Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Department of Conservation, Copenhagen Zoo, Frederiksberg, Denmark.
Epigenetics. 2023 Dec;18(1):2199374. doi: 10.1080/15592294.2023.2199374.
Profiling of circulating cell-free DNA (cfDNA) by tissue-specific base modifications, such as 5-methylcytosines (5mC), may enable the monitoring of ongoing pathophysiological processes. Nanopore sequencing allows genome-wide 5mC detection in cfDNA without bisulphite conversion. The aims of this study were: i) to find differentially methylated regions (DMRs) of cfDNA associated with obesity in Göttingen minipigs using Nanopore sequencing, ii) to validate a subset of the DMRs using methylation-specific PCR (MSP-PCR), and iii) to compare the cfDNA DMRs with those from whole blood genomic DNA (gDNA). Serum cfDNA and gDNA were obtained from 10 lean and 7 obese Göttingen Minipigs both with experimentally induced myocardial infarction and sequenced using Oxford Nanopore MinION. A total of 1,236 cfDNA DMRs (FDR<0.01) were associated with obesity. In silico analysis showed enrichment of the adipocytokine signalling, glucagon signalling, and cellular glucose homoeostasis pathways. A strong cfDNA DMR was discovered in , a gene linked to obesity and type 2 diabetes. The DMR was validated using MSP-PCR and correlated significantly with body weight ( < 0.05). No DMRs intersected between cfDNA and gDNA, suggesting that cfDNA originates from body-wide shedding of DNA. In conclusion, nanopore sequencing detected differential methylation in minute quantities (0.1-1 ng/µl) of cfDNA. Future work should focus on translation into human and comparing 5mC from somatic tissues to pinpoint the exact location of pathology.
通过组织特异性碱基修饰（如 5-甲基胞嘧啶（5mC））对循环无细胞 DNA（cfDNA）进行分析，可能能够监测正在进行的病理生理过程。纳米孔测序允许在 cfDNA 中不经过亚硫酸氢盐转化即可进行全基因组 5mC 检测。本研究的目的是：i）使用纳米孔测序在肥胖的哥廷根小型猪的 cfDNA 中找到与肥胖相关的差异甲基化区域（DMR），ii）使用甲基化特异性 PCR（MSP-PCR）验证 DMR 的一部分，iii）比较 cfDNA DMR 与来自全血基因组 DNA（gDNA）的 DMR。从 10 头瘦型和 7 头肥胖型哥廷根小型猪的血清 cfDNA 和 gDNA 中获得了通过实验诱导心肌梗死的小型猪，并用 Oxford Nanopore MinION 进行了测序。总共发现了 1236 个与肥胖相关的 cfDNA DMR（FDR<0.01）。在计算机分析中，发现了脂肪细胞因子信号、胰高血糖素信号和细胞葡萄糖稳态途径的富集。在基因中发现了一个与肥胖和 2 型糖尿病相关的强 cfDNA DMR。使用 MSP-PCR 验证了 DMR，并且与体重显著相关（ < 0.05）。cfDNA 和 gDNA 之间没有 DMR 相交，这表明 cfDNA 来源于全身 DNA 的脱落。总之，纳米孔测序检测到微量 cfDNA （0.1-1ng/µl）中的差异甲基化。未来的工作应该集中在转化为人类并比较体细胞组织中的 5mC，以精确定位病理的确切位置。
