Functionally Significant Polymorphisms in Serotonin Pathway Genes

描述(申请人提供)：神经递质5-羟色胺(5-羟色胺)是由氨基酸色氨酸的两步酶过程产生的，主要由位于中缝核团的神经元产生。这些神经元投射了太多的大脑区域，包括皮质、丘脑、下丘脑和杏仁核。释放的5-羟色胺可以作用于14种不同的受体，由突触前和突触后神经元表达。囊泡释放后，5-羟色胺转运体(5-HTT，SERT，SLC6A4)将5-羟色胺从突触中移除，从而终止5-羟色胺的作用。5-羟色胺能通路的松弛与许多精神和情感障碍有关，包括抑郁症、自闭症和强迫症。5-HTT是许多抗抑郁药的主要作用部位，这一证明支持了这些联系。5-HTT抑制剂(SRI)不仅在治疗抑郁症方面有效，而且在治疗其他疾病方面也有效，包括焦虑、强迫症(OCD)和自闭症的某些方面。遗传学研究已经报道了5-HTT基因常见和罕见的多态与精神和情感障碍风险的增加有关。这些结果不仅为以5-HTT为靶点治疗这些疾病提供了理论基础，也表明转运蛋白的表达和活性的改变可能有助于疾病本身的发展。事实证明，很难直接测试多态对体内5-羟色胺稳态紊乱的贡献，或者这些紊乱表现为可测量的行为变化的能力。为了开始解决这些问题，我们提出了新的鼠系的产生。这些品系被设计用来检验这样的假设，即5-HTT启动子的多态改变了该基因的表达，并且由此导致的5-羟色胺代谢的变化足以引起可测量的行为变化。为了实现这一目标，我们开发了一种独特的两步法，用于将编码特定老鼠基因的基因与其对应的人类基因进行交换。由于整个小鼠基因，包括启动子以及5‘和3’侧翼区，都与相应的人类序列交换，因此使用这种方法不仅可以解决非同义SNPs的功能，而且可以解决基因内含子和调节区中的非编码多态。利用这个载体系统，我们建议用5-HTT连锁多态区域(HTTLPR)的长(LA)、长(LG)或短(S)等位基因替换内源性5-HTT基因的小鼠。5-HTT的表达，5-羟色胺途径的活性，以及小鼠在行为学测试中的反应将提供关于这三个启动子多态性的功能的信息。用同线人类DNA替换整个小鼠基因，不仅有助于未来测试额外的5-HTT SNPs和变异体，而且通过将这些小鼠品系与在其他基因座表达疾病相关多态的品系杂交，简化了基因-基因相互作用的评估。遗传学研究发现，越来越多的基因多态与情感和精神疾病的风险有关。确定这些遗传变异的功能被证明是困难的，特别是当多态预测不会改变编码蛋白质的一级结构时。在这一应用中，我们提出了一种新的策略，用于将小鼠的基因与其人类同源基因交换。我们证明，在这种交换之后，SNPs可以被导入到小鼠基因组的人类DNA片段中。由于这种遗传操作是在小鼠胚胎干细胞中进行的，因此有可能产生仅在与人类群体中的疾病风险相关的特定多态上存在差异的小鼠品系。这些小鼠可以用来研究这种多态对基因表达、蛋白质功能、发育以及已知对啮齿动物这一特定途径的变化敏感的行为测试/模型的影响。

DESCRIPTION (provided by applicant): The neurotransmitter serotonin (5-HT) is produced by a two step enzymatic process from the amino acid tryptophan, primarily by neurons located in the raphe nuclei. These neurons project too many regions of the brain including the cortex, thalamus, hypothalamus, and amygdala. Released serotonin can act on over 14 different receptors, expressed by both presynaptic and postsynaptic neurons. Following vesicular release, the action of 5-HT is terminated by its removal from the synapse by the serotonin transporter (5-HTT, SERT, SLC6A4). Deregulation of serotonergic pathways has been implicated in many psychiatric and affective disorders including depression, autism, and obsessive compulsive disorder. These associations are supported by the demonstration that 5-HTT is the major site of action of many antidepressants. 5-HTT inhibitors (SRIs) are effective, not only in treating depression, but also in the treatment of other disorders including anxiety, obsessive compulsive disorder (OCD), and some aspects of autism. Genetic studies have reported association of both common and rare polymorphisms in the 5-HTT gene with increased risk for psychiatric and affective disorders. These results not only provide a rationale for targeting 5-HTT in the treatment of these illnesses, but also suggest that altered expression and activity of the transporter may contribute to the development of the illness itself. It has proved difficult to directly test either the contribution of polymorphisms to disturbances in serotonin homeostasis in vivo or the ability of these disturbances to manifest as measurable changes in behavior. To begin to address these questions, we propose the generation of novel mouse lines. These lines are designed to test the hypothesis that polymorphisms in the 5-HTT promoter alter expression of this gene and that the resulting changes in serotonin metabolism are sufficient to cause measurable changes in behavior. To accomplish this goal we have developed a unique two step method for exchange of the locus encoding a specific mouse gene with its human counterpart. Because the entire mouse gene, including the promoter and 5' and 3' flanking regions, is exchanged with the corresponding human sequence, it is possible using this method to address not only the functionality of non-synonymous SNPs but also non- coding polymorphisms in introns and regulatory regions of the gene. Using this vector system we propose to generate mice in which the endogenous 5-Htt gene is excised and replaced with a human 5-HTT gene with either the long (LA), the long (LG), or the short (S) allele of the 5-HTT linked polymorphic region (HTTLPR). The expression of 5-HTT, the activity of the serotonin pathway, and the response of the mice in behavioral tests will provide information concerning the functionality of the three promoter polymorphism. The replacement of the entire mouse gene with the syntenic human DNA will not only facilitate future testing of additional 5-HTT SNPs and variants, but also simplify the evaluation of gene-gene interactions by the intercross of these mouse lines to lines expressing disease related polymorphisms at other loci. Genetic studies are identifying an ever increasing number of polymorphisms linked to risk for affective and psychiatric illness. Determining the functionality of these genetic variations has proved difficult, particularly when no change in the primary structure of the encoded protein is predicted by the polymorphism. In this application, we present the development of a new strategy for the exchange of mouse genes with their human orthologues. We show that after this exchange SNPs can be introduced into the human DNA segment of the mouse genome. As this genetic manipulation is carried out in mouse embryonic stem cells, it is possible to generate mouse lines that differ only in a particular polymorphism associated with risk for disease in the human population. These mice can be used to study the impact of this polymorphism on gene expression, protein function, development, and on the behavioral tests/models known to be sensitive to alterations in this specific pathway in rodents.