Retrotransposition in Health and Disease

描述（由申请人提供）： 科学界仍然对所有生物体基因组中发现的可移动 DNA 感到惊讶。在人类中，主要的移动 DNA 是 L1 逆转录转座子，它将自身与 Alu、SVA 和加工后的假基因一起移动到新的基因组位置。自从大约 23 年前我们在两名甲型血友病患者体内首次发现导致疾病的 L1 插入以来，通过细胞培养测定、生化分析、基因自然插入、转基因动物和生物信息学分析，人们对 L1 和其他人类反转录转座子有了很多了解。最近，PCR技术、高通量测序和生物信息学分析使得发现任何人类基因组中基本上所有非参考反转录转座子成为可能。我们实验室最近的一个令人惊讶的发现是，在转基因动物中，L1 逆转录转座 (Rtn) 主要发生在早期胚胎发育中，而不是生殖系。在本提案中，我们提出了基于这些发现的三个互补的具体目标。首先，我们通过量化胎儿和成人组织中的体细胞 Rtn 频率来研究人类生物学中的 L1 Rtn，并将这些频率与生殖系估计的频率进行比较。我们还确定了新的 L1 插入对附近基因表达的影响。其次，我们最近修改了我们的技术，以找到基本上所有非参考、活跃的鼠标 L1。我们使用此程序来研究不同小鼠品系组织中的 L1 Rtn。然后，在直接测定不同小鼠品系的种系Rtn频率后，我们将比较体细胞和种系频率。我们预计，由于小鼠的活跃 L1 数量是人类的 15-30 倍（1500-3000 vs. 80-100），因此小鼠的 Rtn 频率将显着高于人类。早期的工作已经产生了令人兴奋的新数据，表明 L1 插入小鼠基因组中是非随机地成簇发生的。与人类工作一样，我们将确定新的小鼠 L1 插入对基因表达的影响。第三，我们确定了 Rtn 在人类疾病中的作用，研究了 1) DNA 已完全测序的各种癌症和 2) 患有表型良好的孟德尔疾病（Cornelia de Lange 综合征）的突变阴性患者。通过观察这些患者的 Rtn 事件，我们将了解到它们在人类疾病病因学中被低估的程度。我们预测逆转录转座子对人类健康的影响比目前认为的要大得多。

DESCRIPTION (provided by applicant): The scientific community is still surprised by the mobile DNA found in the genomes of all organisms. In human beings, the master mobile DNA is the L1 retrotransposon which mobilizes itself along with Alu , SVA, and processed pseudogenes to new genomic locations. Since we found the first disease-causing insertions of an L1 in two patients with hemophilia A some 23 years ago, much has been learned about L1 and other human retrotransposons through cell culture assays, biochemical analyses, natural insertions into genes, transgenic animals, and bioinformatic analysis. Recently, PCR techniques, high-throughput sequencing and bioinformatic analysis have made it possible to discover essentially all non-reference retrotransposons in any human genome. A recent surprise finding of our lab was that in transgenic animals L1 retrotransposition (Rtn) predominately occurs in early embryonic development as opposed to the germ line. In this proposal, we propose three complementary Specific Aims that build on these findings. First, we study L1 Rtn in human biology by quantifying the somatic Rtn frequency in fetal and adult tissues and compare these frequencies to the frequency estimated for the germ line. We also determine the effects of new L1 insertions on the expression of nearby genes. Second, we have recently modified our technique to find essentially all non-reference, active mouse L1s. We use this procedure to study L1 Rtn in tissues of different mouse strains. Then, after direct determination of the germ line Rtn frequency in different mouse strains, we will compare the somatic and germ line frequencies. We expect that since the mouse has 15-30 times the number of active L1s as the human (1500-3000 vs. 80- 100), Rtn frequency in the mouse will be significantly greater than in humans. Early work has led to exciting new data, indicating that L1 insertions into the mouse genome occur non-randomly in clusters. As in the human work, we will then determine the effect of new mouse L1 insertions on gene expression. Third, we determine the role of Rtn in human disease, studying 1) various cancers whose DNA has been completely sequenced and 2) mutation-negative patients with a well-phenotyped Mendelian disorder, Cornelia de Lange Syndrome. In observing Rtn events in these patients, we will learn the extent to which they have been underestimated in human disease etiology. We predict that retrotransposon effects on human health are significantly greater than currently believed.