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

项目摘要/摘要： 我们有限的将基因和表型联系起来的能力是生物医学研究和个性化的主要障碍。 医药。目前只有~2%的生殖系错义变体有临床解释，其余的 不确定意义的变异(VU)，不提供任何信息来指导诊断或指导治疗。作为 临床使用的整个外显子组和基因组测序增加，VU的数量将直线上升。大规模 模式生物中的功能分析是目前唯一与之匹配的变异解释方法 变异发现的速度，在这里，我们建议扩大它们的使用来解释遗传复杂性。我们的 方法利用了低成本、大规模基因合成的出现和高通量的发展 对酵母等模型生物中蛋白质功能的活体分析。我们提出了一种可推广的方法 确定人类疾病基因的多态的功能后果，包括个体等位基因， 同一基因中的等位基因组合，以及一条路径中多个基因的等位基因组合 大规模平行的规模。我们化验的数量性质和我们实验设计的结构将 使我们能够将等位基因组合的影响与它们各自的影响进行比较，从而检测出基因 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的 远亲繁殖的人类数量的限制。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存在的或罕见的，但通过将它们置于蛋白质的背景下 和代谢途径结构，我们将深入了解它们的分子基础。我们的研究也将 提供了前所未有的关于单个氨基酸对功能的贡献的信息 在我们的研究中，三种与疾病相关的酶，我们将在它们的上下文中分析我们的结果 已发表的晶体结构。最后，我们将开发新的方法和分析，以扩大产量， 组合学，以及可用于人类变异的功能分析的分析的数量。 我们将使用与一类代谢相关的三个人类基因(OTC、ASS1和ASL)来试验我们的方法 被称为尿素循环障碍(UCD)的疾病。新生儿尿毒症与严重的酶缺乏症有关。 这些婴儿很快就会出现高水平的氨，脑水肿，以及可能包括癫痫发作的症状， 昏迷和死亡。不太严重的形式可能会在儿童或成年后仍未确诊。迟发性UCDS 通常与环境触发因素(如手术、怀孕或化学暴露)有关 酶功能降低。成人发病形式的诊断受到以下事实的阻碍：它通常表现为 如发作性精神病、双相情感障碍和严重抑郁等症状，如果不治疗，预后 很穷。因此，对这些基因中遗传变异的功能影响的了解有可能 减少因延误治疗或诊断不足而导致的发病率和死亡率。

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.