Functionally Significant Polymorphisms in Serotonin Pathway Genes

描述（由申请人提供）：神经递质5-羟色胺（5-HT）主要由位于中缝核中的神经元通过两步酶促过程从氨基酸色氨酸产生。这些神经元投射大脑的太多区域，包括皮质、丘脑、下丘脑和杏仁核。释放的5-羟色胺可以作用于超过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-HT的作用通过5-羟色胺转运蛋白（5-HTT，SERT，SLC 6A 4）将其从突触中清除而终止。多巴胺能通路的失调与许多精神和情感障碍有关，包括抑郁症、自闭症和强迫症。这些关联得到了5-HTT是许多抗抑郁药的主要作用部位的证明的支持。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，包括焦虑症，强迫症（OCD）和自闭症的某些方面也有效。遗传学研究已经报道了5-HTT基因中常见和罕见的多态性与精神和情感障碍风险增加的相关性。这些结果不仅为靶向5-HTT治疗这些疾病提供了理论基础，而且还表明转运蛋白的表达和活性改变可能有助于疾病本身的发展。已经证明难以直接测试多态性对体内5-羟色胺稳态紊乱的贡献或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，我们提出了新的鼠标线的生成。设计这些细胞系是为了检验以下假设：5-HTT启动子中的多态性改变了该基因的表达，并且由此引起的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，用于将编码特定小鼠基因的基因座与其人类对应物交换。因为整个小鼠基因，包括启动子和5'和3'侧翼区，与相应的人序列交换，所以使用该方法不仅可以解决非同义SNP的功能性，而且可以解决基因的内含子和调控区中的非编码多态性。使用该载体系统，我们提出产生小鼠，其中内源性5-Htt基因被切除并被具有5-HTT连锁多态性区域（HTTLPR）的长（LA）、长（LG）或短（S）等位基因的人5-HTT基因替换。5-HTT的表达、5-羟色胺途径的活性以及小鼠在行为测试中的反应将提供关于三个启动子多态性的功能的信息。用同线人DNA替换整个小鼠基因不仅有利于将来对另外的5-HTT SNP和变体的测试，而且还通过这些小鼠品系与在其它基因座表达疾病相关多态性的品系的互交来简化基因-基因相互作用的评估。遗传学研究正在发现越来越多的多态性与情感和精神疾病的风险有关。确定这些遗传变异的功能已被证明是困难的，特别是当通过多态性预测编码蛋白质的一级结构没有变化时。在此应用中，我们提出了一种新的策略，用于交换小鼠基因与人类直系同源物。我们表明，在这种交换后，可以将SNP引入小鼠基因组的人类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疾病风险相关的特定多态性方面不同的小鼠品系。这些小鼠可用于研究这种多态性对基因表达、蛋白质功能、发育以及已知对啮齿动物中这种特定途径的改变敏感的行为测试/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.