DNA Methylation Is Associated with Altered Gene Expression in AMD
DNA Methylation Is Associated with Altered Gene Expression in AMD
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DOI：
10.1167/iovs.11-8449
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发表时间：
2012-04-01
影响因子：
4.4
通讯作者：
Dunaief, Joshua L.
中科院分区：
文献类型：
--
作者：
Hunter, Allan;Spechler, Paul A.;Dunaief, Joshua L.
目的。年龄相关性黄斑变性（AMD）是老年人失明的主要原因。有证据表明氧化应激在该疾病中发挥作用。为了评估与 AMD 发病机制相关的抗氧化基因表观遗传调控的潜在贡献，我们评估了 DNA 甲基化，这是一种影响基因表达的组织特异性遗传调控。方法。使用 Infinium HumanMmethylation27 Illumina 平台，我们进行了 DNA 亚硫酸氢盐测序，以比较 AMD 患者和年龄匹配的对照者死后视网膜色素上皮 (RPE)/脉络膜的甲基化状态。使用 Affymetrix Exon Array 评估基因表达。 TaqMan 基因表达测定用于表达阵列结果的相对定量（RT-PCR）确认。谷胱甘肽 S-转移酶异构体 mu1 (GSTM1) 和 mu5 (GSTM5) 启动子甲基化通过 CpG 岛亚硫酸氢盐焦磷酸测序得到证实。为了评估蛋白质水平和定位，我们对小鼠和人类样本使用了蛋白质印迹分析、免疫组织化学和免疫荧光。结果。与年龄匹配的对照组相比，AMD 中 RPE/脉络膜和神经感觉视网膜 (NSR) 的 GSTM1 和 GSTM5 mRNA 水平显着降低，这与 GSTM1 启动子的高甲基化相对应。 AMD 死后样本中 mRNA 和蛋白质水平均下降（RPE 下降程度高于 NSR），与年龄无关。免疫组织化学和免疫荧光证实 NSR 和 RPE 中存在酶。结论。 DNA 甲基化和 mRNA 水平的比较揭示了 AMD 与正常视网膜之间的显着差异。所提供的证据表明，GSTM1 和 GSTM5 在 AMD RPE/脉络膜中受到表观遗传抑制，这可能会增加 AMD 视网膜对氧化应激的敏感性。 （投资眼科可见科学。2012 年；53：2089-2105）DOI：10.1167/iovs.11-8449
PURPOSE. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Evidence suggests oxidative stress plays a role in the disease. To assess the potential contribution of epigenetic regulation of antioxidant genes relevant to AMD pathogenesis, we evaluated DNA methylation, a tissue-specific genetic modulation that affects gene expression.METHODS. Using the Infinium HumanMethylation27 Illumina platform, we performed DNA bisulfite sequencing to compare the methylation status in postmortem retina pigment epithelium (RPE)/choroid between patients with AMD and age-matched controls. Gene expression was assessed with the Affymetrix Exon Array. TaqMan gene expression assays were used for relative quantification (RT-PCR) confirmation of the expression array results. Glutathione S-transferase isoform mu1 (GSTM1) and mu5 (GSTM5) promoter methylation was confirmed by CpG island bisulfite pyrosequencing. To assess protein levels and localization, we used Western analysis, immunohistochemistry, and immunofluorescence with murine and human samples.RESULTS. The mRNA levels of GSTM1 and GSTM5 were significantly reduced in AMD versus age-matched controls in RPE/choroid and neurosensory retina (NSR), which corresponded to hypermethylation of the GSTM1 promoter. mRNA and protein levels were decreased (RPE to a greater extent than NSR) in AMD postmortem samples, irrespective of age. Immunohistochemistry and immunofluorescence confirm the presence of the enzymes in the NSR and RPE.CONCLUSIONS. Comparison of DNA methylation, together with mRNA levels, revealed significant differences between AMD versus normal retinas. The evidence presented suggests that GSTM1 and GSTM5 undergo epigenetic repression in AMD RPE/choroid, which may increase susceptibility to oxidative stress in AMD retinas. (Invest Ophthalmol Vis Sci. 2012;53:2089-2105) DOI:10.1167/iovs.11-8449