肾综合征出血热患者血浆cf-DNA特征及其深度测序分析

探讨肾综合征出血热(HFRS)患者血浆cf-DNA含量与不同病情、病期等指标的相关性,定性分析游离DNA(cf-DNA)的来源与特征。收集了47例HFRS不同病情患者不同病程的128份血浆标本,以及20例健康志愿者的20份血浆标本。定量分析血浆总cf-DNA水平,提取纯化cf-DNA后进行定性分析,对其中9份HFRS患者血浆标本进行DNA高通量测序和生物信息学分析。统计学分析各参数指标间相关性。健康人血浆cf-DNA水平中位数为919.0 ng/m L,HFRS患者血浆cf-DNA含量明显增高,随着病程进展呈现动态变化的规律。轻型/中型患者与重型/危重型患者有着明显不同的分布格局:发热期cf-DNA水平即明显高于健康对照,在低血压休克期达到峰值,然后逐步下降,至多尿期与健康对照无显著性差异;急性期重型/危重型患者cf-DNA平均水平显著高于同期轻型/中型患者水平(p0.001)。健康人cf-DNA多为高分子量大片段DNA,而患者血浆cf-DNA高度富集于150~200 bp区域,显示为凋亡片段;血浆cf-DNA片段读长(reads)均匀分布于人类基因组的各条染色体,分布频度与染色体长度高度正相关(p0.001),且与HTNV病毒基因组无同源性。HFRS患者,急性期血浆cf-DNA即明显升高,并且随病程进展的不同分期与病情分型,呈现出动态变化的规律。HFRS患者血浆cf-DNA主要来源于疾病状态下,机体细胞发生凋亡后产生的基因组和线粒体DNA断裂片段,不包含HTNV病毒基因组成分。

Characteristics and Deep Sequencing Analysis of Plasma Cell-Free DNA from HFRS Patients

Abstract] Objective To explore the correlation between plasma cf-DNA in HFRS patients and disease severity as well as plasma HTNV load, analyze sources and characteristics of the cf-DNA, and provide evidences for observing the mechanism of HFRS. Methods The 128 plasma samples from 47 patients in different stages of HFRS and 20 plasma samples from 20 cases of healthy volunteers were collected. We quantitatively detected the total plasma cf-DNA and analyzed the depurated cf-DNA qualitatively. The samples of cf-DNA were detected using high-throughput sequencing and studied by bioinformatics. Results In HFRS patients, the plasma cf-DNA levels peaked at the hypotensive stage, and then decreased gradually. Exclusively in the febrile/hypotensive stage, the plasma cf-DNA levels of severe/critical patients were higher than those of the mild/moderate group. Plasma cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA. After sequencing, all the cf-DNA reads can mapped onto human genomic DNA. Conclusion The plasma cf-DNA levels were dynamically elevated during HFRS, and correlated with disease stages and severity, which suggests that plasma cf-DNA may be a potential biomarker for the pathogenesis and prognosis of HFRS.