中国科学院昆明动物研究所机构知识库

自然界存在一类癌细胞，它能打破宿主个体之间异源限制，逃避个体免疫系统识别，在不同个体间感染、传播，以一种“寄生”的方式完成“无限增殖”。目前确定有4种传染性肿瘤，其中犬类生殖器传染性肿瘤，简称为CTVT，是存在时间最长的肿瘤。研究CTVT的遗传机制，包括其产生的原因、进化机制，有助于了解肿瘤或癌症进化，为CTVT治疗提供帮助。通过解析CTVT体细胞突变频谱推测其可能的成因，检测CTVT受正选择的基因，寻找候选driver gene，有助于了解其进化机制。 CTVT是世界上最古老的肿瘤。其核基因组和线粒体DNA（mtDNA）有着截然不同的来源和遗传方式。其中CTVT核基因组起源于一只距今约11,000年前的古老雪橇犬，至今已存在几千年。通过捕获宿主mtDNA占为己有，CTVT mtDNA有5次独立起源，随后分化形成多个群体。 本研究首次检测了野生动物和家养动物——灰狼和家犬germline突变频谱差异。结果发现差异不仅在亚种水平存在，也在亚群体水平存在。本研究讨论了不同群体germline SNPs对CTVT somatic突变的判断无显著影响。本研究首次检测了CTVT核基因组受正选择基因。 自然界中癌症普遍存在。除裸鼹鼠外，几乎所有的哺乳类、鸟类动物都得癌症。一般情况下癌症随患病个体死亡而凋亡，但自然界存在一类传染性肿瘤可以通过活细胞感染不同个体，在群体甚至物种间传播。至今已知4种传染性肿瘤，其中犬类传染性生殖器肿瘤（Canine Transmissible Venereal Tumor, CTVT）是已知的最古老的肿瘤，也是存在时间最久的动物细胞系。单基因、微卫星、全基因组分析结果证明CTVT是单一起源，现今所有CTVT起源于一只距今约11,000年前的古老雪橇犬，并在过去几百年内快速扩散到全球。而CTVT线粒体DNA（mtDNA）有着截然不同的来源和遗传方式。CTVT捕获所寄生的宿主家犬的mtDNA，mtDNA系统发育研究显示CTVT mtDNA分为5个类群，经历了至少5次mtDNA捕获事件。肿瘤的形成不是几个基因，而是整个基因组积累变异的结果。重测序技术的发展为从全基因组水平解析肿瘤发生提供机会。体细胞突变频谱被广泛应用于解析肿瘤形成的原因。肿瘤形成是一个细胞演化成一群细胞的过程，选择压力作用驱动具有更好生存优势的细胞快速增殖，形成亚克隆、最终形成肿瘤。由于CTVT已存在超过1万年，因此CTVT是研究长时间尺度下肿瘤进化的绝佳模型。本研究在昆明采集了2例CTVT肿瘤及其宿主家犬血液样品，并分别对其做基因组重测序。本研究分析了CTVT体细胞突变频谱以及检测了正选择压力，初步探讨了CTVT形成原因以及进化机制。本研究主要分为以下三个部分内容。第一，由于CTVT体细胞突变和生殖细胞突变的判断基于当代犬科动物生殖细胞多态性位点（SNPs），而前人发现当代人群生殖细胞突变存在差异，因此我们首先检测了CTVT宿主群体，突变频谱是否存在差异。结果发现CTVT宿主灰狼和家犬群体间多个突变的频率存在差异，而且差异在家犬亚群——南方家犬和北方家犬群体中也存在。这是首次发现野生动物和家养动物群体之间体细胞突变频谱存在差异，提示驯化可以导致群体间突变频率发生改变。第二，我们比较了分别以201个现代灰狼家犬个体组成的大数据集和31个个体组成的小数据集为背景，解析CTVT体细胞突变频谱。结果发现不同大小的灰狼家犬群体SNPs数据集对CTVT体细胞突变频谱解析结果无显著影响。突变频谱解析出一类与紫外线诱导的突变标记，提示紫外线照射可能与CTVT形成相关或是CTVT长期积累突变的结果。第三，前人认为CTVT mtDNA受负选择。我们对447个CTVT mtDNA分析发现不同群体经历不同选择压力，其中一个亚群CTVT_1A经历强烈正选择。对CTVT核基因组检测正选择，结果发现细胞凋亡相关基因受正选择，这些基因可能是CTVT形成的驱动基因。而细胞粘连相关基因积累大量无义突变，说明CTVT可能通过减少细胞间粘连，增加细胞迁移能力帮助其跨个体传播。本研究首次发现野生群体和家养群体的群体突变频谱存在差异，提示驯化可以造成群体突变频谱改变。群体间突变频率差异应被考虑在今后的群体遗传学研究中。本研究探讨了CTVT mtDNA和核基因组选择压力，发现正选择作用在二者进化过程中起了重要作用。本研究初步探讨了CTVT的成因与选择压力，为研究传染性肿瘤遗传机制打下基础。

Cancer is prevalent in nature. Many types of cancer have been described in almost all mammals and birds, except in naked mole-rat. Generally, cancer dies along with the death of diseased individual. However, there exist a kind of special tumor called transmissible tumor that breaks isolation of individuals and then affects individuals via living tumor cells, resulting in transmission among populations and species as a parasite. One of four, the Canine Transmissible Venereal Tumor (CTVT) is the oldest known tumor and animal cell line. Researches of single gene, microsatellite and whole-genome indicate all the modern CTVTs originate from an ancient sled dog that lived about 11,000 years ago and spread around the world in last several millennia, suggesting CTVT nuclear genomes are from a single origin. While the origin and genetic mechanism of CTVT mitochondrial DNA (mtDNA) differ from that’s of CTVT nuclear genome. Phylogeny results indicate that CTVT mtDNAs diverged into several sub-populations within the past two thousand years, indicating that CTVT mtDNA has undergone at least five independent mtDNA captures from the host dogs. Tumor is a product of variants accumulation of normal cells in whole genome but no single gene level. Re-sequencing provides an opportunity to better understand tumorigenesis in whole genome level. Mutational spectrum is widely applied in tumor tumorigenesis researches to decipher the causes of tumor formation. Tumorigenesis is also an evolution process. Cells with growth advantages are selected and develop to subclones and result in tumor ultimately. Since CTVT has survived for more than 10,000 years, it makes CTVT to be an excellent model to research on tumor evolution during a long time.In this study, we performed whole genome sequencing on two CTVT tumors and their donor dog blood samples collected in Kunming. We discussed the cause and evolution mechanism of CTVT by deciphering CTVT mutational spectrum and positive selection testing. Since detection of somatic mutation in CTVT depends on modern canine germline SNPs and difference was found in modern human populations.Firstly, owing to differences of germline mutation spectrum were observed between modern human populations and deciphering of CTVT somatic mutational spectrum depends on modern canine germline SNPs, we tested if germline spectrum varies among CTVT host populations. We found that mutation frequency vary between wolves and dogs, as well as between southern dogs and north dogs. This is the first time that difference of germline mutation frequency is observed between wild and domestic animal population, suggesting domestication could result in changes in mutation frequency.Secondly, we used two germline SNPs panels of 201 and 31 modern wolves and dogs as background to decipher mutational signatures of CTVT somatic spectrum. The results of mutational signatures of CTVT somatic spectrum kept stable, suggesting background germline SNPs panel doesn’t have a significant effect on CTVT mutational spectrum deciphering. The result suggests exposure to ultraviolet may involve in tumorigenesis or result in mutation accumulation of CTVT.Thirdly, negative selection was detected in CTVT mtDNA in previous studies. Our study found that diverse selections occurred in different CTVT mtDNA sub-level haplogroups after analyzing 447 CTVT mtDNAs. Especially positive selection has been detected in CTVT_1A subclade while others experienced negative selection. Genes involved in many pathways including cell apoptosis were detected under positive selection, suggesting they are candidate driver genes in CTVT. Nonsense mutation enriched in genes affecting cell adhesion, which might benefit infection and transmission of CTVT by reducing cell adhesion between cells and improving migration capacity.This study firstly detects difference of spectrum in wild and domesticated animal population, suggesting domestication could lead to mutation frequency difference. Differences between populations should be taken into consideration during following population genetics researches. Positive selection has operated on CTVT mtDNA and nuclear genome evolution. Adaptive selection took an important role in long evolutionary history of CTVT. Our study provides an early endeavor to the tumorigenesis and evolutionary mechanism of CTVT and established a reference for the study of tumor evolution during long time.