Gene signatures linked to the cell biological phenotypes of familial PD

描述（由申请人提供）：全基因组转录组的高通量测序将产生许多关于神经退行性机制的遗传相互作用的新假设。我们希望生物相关途径的标记基因能够有效地询问实验产生的大量原始数据。这项新的R21提案的目标是帮助验证神经元转录组作为帕金森病研究的工具。来自明确定义的帕金森病家族病例的细胞虽然罕见，但为此类分析提供了一个很好的起点。我们和其他人已经表明，携带LRRK2或PINK1突变的帕金森病患者的细胞表现出线粒体缺陷。重要的是，将LRRK2或PINK1突变与线粒体缺陷联系起来的分子机制仍不清楚。使用一种创新的方法来解释神经元转录组，我们的目标是产生新的假设LRRK2或PINK1相关的线粒体缺陷的分子机制。我们将把我们观察到的携带帕金森病相关LRRK2和PINK1突变的人类神经元的功能性线粒体缺陷与神经元表达的RNA序列联系起来。通过将高通量转录组测序与新的定量PCR阵列相结合，我们提出确定线粒体DNA（SA1）和核DNA（SA2）编码基因的表达水平和序列特征，这些基因标记患者来源的神经元的线粒体缺陷。将使用年龄匹配、同胞和同基因对照iPSC的多个克隆来最小化神经元样品中基因组变异的影响。在实现我们目标的第一步中，对来自诱导多能干细胞（iPSC）衍生的神经元的RNA分子进行了测序，这些神经元携带LRRK2、PINK1突变并显示线粒体缺陷或健康受试者。RNAseq数据的初步分析证实了神经元转录组中存在LRRK2和PINK1突变。此外，我们的分析表明，功能性线粒体缺陷与线粒体DNA编码的转录本的异常加工有关。这些来自人类神经元的数据可用于建立合理和连贯的细胞生物学反应框架，以解释患者的转录组。我们的研究结果将是有益的和关键的未来尝试，有效地分析人类细胞生物学表型与多个潜在的遗传相互作用的散发形式的帕金森氏病。

DESCRIPTION (provided by applicant): High throughput sequencing of the genome-wide transcriptome will generate many new hypotheses regarding the genetic interactions underlying neurodegenerative mechanisms. We expect that marker genes of biologically relevant pathways are needed to efficiently interrogate the large sets of raw data produced by the experiments. The goal of this new R21 proposal is to help validate the neuronal transcriptome as a tool for the study of Parkinson's disease. Cells from well-defined familial cases of Parkinson's disease, although rare, provide an excellent starting point for such analyses. We and others have shown that cells from Parkinson's disease patients carrying LRRK2 or PINK1 mutations demonstrate mitochondrial deficits. Importantly, the molecular mechanisms that connect LRRK2 or PINK1 mutations to the mitochondrial deficits remain unclear. Using an innovative approach to interpreting the neuronal transcriptome, we aim to generate new hypotheses regarding the molecular mechanisms of LRRK2 or PINK1 associated mitochondrial deficits. We will link the functional mitochondrial deficits of human neurons carrying Parkinson's disease associated LRRK2 and PINK1 mutations that we have observed to the RNA sequences expressed by the neurons. By combining high- throughput transcriptome sequencing with new quantitative PCR arrays, we propose to determine the expression level and sequence signatures of mitochondrial DNA (SA1) and nuclear DNA (SA2) encoded genes that mark the mitochondrial deficits of patient-derived neurons. Multiple clones of age-matched, sibling and isogenic control iPSCs will be used to minimize the influence of genomic variation across neuronal samples. In a first step towards our goal, RNA molecules from induced pluripotent stem cell (iPSC)-derived neurons carrying LRRK2, PINK1 mutations and showing mitochondrial deficits or healthy subjects were sequenced. Preliminary analyses of the RNAseq data confirmed the presence of the LRRK2 and PINK1 mutations in the neuronal transcriptome. Furthermore, our analysis suggests that the functional mitochondrial deficits are associated with aberrant processing of mitochondrial DNA-encoded transcripts. These data from human neurons can be used to establish a reasonable and coherent framework of cell biological responses to interpret a patient's transcriptome. Our results will be instructive and critical to future attempts to effectively analyze human cell biological phenotypes with the multiple underlying genetic interactions of sporadic forms of Parkinson's disease.