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

摘要 受试者对药物和环境制剂的反应存在变异性，这对 安全有效地治疗许多人类疾病。药物基因组学试图解决这一挑战 通过将药物反应与患者重要基因座上的基因类型联系起来，称为药物基因，以便更好地 定制患者治疗方案。细胞色素P450(CyP)基因变异是药物不良反应的主要原因 由受试者代谢治疗剂和环境的能力改变引起的反应 毒素，相对于一般人群。Cyps的遗传变异广泛。例如，在12个国家中， 最重要的细胞色素P450(CyP)基因，10%的人至少携带一种罕见的，潜在的 有害的变种。进一步的复杂性是通过由共同变异加上复杂等位基因引入的 连锁的稀有变异，以及这些基因座上广泛的拷贝数变异和基因融合。不幸的是， 只有少数变异被明确地与药物/异种生物反应的改变联系在一起。 显然，需要新的方法来诠释未知的巨大变体池的后果 意义，包括那些已经被现有的大规模测序计划确定的以及将 随着临床测序变得例行公事而被发现。我们已经开发了一套方法来测试所有 在几个CYP基因的所有氨基酸残基上可能存在单一替换。为了实现这一目标，我们 使用深度突变扫描，这是我们开发的一种方法，允许并行、定量 对基因变异库进行测量。我们正在应用这一方法 到CYP2C9和CYP2D6的单部位变异体。我们建议将我们的工作扩展到包括第三个 典型的CYP药物基因，CYP3A4，定量地说是人类肝脏代谢最重要的药物 代谢尼古丁并调节吸烟行为和肺癌风险的酶--CYP2A6，以及 CYP1A1，它可以生物激活多环杂芳烃致癌物。这些努力跨越了主要的 异种生物代谢CYP家族(Cyp1-3)，构成目标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， 这两项努力都集中在药物代谢的CYP2家族上。这个项目的结果将是一个全面的， 对CYP变体的上下文感知、功能分析将导致对 这些关键药物来源的遗传变异的后果。我们还将开发新的、可推广的 用于生成复杂变异库和直接评估酶变异体影响的方法 多元化的时尚。

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.