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Circulating unmethylated CHTOP and INS DNA fragments provide evidence of possible islet cell death in youth with obesity and diabetes.
Clinical Epigenetics ( IF 5.7 ) Pub Date : 2020-07-31 , DOI: 10.1186/s13148-020-00906-5
Farooq Syed 1, 2 , Sarah A Tersey 3 , Jean-Valery Turatsinze 4 , Jamie L Felton 1, 2 , Nicole Jiyun Kang 1, 2 , Jennifer B Nelson 3 , Emily K Sims 1, 2 , Mathieu Defrance 5 , Martin Bizet 5 , Francois Fuks 5 , Miriam Cnop 4, 6 , Marco Bugliani 7 , Piero Marchetti 7 , Anette-Gabriele Ziegler 8 , Ezio Bonifacio 9 , Bobbie-Jo Webb-Robertson 10 , Appakalai N Balamurugan 11, 12 , Carmella Evans-Molina 1, 2, 13, 14 , Decio L Eizirik 4, 15 , Kieren J Mather 1, 13 , Silva Arslanian 16 , Raghavendra G Mirmira 3
Affiliation  

Identification of islet β cell death prior to the onset of type 1 diabetes (T1D) or type 2 diabetes (T2D) might allow for interventions to protect β cells and reduce diabetes risk. Circulating unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of β cell death, but this gene alone may not be sufficiently specific to report β cell death. To identify new candidate genes whose CpG sites may show greater specificity for β cells, we performed unbiased DNA methylation analysis using the Infinium HumanMethylation 450 array on 64 human islet preparations and 27 non-islet human tissues. For verification of array results, bisulfite DNA sequencing of human β cells and 11 non-β cell tissues was performed on 5 of the top 10 CpG sites that were found to be differentially methylated. We identified the CHTOP gene as a candidate whose CpGs show a greater frequency of unmethylation in human islets. A digital PCR strategy was used to determine the methylation pattern of CHTOP and INS CpG sites in primary human tissues. Although both INS and CHTOP contained unmethylated CpG sites in non-islet tissues, they occurred in a non-overlapping pattern. Based on Naïve Bayes classifier analysis, the two genes together report 100% specificity for islet damage. Digital PCR was then performed on cell-free DNA from serum from human subjects. Compared to healthy controls (N = 10), differentially methylated CHTOP and INS levels were higher in youth with new onset T1D (N = 43) and, unexpectedly, in healthy autoantibody-negative youth who have first-degree relatives with T1D (N = 23). When tested in lean (N = 32) and obese (N = 118) youth, increased levels of unmethylated INS and CHTOP were observed in obese individuals. Our data suggest that concurrent measurement of circulating unmethylated INS and CHTOP has the potential to detect islet death in youth at risk for both T1D and T2D. Our data also support the use of multiple parameters to increase the confidence of detecting islet damage in individuals at risk for developing diabetes.

中文翻译:

循环未甲基化的 CHTOP 和 INS DNA 片段提供了肥胖和糖尿病青年可能发生胰岛细胞死亡的证据。

在 1 型糖尿病 (T1D) 或 2 型糖尿病 (T2D) 发作之前识别胰岛 β 细胞死亡可能允许采取干预措施来保护 β 细胞并降低糖尿病风险。源自人类 INS 基因的循环未甲基化 DNA 片段已被提议作为 β 细胞死亡的生物标志物,但仅此基因可能不足以报告 β 细胞死亡。为了识别其 CpG 位点可能对 β 细胞表现出更高特异性的新候选基因,我们使用 Infinium HumanMethylation 450 阵列对 64 个人类胰岛制剂和 27 个非胰岛人类组织进行了无偏倚的 DNA 甲基化分析。为了验证芯片结果,对人类 β 细胞和 11 种非 β 细胞组织进行了亚硫酸氢盐 DNA 测序,对发现差异甲基化的前 10 个 CpG 位点中的 5 个进行了测序。我们将 CHTOP 基因鉴定为候选基因,其 CpG 在人类胰岛中显示出更高频率的非甲基化。使用数字 PCR 策略来确定原代人体组织中 CHTOP 和 INS CpG 位点的甲基化模式。尽管 INS 和 CHTOP 在非胰岛组织中都含有未甲基化的 CpG 位点,但它们以非重叠模式出现。基于朴素贝叶斯分类器分析,这两个基因共同报告了 100% 的胰岛损伤特异性。然后对来自人类受试者血清的无细胞 DNA 进行数字 PCR。与健康对照组 (N = 10) 相比,新发 T1D 青年 (N = 43) 的差异甲基化 CHTOP 和 INS 水平更高,而且出乎意料的是,在具有一级亲属患有 T1D 的健康自身抗体阴性青年 (N = 23). 在瘦 (N = 32) 和肥胖 (N = 118) 青年中进行测试时,在肥胖个体中观察到未甲基化 INS 和 CHTOP 水平升高。我们的数据表明,同时测量循环中未甲基化的 INS 和 CHTOP 有可能检测出处于 T1D 和 T2D 风险的青年人的胰岛死亡。我们的数据还支持使用多个参数来增加检测有患糖尿病风险的个体胰岛损伤的置信度。
更新日期:2020-07-31
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