Genomic Region Influencing Blood Pressure in SS-13bn Rat

影响 SS-130 亿大鼠血压的基因组区域

• 批准号: 7217709
• 负责人: Richard J. Roman
• 金额: $ 33.28万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217709
• 关键词: angiotensin /renin /aldosterone hypertension; artificial chromosomes; blood pressure; dietary sodium; functional /structural genomics; gene expression; genetic manipulation; genetic mapping; genetic models; genetic polymorphism; genetic strain; genetic susceptibility; genetically modified animals; inbreeding; laboratory rat; nucleic acid sequence; nutrition related tag; phenotype; polymerase chain reaction; protein structure function; proteinuria

Hypertension affects more than 50 million Americans. Despite many treatments, blood pressure remains largely uncontrolled in large number of patients in North America leading to increased incidence of stroke, heart and renal disease and escalating health care costs. Thus, there is considerable interest in better defining the genetic basis of hypertension in humans and experimental animal models. In preliminary experiments, we found that introgression of chromosome 13 (Chr 13) from the BN into the SS genetic background (SS-13BN) attenuates the development of hypertension. We subsequently developed 26 and phenotyped 23 overlapping congenic strains with various regions of Chr 13 from a BN rat introgressed into the SS genetic background and found that 4 of these congenic strains (Strains 1, 5, 9 and 26) had lower blood pressure and proteinuria than SS rats. We selected the Chr 13 congenic strain 5 for further study and confirmed by telemetry that this strain was protected from the development of hypertension and proteinuria when fed a high salt diet for 4 weeks. In contrast, an overlapping congenic strain 6, with a shorter introgressed region, exhibited no protection. These results narrow the region of interest to a 10 Mbp segment on Chr 13 (32.4-42.5Mbp) that contains between 110 and 159 named and predicted genes. This region is homologous to a region on human chromosome 2 linked to blood pressure in the QuAbec Family Study and the Family Blood Pressure Program. The goal of Project 2 is to identify and prioritize the candidate genes in the region responsible for the protection from the development of salt-sensitive hypertension in the Chr 13 congenic strain 5 and then to test whether they can alter blood pressure in the SS genetic background using transgenic techniques. The Specific Aims are: 1) To create and phenotype subcongenic strains to narrow the region responsible for the protection from the development of salt-sensitive hypertension to 1-2 Mbp. We have already created 12 overlapping subcongenic lines from the Chr 13 strain 5 congenic strain. These strains will be challenged with a high salt diet and sequentially phenotyped for blood pressure and proteinuria to narrow the region responsible for the lowering of blood pressure from 10 Mbp containing from 110 to 159 genes to a region less than 2 Mbp containing 10-20 genes. 2) To prioritize, using sequencing and gene expression analysis, which of the positional candidate genes (1-5 expected) to evaluate further in functional studies. We will compare the cDNA sequence of all of the genes in the region of interest that are present in several tissues of the subcongenic rats protected from hypertension versus the susceptible SS strain to identify sequence variants that could alter the function of the protein. In parallel, we will perform real time PCR expression studies to look for differentially expressed genes in the interval in subcongenic rats versus the susceptible SS strain. Finally, we will sequence through the entire region of interest in SS rats and the congenic strain to identify potential causal sequence variants in the 5' or 3' regions of the differential expressed genes or in highly conserved flanking regions. 3) To test which of the prioritized candidate genes alter blood pressure in the SS genetic background using transgenic techniques. If the expression of the gene is downregulated or there is an inactivating mutation in SS rats, we will test if global upregulation of the expression of the gene using lentiviral transgenesis can reduce blood pressure in SS rats. Alternatively, we will overexpress the gene in the subcongenic strain if the expression of the gene or the activity of the protein is found to be upregulated in SS rats. Final functional validation of candidate genes with a potential causal mutation that alters blood pressure in the screen will rely on the creation of a BAG transgenic line that expresses the correct allele under the control of the native promoter in a permissive genetic background (SS or the subcongenic strain) using pronuclear injection. Characterization of candidate gene(s) in this region that influence blood pressure may identify novel pathways contributing to the control of arterial pressure and the development of new therapeutic approaches for the treatment of hypertension.

高血压影响着5000多万美国人。尽管有许多治疗方法，血压仍然 在北美的大量患者中基本上不受控制，导致中风发病率增加， 心脏病和肾病以及不断上升的医疗费用。因此，人们对更好地界定 人类和实验动物模型中高血压的遗传基础。在初步实验中，我们 发现13号染色体（Chr 13）从BN渗入SS遗传背景（SS-13 BN）中， 减弱高血压的发展。我们随后开发了26个和表型23重叠 具有来自BN大鼠的Chr 13的不同区域的同源株渗入SS遗传背景， 发现这些同源菌株中的4个（菌株1、5、9和26）具有比其他菌株更低的血压和蛋白尿。 党卫军老鼠。我们选择了Chr 13同源株5进行进一步研究，并通过遥测证实该株 当喂食高盐饮食4周时，保护免于发生高血压和蛋白尿。在 相反，具有较短渗入区域的重叠同源菌株6没有表现出保护作用。这些 结果将感兴趣的区域缩小到Chr 13上的10 Mbp片段（32.4-42.5Mbp），其含有110个 和159个命名和预测的基因。该区域与人类2号染色体上的一个区域同源， 魁北克家庭研究和家庭血压计划中的血压。项目2的目标 是识别和优先考虑负责保护区域中的候选基因， 在Chr 13同源株5中发展盐敏感性高血压，然后测试是否 他们可以使用转基因技术在SS遗传背景下改变血压。具体 目的是：1）创建和表型亚同源菌株，以缩小负责该区域的区域。 防止盐敏感性高血压发展至1-2 Mbp。我们已经创建了12个 来自Chr 13株系的重叠亚同源系5同源株系。这些菌株将用 高盐饮食和随后的血压和蛋白尿的表型，以缩小区域负责 用于将血压从含有110至159个基因的10 Mbp降低至小于2 Mbp的区域 含有10-20个基因。2)为了优先考虑，使用测序和基因表达分析， 定位候选基因（预期1-5个），以在功能研究中进一步评估。我们将比较 在感兴趣的区域中存在于几种组织中的所有基因的cDNA序列， 保护亚同源大鼠免于高血压与敏感SS品系以鉴定序列变体 可以改变蛋白质的功能同时，我们将进行真实的时间PCR表达研究， 差异表达基因的间隔在亚同源大鼠与敏感的SS株。最后， 我们将对SS大鼠和同类品系的整个感兴趣区域进行测序，以确定潜在的 在差异表达基因的5'或3'区域中或在高度保守区域中的致病序列变体 侧翼区3)为了测试哪些优先候选基因改变SS中的血压， 遗传背景的转基因技术。如果基因的表达下调， 在SS大鼠的失活突变，我们将测试是否整体上调的基因表达，使用 慢病毒转基因可降低SS大鼠血压。或者，我们将在细胞中过表达该基因。 如果发现SS中基因表达或蛋白质活性上调，则为亚同源菌株 大鼠最终功能验证候选基因与潜在的因果突变，改变血压， 筛选将依赖于在对照下表达正确等位基因的BAG转基因系的产生 在允许的遗传背景（SS或亚同源菌株）中使用原核表达载体（pronuclear） 注射表征该区域中影响血压的候选基因可以鉴定新的高血压基因。 有助于控制动脉压和开发新治疗方法的途径 用于治疗高血压。