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

描述(由申请人提供)： 这项建议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)的新方法应用于大样本集，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。这里开发的廉价而快速的方法将使大规模流行病学研究线粒体DNA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，我们将开发有效地对线粒体DNA进行测序的技术。我们将对从常用的人类细胞系和人类血液样本中分离的线粒体DNA进行测序，以便建立与未来研究比较的基线。这也将帮助我们理解线粒体DNA支持的变异的性质。我们还将开发分析技术，以确定线粒体DNA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。在特定目标2中，我们将修改并应用特定目标1的技术来处理亚硫酸氢盐处理的mtDNA。这将使我们能够以公正的方式分析线粒体DNA上的DNA甲基化。我们将使用与特定目标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，无论是在细胞系内还是在个体血液样本之间。在具体目标3中，将把具体目标1和2的技术应用于西奈山医学院建立的新生儿队列中的胎盘样本。由于胎盘含有丰富的线粒体，并掌握着早期发育的关键，我们相信这将使我们深入了解新生儿之间的发育差异。我们将把表型(出生体重、身高和头围)与线粒体DNA图谱相关联，并产生关于变异在决定表型中的作用的假设。这将是线粒体DNA图谱的首次流行病学应用。这一提议的结果将对一系列领域产生广泛影响，从植物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.