Anwar Sumadi Lukman, Lehmann Ulrich
Department of Surgery, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.
Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany.
Methods Mol Biol. 2018;1726:35-47. doi: 10.1007/978-1-4939-7565-5_5.
The retinoblastoma protein (pRb) plays a central role in the regulation of cell cycle by interaction with members of the E2F transcription factor family. As a tumor suppressor protein, pRb is frequently dysregulated in several major cancers. In addition to mutations, inactivation of pRb is also caused by epigenetic mechanisms including alterations of DNA methylation. There are three CpG islands located within the RB1 gene that encodes pRb that are closely associated with the regulation of pRb expression. Aberrant DNA methylation at the RB1 gene has been reported in sporadic retinoblastoma as well as other cancers including glioblastoma, hepatocellular carcinoma, and breast cancer. Recent studies have revealed that the RB1 gene is imprinted. Therefore, quantitative analysis is required to detect aberrations in DNA methylation associated with imprint deregulation. Pyrosequencing is considered as the method of choice for quantitative and reproducible analysis of DNA methylation with single base resolution. In this chapter, we provide a detailed protocol for the quantitative analysis of RB1 gene methylation using bisulfite Pyrosequencing.
视网膜母细胞瘤蛋白（pRb）通过与E2F转录因子家族成员相互作用，在细胞周期调控中发挥核心作用。作为一种肿瘤抑制蛋白，pRb在几种主要癌症中经常发生失调。除了突变外，pRb的失活还由包括DNA甲基化改变在内的表观遗传机制引起。在编码pRb的RB1基因内有三个与pRb表达调控密切相关的CpG岛。散发性视网膜母细胞瘤以及包括胶质母细胞瘤、肝细胞癌和乳腺癌在内的其他癌症中均已报道RB1基因存在异常DNA甲基化。最近的研究表明RB1基因是印记基因。因此，需要进行定量分析以检测与印记失调相关的DNA甲基化异常。焦磷酸测序被认为是用于单碱基分辨率的DNA甲基化定量和可重复分析的首选方法。在本章中，我们提供了使用亚硫酸氢盐焦磷酸测序对RB1基因甲基化进行定量分析的详细方案。
