Retrotransposition in Health and Disease

健康与疾病中的逆转录转座

• 批准号: 8296918
• 负责人: HAIG H. KAZAZIAN
• 金额: $ 47.73万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8296918
• 关键词: Adult; Affect; Autopsy; Biochemical; Bioinformatics; Biological Assay; Biology; Brain; Bruck-de Lange syndrome; Cancer Etiology; Cell Culture Techniques; Chromatin; Collaborations; Communities; Computer Analysis; Computing Methodologies; DNA; Data; Disease; Embryonic Development; Etiology; Event; Frequencies; Gene Expression; Gene Expression Profile; Gene Frequency; Gene Targeting; Generations; Genes; Genetic Heterogeneity; Genetic Polymorphism; Genome; Genomics; Germ Lines; Health; Heart; Hemophilia A; Human; Human Biology; Human Genome; Individual; Learning; Limb structure; Liver; Location; Malignant Neoplasms; Methods; Molecular Conformation; Mosaicism; Mouse Strains; Mus; Mutation; Neuroblastoma; Normal tissue morphology; Organism; Patients; Phenotype; Placenta; Positioning Attribute; Procedures; Process; Pseudogenes; Retrotransposition; Retrotransposon; Role; Sampling; Somatic Mutation; Tail; Techniques; Testis Brain; Time; Tissues; Transgenic Animals; Variant; Work; fetal; human disease; innovation; mammalian genome; mouse genome; transposon/insertion element

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. PUBLIC HEALTH RELEVANCE: L1 retrotransposons are the major mobile DNA of human and mammalian genomes. Here we study the effects of L1 in human health and disease. We search for somatic mutations in human and mice, determine the effects of retrotransposons on gene expression in humans and mice, and analyze both human cancers and Mendelian disorders for causative retrotransposition events.

描述（由申请人提供）： 科学界仍然对所有生物的基因组中发现的移动DNA感到惊讶。在人类中，主移动DNA是L1逆转录座子，它与Alu，SVA和经过加工的假基因一起动员到新的基因组位置。自从我们发现23年前两名血友病患者中首次引起疾病的L1插入，因此通过细胞培养试验，生化分析，自然插入基因，转基因动物和生物信息分析，已经了解了L1和其他人逆转录座子。最近，PCR技术，高通量测序和生物信息学分析使得在任何人类基因组中基本上都可以发现所有非引用逆转录子。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（RTN）主要发生在早期的胚胎发育中，而不是种系。在此提案中，我们提出了以这些发现为基础的三个补充特定目标。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，并将这些频率与估计生殖系的频率进行比较。我们还确定了新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，以找到所有非参考的活性小鼠L1。我们使用此过程研究不同小鼠菌株组织中的L1 RTN。然后，在直接确定不同小鼠菌株中种系RTN频率之后，我们将比较体细胞和生殖线频率。我们预计，由于小鼠的活性L1数量是人类的15-30倍（1500-3000 vs. 80-100），因此小鼠的RTN频率将大大高于人类。早期工作导致了令人兴奋的新数据，表明L1插入小鼠基因组中的群集中非随机插入。与人类的工作一样，我们将确定新小鼠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1）各种DNA已完全测序的各种癌症，以及2）具有良好型的Mendelian疾病Cornelia de Lange综合征的突变阴性患者。在观察这些患者的RTN事件时，我们将了解人类病病因中低估的程度。我们预测，逆转座子对人类健康的影响明显大于目前的想象。 公共卫生相关性： L1逆转座子是人和哺乳动物基因组的主要移动DNA。在这里，我们研究L1对人类健康和疾病的影响。我们在人和小鼠中搜索体细胞突变，确定逆转座子对人类和小鼠基因表达的影响，并分析人类癌症和孟德尔疾病的因果逆转录事件。