Environmental Risk Factors for Copy Number Variation in Human Chromosomes

描述(由申请人提供)： 这项申请涉及广泛的挑战领域，(08)基因组学和特定挑战主题，08-ES-106：环境暴露在拷贝数变异(CNV)中的作用：“基因组的微观缺失和复制因其在许多复杂人类疾病中的潜在作用而引起越来越多的关注。特别令人感兴趣的是自发的CNV，定义为存在于受影响个体中的CNV，但在父母双方中不存在。对于自发的CNV是如何产生的，人们的理解有限。需要进行研究，以确定环境暴露是否会影响与复杂疾病有关的拷贝数变异和其他结构变异的风险。鉴于这一研究领域还处于早期阶段，研究应侧重于体外暴露的细胞的变化。近年来，拷贝数变异(CNV)被发现广泛分布于人类基因组中，是遗传变异的重要组成部分，在表型多样性、复杂疾病和进化中发挥着不可或缺的作用。在健康个体中，超过1300个CNV的频率为1%，这些CNV的大小从几Kb到超过1Mb不等，存在缺失或复制，随着更多数据的获得，这个数字肯定会增加。众所周知，类似的自发CNV是遗传和发育障碍的主要原因，包括智力低下、自闭症、精神分裂症、癫痫、腭裂等许多其他疾病。对特发性精神发育迟滞和发育障碍的研究发现，在5-17%的受影响个体中存在新生CNV，这表明有很高的突变率。类似的拷贝数改变也在许多癌症中被发现，在启动或进展中可能起到作用。因此，CNV是正常遗传变异和进化的关键因素，是遗传和发育障碍以及癌症中非常重要的一类突变。对于CNV是如何产生的，所涉及的细胞机制和风险因素，以及环境因素对其形成的影响，人们的了解非常有限。与所有突变类别一样，几乎可以肯定的是，环境侮辱会诱发或增加新的和有害的CNV的风险，但我们对此类事件的机制和频率知之甚少。通过侧翼重复序列或节段性复制介导的减数分裂不平等重组[或非等位同源重组(Nahr)]导致许多复发的、与疾病相关的CNV。然而，越来越多的证据表明，许多或大多数正常和散发性的、非复发的CNV是通过与DNA异常复制和/或DNA损伤的非同源修复有关的机制产生的，这些CNV占人类和癌症中与疾病相关的CNV的大部分。这表明许多CNV是意外的有丝分裂而不是减数分裂的细胞起源，并对环境暴露在其形成中的作用和为其研究开发体外模型系统具有许多重要意义。这项挑战，也是我们的目标，是确定环境因素在CNV形成中的作用，并对这些频繁突变的产生机制获得新的见解。我们组建了一支强大的研究团队，并开发了一个正常的人类细胞培养模型系统，结合先进的基因组分析技术，使我们处于独特的地位，以应对这一及时的挑战。利用这一系统，我们发现，蜂毒灵诱导的复制应激导致从头开始的CNV的频率非常高。这些发现使我们假设，环境诱导的复制应激和/或DNA双链断裂是导致人类生殖系和癌细胞有丝分裂期间CNV的两个主要因素。为了验证这一假设，我们将在全基因组水平上表征环境毒剂诱导的CNV，并直接比较这两种非排他性CNV形成模型的强度，从而首次提供最有可能与CNV形成相关的两种不同类别环境毒剂的基因组表现的高分辨率目录。它们是：(1)导致复制应激的药物，可能通过继发性断裂或复制模板转换而导致CNV；(2)直接诱导DNA双链断裂(DNA DSB)的药物，可能通过断裂末端的不适当连接而导致CNV。具体地说，我们将研究羟基尿素和叶酸胁迫，它们通过不同于Ahidiclin的机制抑制复制，以及电离辐射和博莱霉素，它们直接导致DNA双链断裂。我们“准备好铲”的体外模型系统与高分辨率基因组微阵列和下一代测序相结合，将使我们能够确定这些药物对CNV和其他亚微观结构变异的频率、光谱、分布和结构的影响。合并后的结果将解决我们对一类非常重要的突变的知识中的一个重大缺口，并使我们能够预测哪些环境因素会带来正在进行的人类基因组结构变化的最大风险。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area, (08) Genomics and Specific Challenge Topic, 08-ES-106: The role of environmental exposure in copy number variation (CNV): "Microscopic deletions and replications of the genome have attracted increasing attention for their potential role in many complex human diseases. Of particular interest are spontaneous CNVs, defined as those present in an affected individual, but absent in both parents. There is limited understanding of how spontaneous CNVs arise. Studies are needed that will determine whether environmental exposures can affect risk for copy number variation and other structural variations that have been implicated in complex diseases. Given the early stage of this research area, studies should focus on changes in cells exposed in vitro". In recent years, copy number variants (CNVs) have been found to be widely distributed throughout the human genome where they represent an important component of genetic variation and play an integral role in phenotypic diversity, complex disease and evolution. Over 1300 CNVs with frequencies of >1% have been described in healthy individuals that exist as deletions or duplications ranging in size from a few Kb to over a Mb, and this number will surely increase as more data become available. Similar spontaneous CNVs are now well known to be a major cause of genetic and developmental disorders, including mental retardation, autism, schizophrenia, epilepsy, cleft palate and many others. Studies of idiopathic mental retardation and developmental disorders have found de novo CNVs in 5-17% of affected individuals, suggesting a high mutation rate. Similar copy number alterations are also found at high frequency in many cancers where a role in initiation or progression is likely. CNVs are thus a key factor in normal genetic variation and evolution and are a very important class of mutation in genetic and developmental disorders and cancer. There is very limited understanding of how CNVs arise, the cellular mechanisms and risk factors that are involved, and the effects of environmental agents on their formation. As with all mutation classes, it is almost certain that environmental insults can induce or increase the risk for new and deleterious CNVs, however we have little knowledge of the mechanisms and frequency of such events. Meiotic unequal recombination [or non-allelic homologous recombination (NAHR)] mediated by flanking repeated sequences or segmental duplications leads to many recurrent, disease-related CNVs. However, there is growing evidence that many or most normal and sporadic, nonrecurrent CNVs, which account for the majority of disease-associated CNVs in humans and those in cancers arise via mechanisms coupled to aberrant DNA replication and/or non-homologous repair of DNA damage. This suggests an unexpected mitotic, rather than meiotic, cell origin for many CNVs and has a number of important implications for the role of environmental exposures in their formation and the development of in vitro model systems for their study. The Challenge, and our goal, is to determine the role of environmental factors in the formation of CNVs and to gain novel insight into the mechanisms by which these frequent mutations are generated. We have assembled a strong team of investigators and have developed a normal human cell culture model system coupled with leading edge genome analysis technologies, placing us in a unique position to address this timely Challenge. Using this system we have found that aphidicolin-induced replication stress leads to a remarkably high frequency of de novo CNVs. These findings lead us to hypothesize that environmentally-induced replication stress and/or DNA double strand breaks are two major factors leading to CNVs during mitotic cell divisions in the human germline and in cancer cells. To test this hypothesis, we will characterize environmental agent-induced CNVs at the genome-wide scale and directly compare the strength of these two non-exclusive models for CNV formation, thus providing for the first time a high resolution catalog of genomic manifestations of two different categories of environmental agents most likely to be associated with CNV formation. These are (1) agents that lead to replication stress, which might lead to CNVs through secondary breakage or replicative template switching, and (2) agents that directly induce DNA double-strand breaks (DNA DSBs), which might lead to CNVs through inappropriate joining of broken ends. Specifically, we will examine hydroxyurea and folate stress, which inhibit replication through different mechanisms than aphidicolin, and on ionizing radiation and bleomycin, which lead directly to DNA DSBs. Our "shovel ready" in vitro model system coupled with high resolution genomic microarrays and next-generation sequencing will allow us to determine the effects of these agents on the frequency, spectrum, distribution and structure of CNVs and other submicroscopic structural variations. The combined results will address a major gap in our knowledge about a very important class of mutations and allow predictions of the environmental agents that confer the greatest risk for ongoing structural alteration of the human genome.