Weisenberger Daniel J, Campan Mihaela, Long Tiffany I, Kim Myungjin, Woods Christian, Fiala Emerich, Ehrlich Melanie, Laird Peter W
Department of Surgery, Keck School of Medicine, USC/Norris Comprehensive Cancer Center, University of Southern California Los Angeles, CA, USA.
Nucleic Acids Res. 2005 Dec 2;33(21):6823-36. doi: 10.1093/nar/gki987. Print 2005.
Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satalpha) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.
重复元件占人类基因组的很大一部分，并且包含在正常人类出生后体细胞组织中发现的大部分CpG甲基化。这些序列中DNA甲基化的缺失可能是大多数已研究的人类癌症所具有的全基因组低甲基化的主要原因。人们广泛关注将基因组5-甲基胞嘧啶含量与临床结果、饮食史、生活方式等相关联。然而，一种高通量、准确且易于获取的技术，甚至可应用于石蜡包埋组织DNA的技术尚未出现。在此，我们报告了定量MethyLight分析方法的开发，用于确定甲基化和未甲基化重复序列的水平，即Alu和LINE-1序列以及着丝粒卫星α（Satalpha）和近着丝粒卫星2（Sat2）DNA序列。通过高效液相色谱法测量，Alu、Sat2和LINE-1重复序列的甲基化水平与全基因组DNA甲基化显著相关，并且Alu和Sat2甲基化的联合测量与全基因组DNA甲基化测量高度相关。这些MethyLight分析仅依赖于实时PCR，并为全基因组DNA甲基化分析提供替代标志物。我们还描述了一种基于Alu序列的新型设计策略，用于开发不依赖甲基化的MethyLight对照反应，该Alu序列在一条链上通过进化脱氨去除了CpG二核苷酸。我们表明，一种这样的基于Alu的反应在MethyLight应用中能大大改善DNA检测的标准化，并且不易受到癌症相关非整倍性和拷贝数变化引起的标准化误差的影响。
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