Functionally Significant Polymorphisms in Serotonin Pathway Genes

血清素途径基因中具有功能意义的多态性

• 批准号: 7493302
• 负责人: Beverly H Koller
• 金额: $ 14.73万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-07 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7493302
• 关键词: 17q11; 3' Flanking Region; Abbreviations; Address; Affective; Alleles; Amino Acids; Aminopterin; Amygdaloid structure; Antidepressive Agents; Anxiety; Autistic Disorder; BAC (bacterial artificial chromosome); Backcrossings; Behavior; Behavioral; Brain region; Breeding; C57BL/6 Mouse; Carrier Proteins; Cell Line; Chimera organism; Chromosome Pairing; Chromosomes; Chromosomes, Human, Pair 11; Cyclization; DNA; Data; Development; Disease; ES Cell Line; Embryo; Evaluation; Excision; First Births; Functional RNA; Future; Gene Expression; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genetic Variation; Genome; Goals; Homeostasis; Human; Human Genetics; Human Genome; Hypothalamic structure; Injection of therapeutic agent; Introns; Link; Measurable; Mental Depression; Metabolism; Methods; Minisatellite Repeats; Modeling; Mood Disorders; Mus; Neomycin; Neonatal; Neurons; Neurotransmitters; Nucleic Acid Regulatory Sequences; Numbers; Obsessive-Compulsive Disorder; Pathway interactions; Performance; Plasmids; Population; Positron-Emission Tomography; Process; Proteins; Reporting; Research Design; Research Personnel; Risk; Rodent; Role; Scheme; Selective Serotonin Reuptake Inhibitor; Serotonin; Single Nucleotide Polymorphism; Site; Structure; Synapses; System; Testing; Thalamic structure; Thymidine; Tryptophan; Tryptophan 5-monooxygenase; Variant; Work; base; behavior test; blastocyst; design; disorder risk; embryonic stem cell; gene interaction; genetic manipulation; in vivo; inhibitor/antagonist; mouse genome; novel; postsynaptic; prenatal stress; presynaptic; promoter; protein function; raphe nuclei; receptor; receptor expression; recombinase; response; serotonin receptor; serotonin transporter; stem; vector

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.

描述（由申请人提供）：神经递质5-羟色胺（5-HT）是由氨基酸色氨酸的两步酶促过程产生的，主要由位于Raphe核中的神经元。这些神经元的大脑区域太多，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，并由突触前和突触后神经元表达。囊泡释放后，5-HT的作用是通过5-羟色胺转运蛋白（5-HTT，SERT，SLC6A4）从突触中删除的。血清素能途径的放松管制与许多精神病和情感障碍有关，包括抑郁症，自闭症和强迫症。这些关联得到了以下证明，即5-HTT是许多抗抑郁药的主要作用部位。 5-HTT抑制剂（SRI）不仅有效，不仅可以治疗抑郁症，而且还可以治疗其他疾病，包括焦虑，强迫症（OCD）和自闭症的某些方面。遗传研究报告了5-HTT基因中常见和罕见多态性的关联，对精神病和情感障碍的风险增加。这些结果不仅为靶向5-HTT治疗这些疾病提供了理由，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。事实证明，很难直接测试多态性对体内脑稳态的障碍的贡献，或者这些障碍表现为可测量的行为变化的能力。为了开始解决这些问题，我们提出了新型鼠标线条的产生。这些线旨在检验以下假设：5-HTT启动子中的多态性改变该基因的表达，而羟色胺代谢的结果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方法，用于交换编码特定鼠标基因的基因座。由于整个小鼠基因，包括启动子以及5'和3'侧翼区域，都与相应的人类序列交换，因此可以使用这种方法不仅可以解决非同义SNP的功能，还可以解决该基因内含子和调节区域中非编码多态性的功能。我们建议使用此矢量系统生成小鼠，其中切除内源性5-HTT基因并用长（LA），长（LG）或5-HTT链接的多晶型多型区域（HTTLPR）的人类5-HTT基因代替。 5-HTT的表达，血清素途径的活性以及小鼠在行为测试中的反应将提供有关三个启动子多态性功能的信息。用同义人DNA替换整个小鼠基因不仅会促进对其他5-HTT SNP和变体的未来测试，而且还可以简化这些小鼠线中的跨基因 - 基因相互作用的评估。遗传研究正在确定与情感和精神病风险有关的多态性越来越多。事实证明，确定这些遗传变异的功能已被证明很困难，尤其是当多态性预测编码蛋白的主要结构没有变化时。在此应用中，我们介绍了一种新的策略，用于将小鼠基因与人类直系同源物交换。我们表明，在这种交换后，SNP可以引入小鼠基因组的人DNA段。由于这种遗传操作是在小鼠胚胎干细胞中进行的，因此可能会产生仅在人群中与疾病风险相关的特定多态性中不同的小鼠系。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特定途径改变的行为测试/模型的影响。