Comprehensive approaches for understanding the functional impact of genetic variation and genetic complexity

项目概要/摘要： 我们将基因型与表型联系起来的能力有限，这是生物医学研究和个性化治疗的主要障碍。 药目前，只有约2%的种系错义变异具有临床解释，其余的， 重要性不确定的变异（VUS）不能提供诊断或指导治疗的信息。为 随着全外显子组和基因组测序的临床应用增加，VUS的数量将激增。大规模 在模式生物中进行功能测定是目前唯一准备与之匹配的变异解释方法 变异发现的速度，在这里，我们建议将其用于解释遗传复杂性。我们 这种方法利用了低成本、大规模基因合成的出现和高通量 在模型生物体如酵母中进行蛋白质功能的体内测定。我们提出了一个可推广的方法， 确定人类疾病基因中多态性的功能后果，包括个体等位基因， 同一基因中的等位基因的组合，以及途径中多个基因中的等位基因的组合， 大规模并行我们的分析的定量性质和我们的实验设计的结构将 使我们能够比较等位基因组合的影响与其个体效应，从而检测遗传 上位性（非线性遗传相互作用），由自然发生的人类遗传变异引起， 远系繁殖的人类种群的极限。通过这种新颖的方法，我们不仅将探索 人类基因之间的非线性相互作用是普遍的或罕见的，但通过将它们放在蛋白质的背景下， 和代谢途径结构，我们将深入了解它们的分子基础。我们的研究还将 提供了关于单个氨基酸对功能的贡献的前所未有的大量信息 在我们的研究中，这三种疾病相关的酶，我们将在他们的背景下分析我们的结果。 发表的晶体结构。最后，我们将开发新的方法和测定，将扩大吞吐量， 组合学和可用于人类变异的功能分析的测定的数量。 我们将使用与一类代谢相关的三种人类基因（OTC，ASS 1和ASL）来试验我们的方法。 尿素循环障碍（UCD）。新生儿UCD与严重的酶缺乏症有关。 这些婴儿迅速发展高水平的氨，脑水肿，和症状，可包括癫痫发作， 昏迷和死亡不太严重的形式可能在儿童期或成年期仍未被诊断出来。迟发性UCD 通常涉及环境触发因素（例如手术、妊娠或化学品暴露）， 降低酶的功能。成人发病形式的诊断受到阻碍的事实，它往往提出与 发作性精神病、双相情感障碍和重度抑郁症等症状，如果不治疗，预后 差因此，了解这些基因中遗传变异的功能意义有可能 减少与延误治疗或诊断不足相关的发病率和死亡率。

Project Summary/ Abstract: Our limited ability to relate genotype to phenotype is a major obstacle for biomedical research and personalized medicine. Currently only ~2% of germline missense variants have clinical interpretations, and the remainder, variants of uncertain significance (VUS), offer no information to inform diagnosis or guide treatment. As the clinical use of whole exome and genome sequencing increases, the number of VUS will skyrocket. Large-scale functional assays in model organisms are the only methods for variant interpretation currently poised to match the pace of variant discovery, and here we propose to extend their use to interpret genetic complexity. Our approach leverages the advent of low-cost, large-scale gene synthesis and the development of high throughput in vivo assays of protein function in model organisms, such as yeast. We propose a generalizable approach for determining the functional consequences of polymorphisms in human disease genes, including individual alleles, combinations of alleles in the same gene, and combinations of alleles in multiple genes in a pathway, on a massively parallel scale. The quantitative nature of our assay and the structure of our experimental design will allow us to compare the impact of allele combinations with their individual effects, and thus detect genetic epistasis (nonlinear genetic interactions) arising from naturally occurring human genetic variation outside of the limits of outbred human populations. Through this novel approach, we will not only explore the extent to which nonlinear interactions between human genes are pervasive or rare, but by placing them in the context of protein and metabolic pathway structure, we will gain insight into their molecular underpinnings. Our study will also provide an unprecedented amount of information about the contribution of individual amino acids to the function of the three disease-relevant enzymes in our study, and we will analyze our results in the context of their published crystal structures. Finally, we will develop new methods and assays that will expand the throughput, combinatorics, and number of assays available for functional analysis of human variation. We will pilot our approach using three human genes (OTC, ASS1, and ASL) associated with a class of metabolic disorders known as urea cycle disorders (UCD). Neonatal UCD is associated with severe enzyme deficiency. These infants rapidly develop high levels of ammonia, cerebral edema, and symptoms that can include seizures, coma, and death. Less severe forms may remain undiagnosed into childhood or adulthood. Late onset UCDs generally involve an environmental trigger (e.g. surgery, pregnancy, or chemical exposure) in individuals with reduced enzyme function. Diagnosis of the adult onset form is hampered by the fact that it often presents with symptoms such as episodic psychosis, bipolar disorder and major depression, and without treatment, prognosis is poor. Thus, knowledge of the functional implications of genetic variation in these genes has the potential to reduce the morbidity and mortality associated with delayed treatment or underdiagnosis.