Punia Sohan, Juran Brian D, Ali Ahmad H, Schlicht Erik M, Moore Raymond M, Sun Zhifu, Lazaridis Konstantinos N
Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA.
Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, 55905, USA.
BMC Gastroenterol. 2021 Apr 1;21(1):149. doi: 10.1186/s12876-021-01741-5.
Quantification of circulating organ-specific cell-free DNA (cfDNA) provides a sensitive measure of ongoing cell death that could benefit evaluation of the cholestatic liver diseases primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), which lack reliable non-invasive biomarkers. Our goal in this pilot study was to determine whether liver-specific cfDNA levels are increased in PBC and PSC patients relative to controls and in advanced versus early disease, to evaluate their potential as novel disease biomarkers.
Peripheral blood derived bisulfite-treated DNA was PCR amplified from patients with PBC (n = 48), PSC (n = 48) and controls (n = 96) to evaluate methylation status at 16 CpG sites reported to be specifically unmethylated in liver tissue near the genes IGF2R, ITIH4 and VTN. Amplicons were used to prepare paired end libraries which were sequenced on a MiSeq sequencer. Trimmed reads were aligned and used to determine unmethylation ratios and to calculate concentration of liver-specific cfDNA. Comparisons between groups were performed using the two-tailed Mann-Whitney Test and relationships between variables were evaluated using Pearson's Correlation.
Levels of liver-specific cfDNA, as measured at the 3 genetic loci, were increased in PBC and PSC patients relative to controls and in late-stage relative to early-stage patients. As well, cfDNA levels were correlated with levels of alkaline phosphatase, a commonly used biochemical test to evaluate disease severity in liver disease, in patients, but not in controls.
cfDNA offers promise as a non-invasive liquid-biopsy to evaluate liver-specific cell-death in patients with cholestatic liver diseases.
循环器官特异性游离DNA（cfDNA）的定量分析提供了一种敏感的方法来衡量正在发生的细胞死亡，这可能有助于对缺乏可靠非侵入性生物标志物的胆汁淤积性肝病——原发性胆汁性胆管炎（PBC）和原发性硬化性胆管炎（PSC）进行评估。我们这项初步研究的目的是确定PBC和PSC患者相对于对照组以及疾病晚期与早期患者的肝脏特异性cfDNA水平是否升高，以评估其作为新型疾病生物标志物的潜力。
从PBC患者（n = 48）、PSC患者（n = 48）和对照组（n = 96）中提取经亚硫酸氢盐处理的外周血DNA，进行PCR扩增，以评估在IGF2R、ITIH4和VTN基因附近的肝脏组织中报道的16个CpG位点的甲基化状态。扩增子用于制备双端文库，并在MiSeq测序仪上进行测序。修剪后的读段进行比对，用于确定未甲基化比率并计算肝脏特异性cfDNA的浓度。使用双尾曼-惠特尼检验进行组间比较，并使用皮尔逊相关性评估变量之间的关系。
在3个基因位点测量的肝脏特异性cfDNA水平，PBC和PSC患者相对于对照组升高，晚期患者相对于早期患者升高。此外，患者的cfDNA水平与碱性磷酸酶水平相关，碱性磷酸酶是评估肝病疾病严重程度常用的生化检测指标，但对照组中无此相关性。
cfDNA有望作为一种非侵入性液体活检方法，用于评估胆汁淤积性肝病患者肝脏特异性细胞死亡情况。
