PO.TB09.03 · 肿瘤生物学
利用原钙黏蛋白基因簇甲基化条形码进行体内克隆谱系追踪
In vivo clonal lineage tracing using methylation barcodes in the protocadherin gene cluster
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
解析细胞的谱系历史对于理解组织发育、干细胞动态和癌症至关重要，但现有方法具有侵入性、分辨率低，或对人类研究而言成本过高，从而制约了这一目标的实现。单一干细胞谱系的克隆性扩增是实体组织肿瘤和血液系统恶性肿瘤癌前病变的共同标志。在此，我们描述了一种天然的、体内谱系追踪系统，它利用原钙黏蛋白（PCDH）基因簇中CpG位点的随机（去）甲基化。通过在批量甲基化测序中对这些CpG进行相位分析，我们构建了可原位演化的甲基化“条形码”，用以唯一性地标记干细胞克隆。我们对50名个体及年龄匹配对照的年度血液样本进行了纵向追踪，最长可追溯至急性髓系白血病（AML）确诊前十年。我们发现，多克隆组织具有高条形码多样性——反映出广泛的干细胞库——随后在克隆性清扫过程中该多样性坍缩。优势条形码的分数丰度提供了克隆大小的定量测量，能够精确追踪基于体细胞突变的遗传学估计。然而，10%的AML前期供者虽携带扩增的条形码，却缺乏可检测到的体细胞驱动突变，提示在确诊前数年即存在具有隐匿性驱动因素的克隆扩增。条形码轨迹还揭示了亚克隆之间的竞争与干扰。由于这些条形码以每个CpG每年约1%的速率通过随机（去）甲基化演化，克隆群体中会产生可遗传的多样性，从而能够追踪和量化亚克隆生长动态。随后我们证明，由长读长测序生成的、包含数十至数百个PCDH CpG位点的条形码，可从单一时间点实现深度系统发育重建。最后，我们在包括前列腺和肾脏在内的实体瘤组织中观察到了扩增的条形码。因此，PCDH甲基化条形码代表了一种可扩展、经济高效且泛组织适用的工具，可用于人体高分辨率谱系追踪，为研究健康与疾病中的体细胞演化开辟了新途径。
查看英文原文 English abstract
Resolving the lineage history of cells is crucial for understanding tissue development, stem cell dynamics, and cancer, but is hampered by methods that are invasive, low-resolution, or prohibitively expensive for human studies. Clonal expansion of a single stem cell lineage is a hallmark of pre-cancer in both solid tissue tumors and hematological malignancies. Here, we describe a native, in vivo lineage tracing system that utilizes the stochastic (de)methylation of CpGs in the protocadherin (PCDH) gene cluster. By phasing these CpGs in bulk methylation sequencing we construct in situ evolvable methylation “barcodes” that uniquely mark stem cell clones. We longitudinally tracked these barcodes in annual blood samples from 50 individuals and age-matched controls up to a decade prior to acute myeloid leukemia (AML) diagnosis. We show that polyclonal tissues have high barcode diversity-reflecting a broad stem-cell pool-which then collapses during a clonal sweep. The fractional abundance of dominant barcodes provides a quantitative measure of clone size that precisely tracks genetic estimates from somatic mutations. However, 10% of pre-AML donors harbored expanded barcodes but lacked detectable somatic driver mutations, suggesting the presence of clonal expansions with cryptic drivers years before diagnosis. Barcode trajectories also reveal subclonal competition and interference. Because these barcodes evolve by stochastic (de)methylation at approximately 1% per CpG per year, heritable diversity is generated in clonal populations enabling the tracking and quantification of subclonal growth dynamics. We then demonstrate that barcodes containing dozens to hundreds of PCDH CpGs produced from long-read sequencing allow for deep phylogenetic reconstruction from a single time point. Finally, we observed expanded barcodes in solid tumor tissues, including prostate and kidney. PCDH methylation barcodes therefore represent a scalable, cost-effective, and pan-tissue tool for high-resolution lineage tracing in humans, opening new avenues for studying somatic evolution in health and disease.
利益披露 Disclosure
C. T. Boniface, None..
S. Hackett, None..
A. V. A. Fonseca, None..
A. Ramos-Yamasaki, None..
C. Watson, None..
J. Baxter, None..
J. Nangalia, None..
S. Esener, None..
H. Mohammed, None..
J. Blundell, None.