Development of a quantitative methylation-specific polymerase chain reaction method for monitoring beta cell death in type 1 diabetes.
Development of a quantitative methylation-specific polymerase chain reaction method for monitoring beta cell death in type 1 diabetes.
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DOI：
10.1371/journal.pone.0047942
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Ferreri K
中科院分区：
文献类型：
--
作者：
Husseiny MI;Kuroda A;Kaye AN;Nair I;Kandeel F;Ferreri K
DNA甲基化是细胞控制基因表达的一种机制，细胞特异性基因通常表现出独特的DNA甲基化模式。我们以前报道过小鼠胰岛素-2基因（Ins 2）启动子有三个潜在的甲基化（CpG）位点，所有这些位点在胰岛素产生细胞中都未甲基化，但在其他组织中甲基化。在这项研究中，我们检查了Ins 2外显子2，发现了类似的组织特异性甲基化模式。这些甲基化模式可以区分来自产生胰岛素的β细胞和其他组织的DNA。我们假设受损的β细胞在1型糖尿病（T1 DM）发作时将其DNA释放到循环中，并试图开发一种定量甲基化特异性聚合酶链反应（qMSP）测定循环β细胞DNA以监测β细胞的损失。设计甲基化特异性引物以在同一测定中询问两个或更多个CpG。克隆的小鼠Ins 2基因在体外甲基化，并用于开发qMSP试验。我们发现qMSP方法是敏感和特异性的，以区分胰岛素产生细胞和其他组织，在非特异性基因组DNA背景的存在下，检测限为10个拷贝。我们还比较了不同的数据分析方法，发现相对表达比方法是最稳健的方法，因为它既包含了标准化日常变异性的参考值，又包含了标准化甲基化特异性和亚硫酸氢盐特异性计算组分之间的PCR反应效率。将该测定应用于链脲佐菌素治疗的糖尿病小鼠模型，并在血糖水平升高之前检测到循环β细胞DNA的显著增加。这些结果表明，该qMSP检测可用于监测胰岛素产生细胞的循环DNA，这将为开发检测早期T1 DM中β细胞破坏的检测方法提供基础。
DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (Ins2) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined Ins2 exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse Ins2 gene was methylated in vitro and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM.
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实时甲基化特异性PCR（SMART-MSP）之后的敏感熔解分析：高通量和无探针定量DNA甲基化检测。
影响因子：
14.9
作者：
Kristensen, Lasse S.;Mikeska, Thomas;Krypuy, Michael;Dobrovic, Alexander
通讯作者：
Dobrovic, Alexander
DOI：
10.1073/pnas.87.4.1576
发表时间：
1990-02-01
影响因子：
11.1
作者：
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通讯作者：
COHEN, IR
DOI：
10.1073/pnas.0508621102
发表时间：
2005-12-20
影响因子：
11.1
作者：
Pinkse, GGM;Tysma, OHM;Roep, BO
通讯作者：
Roep, BO
影响因子：
16.2
作者：
Knip M;Korhonen S;Kulmala P;Veijola R;Reunanen A;Raitakari OT;Viikari J;Akerblom HK
通讯作者：
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影响因子：
2.5
作者：
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通讯作者：
Yamagishi, H