Retrotransposition in Health and Disease

描述(由申请人提供)： 科学界仍然对在所有生物体的基因组中发现的可移动DNA感到惊讶。在人类中，主要的可移动DNA是L1反转录转座子，它与Alu、SVA和加工的假基因一起动员自己到新的基因组位置。自从我们在大约23年前在两名血友病A患者中首次发现L1致病插入以来，通过细胞培养分析、生化分析、自然插入基因、转基因动物和生物信息学分析，已经对L1和其他人类反转录转座子有了很多了解。最近，聚合酶链式反应技术、高通量测序和生物信息学分析已经使发现任何人类基因组中几乎所有的非参考反转录转座子成为可能。我们实验室最近的一项令人惊讶的发现是，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。在这项提案中，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的估计频率进行比较。我们还确定了新的L1插入对邻近基因表达的影响。其次，我们最近修改了我们的技术，基本上找到了所有非参考的、活动的小鼠L1。我们使用这个程序来研究不同品系小鼠组织中的L1 RTN。然后，在直接测定不同品系小鼠的生殖系RTN频率后，我们将比较体细胞和生殖系的频率。我们预计，由于小鼠的L1活动数量是人类的15-30倍(1500-3000对80-100)，小鼠的RTN频率将显著高于人类。早期的工作带来了令人兴奋的新数据，表明L1插入小鼠基因组的过程是非随机的。与人类的工作一样，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，以及2)突变阴性的孟德尔疾病患者，科妮莉亚·德兰格综合征。在观察这些患者的RTN事件时，我们将了解在人类疾病病因学中它们被低估的程度。我们预测，反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.