Dept of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Hadassah Medical Center and Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel.
Equal contributors.
Eur Respir J. 2022 Nov 3;60(5). doi: 10.1183/13993003.03056-2021. Print 2022 Nov.
Circulating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death.
We sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease.
Loci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients.
Universal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.
目前缺乏用于肺部损伤的循环生物标志物。肺上皮细胞特异性 DNA 甲基化模式可潜在地报告血液中存在源自肺的无细胞 DNA（cfDNA），这是肺细胞死亡的一个指征。
我们从手术标本中分拣人肺肺泡和支气管上皮细胞，并使用全基因组亚硫酸氢盐测序获得它们的甲基组。我们开发了一种 PCR 测序检测方法，用于确定具有肺特异性甲基化模式的 17 个位点的甲基化状态，并将其用于评估健康志愿者和肺部疾病患者血浆中的肺源性 cfDNA。
在肺泡或支气管上皮细胞中唯一非甲基化的位点富含控制肺特异性基因的增强子。从这些甲基组中提取的甲基化标志物表明，正常的肺细胞更新可能会将 cfDNA 释放到肺泡腔中，而不是进入血液。晚期肺癌患者的血液中肺 cfDNA 浓度显著升高。在接受支气管镜检查的个体中，在后来被诊断患有肺癌的患者的血浆中观察到肺源性 cfDNA，而在被诊断患有其他肺部疾病的患者中则较少见。与稳定期患者相比，COPD 急性加重患者的肺 cfDNA 水平升高，并且与这些患者未来的恶化和死亡相关。
正常肺上皮的通用 cfDNA 甲基化标志物可实现对肺源性 cfDNA 的非突变依赖、敏感和特异性检测，从而报告持续的肺损伤。这些标志物可广泛应用于正常和病理性人类肺部动态的研究。
