A pipeline for rapid functional determination and drug discovery of UDP genes

描述(申请人提供)：患者DNA的高通量基因组测序带来的遗传知识的快速爆炸正在产生前所未有的数量的与罕见和常见疾病相关的新等位基因。这些新发现的等位基因中有许多位于功能未知的基因中。为了建立这些遗传病的模型，遗传学家在基因中产生敲除突变，并研究由此产生的表型。表型分析可以证实特定的基因可以影响疾病状态，并提供更好地了解疾病的工具。然而，要更好地了解基因的功能，就需要确定疾病基因所需的遗传途径。为了识别这些遗传途径，可以采取许多方法，但无偏筛选方法的优势在于它们是独立于模型的，可以应用于分析管道中的许多不同的疾病相关基因。传统上，基因抑制子/增强子筛查已被用来识别途径和遗传相互作用，然而这种方法既耗时又费力。最近，已经开发了化学相互作用筛选，其中特征良好的、具有药理活性的小分子文库被筛选出来对抗突变体。这种方法适用于高输量管道。相互作用因子是那些改善或特别加剧表型的小分子。由于这些文库的蛋白质靶标都有很好的特征，因此可以从这些筛选技术中识别出该途径。然后，这些结果可以通过与药物靶基因的遗传相互作用来证实。我们的实验室已经开发出新的基因组工程技术，允许快速产生斑马鱼基因组的突变。我们将利用这个CRISPR指导的突变系统，在美国国立卫生研究院未诊断疾病计划确定的基因的每个斑马鱼同源基因中产生突变。为了开始描述这些基本上没有特征的基因，将通过原位杂交来分析这些基因在生命的头5天的表达模式。产生的突变体将被分析解剖/发育缺陷以及行为和生理上的功能差异。许多UDP确定的等位基因与神经系统疾病有关，这些基因将特别关注中脑和后脑的神经解剖学、视动反应、运动、心脏自主调节和SS细胞质量变化，以适应疾病的表型。然后，将测试最适合小分子文库筛选的表型与具有药理活性和良好特征的化合物的小分子文库的相互作用。这种复合筛选将用于两个目的：首先，筛选将识别与等位基因相互作用的化合物，这将有助于基因功能模型的建立。其次，复合筛查可以识别可能对患者有直接临床用途的药物。

DESCRIPTION (provided by applicant): The rapid explosion in genetic knowledge coming from high-throughput genome sequencing of patient DNA is generating unprecedented numbers of novel alleles correlated with both rare and common diseases. Many of these newly discovered alleles are in genes whose function is not known. In order to build models of these genetic diseases, geneticists generate knockout mutations in the gene and study the resulting phenotype. Phenotypic analysis can provide confirmation that a particular gene can contribute to the disease state and provide a tool to better understand the disease. However, a better understanding of gene function requires identification of the genetic pathways that the disease gene is required for. To identify these genetic pathways many approaches can be taken, but unbiased screening approaches have the advantage that they are model independent and can be applied to many different disease-associated genes in an analytical pipeline. Classically, genetic suppressor/enhancer screens have been used to identify pathways and genetic interactions, however this approach is time consuming and laborious. More recently, chemical interaction screens have been developed in which well-characterized, pharmacologically-active small molecule libraries are screened against the mutants. This approach is amenable to high throughput pipelines. Interactors are those small molecules that ameliorate or specifically exacerbate the phenotype. Since the protein targets of these libraries are well characterized, the pathway can be identified from these screening techniques. These results can then be confirmed through genetic interaction with the drug target genes. Our laboratories have developed new genome engineering technologies that allow for the rapid generation of mutations in the zebrafish genome. We will utilize this crispr-directed mutagenesis system to generate mutations in each of the zebrafish orthologs of the genes identified by the NIH Undiagnosed Disease Program. To begin to characterize these largely uncharacterized genes, the expression pattern of the genes will be analyzed during the first 5 days of life by in situ hybridization. The generated mutants will be analyzed for anatomical/developmental defects as well as functional differences in behavior and physiology. Many of the UDP-identified alleles are associated with neurological disease and particular attention for those genes will be placed on midbrain and hindbrain neuroanatomy, optokinetic response, motility, autonomic regulation of the heart, and ss cell mass changes as appropriate for the disease phenotype. The phenotypes most amenable to small molecule library screening will then be tested for interaction against a small molecule library of pharmacologically active and well characterized compounds. This compound screen will serve two purposes: First, the screen will identify compounds that interact with the allele which will assist in gene function model building. Second, the compound screen may identify pharmaceuticals that may be of direct clinical use to the patient.