Comprehensive, context-aware, functional analysis of Cytochrome P450 variants

摘要 受试者对药物和环境因子的反应的差异性对药物和环境因子产生了重大挑战。 安全有效地治疗许多人类疾病。药物基因组学旨在应对这一挑战 通过将药物反应与患者重要基因座的基因型（称为药物基因）联系起来， 定制患者治疗。细胞色素P450（CYP 1A 1）基因变异是药物不良反应的主要原因 由受试者代谢治疗药物和环境的能力改变引起的反应 毒素，相对于整个人口。CYP的遗传变异是广泛的。例如，在12个 最重要的细胞色素P450（CYP 450）基因，10%的人携带至少一个罕见的，潜在的 有害变体。进一步的复杂性通过由共同变异加上 连锁的罕见变异，并通过广泛的拷贝数变异和这些基因座的基因融合。不幸的是， 只有少数变异体与药物/异生物质反应的改变明确相关。 显然，需要新的方法来注释未知的巨大变体池的后果。 重要性，包括那些已经通过现有的大规模测序计划，以及那些将 随着临床测序变得常规，人们会发现。我们开发了一套方法来测试所有 可能在几个cDNA基因中的所有氨基酸残基上进行单取代。为了做到这一点，我们 使用深度突变扫描，我们开发的一种方法， 在一些实施方案中，可以对遗传变体的文库进行测量。我们正在应用这种方法， CYP 2C 9和CYP 2D 6的单位点变体。我们建议将我们的工作扩展到包括CYP 2C 19，第三个 原型药物代谢基因CYP 3A 4，在数量上是最重要的人类肝脏药物代谢 代谢尼古丁并调节吸烟行为和肺癌风险的酶CYP 2A 6，以及 CYP 1A 1，可生物活化多环杂芳族致癌物。这些努力，其中跨越主要 异生物质代谢酶家族（CYP 1 -3）构成Aim 1。在目标2中，我们将评估更复杂的 等位基因，包括新的嵌合体，在目标3中，我们将剖析遗传变异的底物依赖性， 这两项工作都集中在药物代谢CYP 2家族上。这一项目的结果将是一个全面、 上下文感知的，功能性的分析，这将导致更深入地了解 这些关键药物基因的遗传变异的后果。我们还将开发新的、可推广的 用于产生复杂变体文库和用于直接评估酶变体在 多元化的方式。

ABSTRACT Subject-to-subject variability in response to drugs and environmental agents creates a significant challenge for the safe and effective treatment of many human diseases. Pharmacogenomics seeks to address this challenge by linking drug response to patient genotypes at important loci, termed pharmacogenes, in order to better customize patient treatments. Cytochrome P450 (CYP) gene variation is a major contributor to adverse drug reactions resulting from alterations in a subject's ability to metabolize therapeutic agents and environmental toxins, relative to the population at large. Genetic variation in CYPs is extensive. For example, amongst 12 of the most important cytochrome P450 (CYP) genes, 10% of people carry at least one rare, potentially deleterious variant. Further complexity is introduced via complex alleles consisting of common variation plus linked rare variants, and by extensive copy number variation and gene fusions at these loci. Unfortunately, only a small number of variants have been unambiguously linked to alterations in drug/xenobiotic response. Clearly, new approaches are needed to annotate the consequences of the huge pool of variants of unknown significance, including those already identified by existing large-scale sequencing programs, and those that will be discovered as clinical sequencing becomes routine. We have developed a suite of methods to test all possible single substitutions at all amino acid residues in several CYP genes. In order to accomplish this, we use deep mutational scanning, a method we have developed that allows parallelized, quantitative measurements to be performed on libraries of genetic variants. We are in the midst of applying this approach to single site variants of CYP2C9 and CYP2D6. We propose to extend our work to include CYP2C19, the third prototypic CYP pharmacogene, CYP3A4, quantitatively the most important human liver drug metabolizing enzyme, CYP2A6, which metabolizes nicotine and modulates smoking behaviors and lung cancer risk, and CYP1A1, which bioactivates polycyclic heteroaromatic carcinogens. These efforts, which span the major xenobiotic metabolizing CYP families (CYP1-3), constitute Aim 1. In Aim 2, we will evaluate more complex alleles, including novel chimeras, and in Aim 3 we will dissect the substrate-dependency of genetic variation, both efforts focusing on the drug-metabolizing CYP2 family. The result of this project will be a comprehensive, context aware, functional analysis of CYP variants that will lead to a deeper understanding of the consequences of genetic variation in these key pharmacogenes. We will also develop new, generalizable methods for generating complex variant libraries and for directly assessing the effects of enzyme variants in a multiplex fashion.