Detection of Islet β-Cell Death in Vivo by Multiplex PCR Analysis of Differentially Methylated DNA
Detection of Islet β-Cell Death in Vivo by Multiplex PCR Analysis of Differentially Methylated DNA
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DOI：
10.1210/en.2013-1223
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发表时间：
2013-09-01
期刊：
影响因子：
4.8
通讯作者：
Tersey, Sarah A.
中科院分区：
文献类型：
--
作者：
Fisher, Marisa M.;Chumbiauca, Cristina N. Perez;Tersey, Sarah A.
1型糖尿病早期β细胞死亡的非侵入性检测可能会识别出治疗干预措施可以保护β细胞质量并预防高血糖的个体。最近在小鼠中的研究表明，β细胞死亡导致血清中未甲基化的前胰岛素原（PPI）DNA相应增加。在这里，我们报告了一种新的检测方法，使用双荧光探针多重PCR（TaqMan）检测循环PPI DNA的差异甲基化。主要检测功能包括低背景信号、大范围数值的线性检测输出以及在单个反应中同时检测甲基化和非甲基化PPI DNA。我们将“非甲基化指数”定义为反映未甲基化与甲基化PPI DNA相对量的汇总参数。为了验证该测定法在体内检测β细胞死亡的能力，我们测量了糖尿病小鼠模型血清中的非甲基化指数，包括高剂量和多次低剂量链脲霉素诱导的糖尿病，以及1型糖尿病的非肥胖糖尿病小鼠模型。我们的数据显示，显著增加的非甲基化指数与已知的高血糖症发作前β细胞死亡的时间轴一致。随后，我们观察到糖尿病发展后非甲基化指数下降，可能反映了胰腺中没有进一步的β细胞死亡。我们的结论是，同时测量甲基化和未甲基化的PPI DNA使用这里描述的多重PCR方法是一个容易获得的和敏感的指标垂死的β细胞，可能是有用的跟踪糖尿病的进展和治疗干预的反应。
Noninvasive detection of early beta-cell death in type 1 diabetes might identify individuals in whom therapeutic interventions would preserve beta-cell mass and prevent hyperglycemia. Recent studies in mice have shown that beta-cell death produces a corresponding increase in unmethylated preproinsulin (PPI) DNA in serum. Here, we report the development of a novel assay using dual fluorescent-probe multiplex PCR (TaqMan) to detect differential methylation of circulating PPI DNA. Key assay features include low background signals, linear assay output across a large range of values, and simultaneous detection of methylated and unmethylated PPI DNA in a single reaction. We defined the "unmethylation index" as a summary parameter that reflects the relative amounts of unmethylated vs methylated PPI DNA. To validate this assay's ability to detect beta-cell death in vivo, we measured the unmethylation index in the serum of diabetic mouse models, including high-and multiple low-dose streptozotocin-induced diabetes, and the nonobese diabetic mouse model of type 1 diabetes. Our data show a significantly increased unmethylation index concordant with the known timeline of beta-cell death that precedes the onset of hyperglycemia. Subsequently, we observed a decrease in the unmethylation index following diabetes development, likely reflecting the absence of further beta-cell death in the pancreas. We conclude that simultaneous measurement of methylated and unmethylated PPI DNA using the multiplex PCR method described here is a readily available and sensitive indicator of dying beta-cells that may be useful to track diabetes progression and response to therapeutic intervention.