Rapid and inexpensive epi/genetic profiling of the human mitochondrial genome

描述（由申请人提供）： 该提案（“人类线粒体基因组的快速和廉价的表观/遗传分析”）的主要目标是将我们分离和测序线粒体基因组（mtDNA）和表观基因组（mtDNA-甲基化）的新方法应用于大量样品，以获得生物学和临床见解。我们将描述线粒体基因组和表观基因组的变异性。这种廉价而快速的方法将使大规模的流行病学研究mtDNA在衰老和各种人类疾病的发生和发展中的作用成为可能。在具体目标1中，我们将开发有效测序mtDNA的技术。我们将对从常用的人类细胞系和人类血液样本中分离的mtDNA进行测序，以建立与未来研究进行比较的基线。这也将有助于我们理解mtDNA支持的变异的性质。我们还将开发分析技术，以确定mtDNA分离的质量，将读数组装成完整的基因组并识别基因组中的变异。在具体目标2中，我们将修改具体目标1的技术并将其应用于亚硫酸氢盐处理的mtDNA。这将使我们能够以公正的方式分析mtDNA的DNA甲基化。我们将使用与具体目标1相同的样本来量化mtDNA甲基化水平及其在样本之间的变异性，包括细胞系内和个体血液样本之间。在具体目标3中，将具体目标1和2的技术应用于西奈山医学院建立的新生儿队列的胎盘样本。由于胎盘富含线粒体，并掌握着早期发育的关键，我们相信这将使我们了解新生儿之间的发育差异。我们将把表型（出生体重、身长和头围）与mtDNA谱相关联，并对变异在决定表型中的作用提出假设。这将是mtDNA谱的首次流行病学应用。该提案的结果将对一系列领域产生广泛影响，从植物中的细菌基因组学和叶绿体遗传学到人类的癌症和糖尿病等疾病。

DESCRIPTION (provided by applicant): The primary goal of this proposal ("Rapid and inexpensive epi/genetic profiling of the human mitochondrial genome") is to apply our new method of isolating and sequencing the mitochondrial genome (mtDNA) and epigenome (mtDNA-methylation) to large sets of samples in order to derive biological and clinical insights. We will characterize the variability in the mitochondrial genome and epigenome. The inexpensive and rapid method developed here will enable large-scale epidemiological studies of the role of mtDNA in aging and in the initiation and progression of a variety of human disorders. In specific aim 1 we will develop the techniques to efficiently sequence the mtDNA. We will sequence the mtDNA isolated from commonly used human cell-lines and human blood samples in order to establish a baseline for comparison with future studies. This will also help us understand the nature of variations supported by mtDNA. We will also develop analytical techniques to determine the quality of mtDNA isolation, assemble the reads into a complete genome and identify variations in the genome. In specific aim 2, we will modify and apply the technique of specific aim 1 to bisulfite treated mtDNA. This will allow us to profile DNA-methylation across the mtDNA in an unbiased manner. We will use the same samples as in specific aim 1 to quantify the levels of mtDNA methylation and its variability between samples, both within cell-lines and between individual blood samples. In specific aim 3 will apply the techniques of specific aims 1 and 2 to placental samples from a newborn cohort established at Mount Sinai School of Medicine. Since placentas are rich in mitochondria and hold the key to early development, we believe this will give us insights into developmental differences between newborns. We will correlate the phenotypes (birth weight, length and head circumference) to mtDNA profiles and generate hypotheses on the role of variants in determining the phenotypes. This will be the first epidemiological use of mtDNA profiling. The results of this proposal will have a broad impact on a range of fields, from bacteril genomics and genetics of chloroplasts in plants to disorders such as cancer and diabetes in humans.