Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.
Clin Chem. 2021 Mar 31;67(4):621-630. doi: 10.1093/clinchem/hvaa325.
Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends.
Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing.
The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83.
Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.
已知血浆中的双链 DNA 带有单链末端，称为锯齿状末端。血浆 DNA 锯齿状末端是妊娠和癌症等病理生理状态的生物标志物。尚不清楚尿液无细胞游离 DNA（cfDNA）分子是否具有锯齿状末端。
通过在 DNA 末端修复过程中掺入未甲基化的胞嘧啶来检测 cfDNA 的锯齿状末端，然后进行亚硫酸氢盐测序。在修复锯齿状末端时掺入未甲基化的胞嘧啶会降低亚硫酸氢盐测序测量的表观甲基化水平，并用于计算锯齿状末端指数。这种方法称为测序的锯齿状末端分析。
尿 cfDNA 的锯齿状末端指数高于血浆 DNA。血浆 DNA 的锯齿状末端指数谱显示出几个强烈振荡的主要峰值，间隔约 165 bp（即核小体大小），以及周期性约 10 bp 的较弱振荡的次要峰值。相比之下，尿液 DNA 的锯齿状末端指数谱显示出较弱振荡的主要峰值和强烈振荡的次要峰值。锯齿状末端指数通常在核小体连接 DNA 区域较高。膀胱癌患者（n=46）的尿液 DNA 锯齿状末端指数低于无膀胱癌患者（n=39）。区分膀胱癌患者和无膀胱癌患者的曲线下面积为 0.83。
锯齿状末端代表尿液 cfDNA 的一种特性。锯齿状末端的产生可能与核小体结构有关，在连接 DNA 区域富集。尿液 DNA 的锯齿状末端可能有潜力成为膀胱癌检测的新生物标志物。
