相关。IDH-1突变的肿瘤具有同源重组缺陷，表现为细胞代谢及烷化剂类化疗加重DNA损伤。[9]
因此全局低甲基化事件存在就可以猜测重复序列在GBM中应当是高表达的
(IDH) mutational status in gliomas. IDH-mutant gliomas manifest the cytosine-phosphate-guanine (CpG) island methylator phenotype (G-CIMP).这种表型仅仅指岛甲基化高，整体还是甲基化降低的，因此在分析重复序列表达水平的时候没有必要单独列出这个表型。[10]

G-CIMP

CIMP subtypes in human cancer. This illustration depicts aberrant DNA methylation changes at specific genomic loci in normal and tumor cells, especially in CIMP tumors. Each DNA strand represents one individual methylome. Methylated CpG sites in normal state are represented in blue, non-CIMP tumor DNA methylation gain in yellow, and aberrant DNA hypermethylation in CIMP tumors in red (modified from Weisenberger).
在癌症中，DNA甲基化变得异常，其主要特征是基因和基因体启动子周围的局灶性高甲基化以及非启动子元件之间的全局低甲基化.[10]

[1] Bariol C, Suter C M, Cheong K, et al. The relationship between hypomethylation and CpG island methylation in colorectal neoplasia[J]. American Journal of Pathology, 2003, 162(4): 1361-1371.
[2] https://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/appnote-methylseq-wgbs.pdf
[3] 诺禾
[4] 生信技能树论坛-组学大全版块-表观组
[5] 甲基化芯片 陈巍学基因
[6] https://mp.weixin.qq.com/s?src=11×tamp=1589022589&ver=2328&signature=3JaYIARxyUXqcn2KpQ6GrG02jxI0zEEDVhYiD8StERPhqVnIcafotEBIkjrRJ-TuWzJ6A7Q2-ioQqACjziyYX3Kh1JlMraoHFpsL6uUYDI0GNhK4dXBbYXCiQzWqUHWF&new=1
[7] Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer
[8] Transposable element expression in tumors is associated with immune infiltration and increased antigenicity
[8.1] Pervasive H3K27 Acetylation Leads to ERV Expression and a Therapeutic Vulnerability in H3K27M Gliomas
[9] Cadieux B, Ching T T, Vandenberg S R, et al. Genome-wide Hypomethylation in Human Glioblastomas Associated with Specific Copy Number Alteration, Methylenetetrahydrofolate Reductase Allele Status, and Increased Proliferation[J]. Cancer Research, 2006, 66(17): 8469-8476.
[10] Malta T M, Souza C, Sabedot T S, et al. Glioma CpG island methylator phenotype (G-CIMP): biological and clinical implications[J]. Neuro-oncology, 2018, 20(5): 608-620.