Genomic Region Influencing Blood Pressure in SS-13bn Rat

高血压影响着5000多万美国人。尽管进行了许多治疗，血压仍保持不变 北美的大量患者基本上得不到控制，导致中风发病率增加， 心脏病和肾脏疾病，以及不断上升的医疗保健费用。因此，人们对更好地定义 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向SS遗传背景(SS-13BN)的导入 延缓高血压的发展。随后，我们开发了26个和表型23个重叠 来自BN大鼠的具有不同Chr 13区域的同源菌株被导入SS遗传背景并 发现这些同源菌株中的4种(菌株1、5、9和26)的血压和蛋白尿低于 SS大鼠。我们选择了Chr 13同源菌株5进行进一步的研究，并通过遥测证实该菌株 高盐饮食4周可预防高血压和蛋白尿的发生。在……里面 相反，重叠的同源菌株6，具有较短的导入区域，没有表现出保护作用。这些 结果将感兴趣区域缩小到Chr 13上10Mbp的片段(32.4-42.5Mbp)，包含110 159个命名和预测的基因。该区域与人类2号染色体上连接的区域同源。 QuAbec家庭研究和家庭血压计划中的血压。项目2的目标 就是识别并优先考虑该区域中负责保护人类免受 在Chr 13同源株5中发生盐敏性高血压，然后测试 他们可以使用转基因技术改变SS遗传背景下的血压。具体的 目标是：1)创造和表型亚基因菌株，以缩小负责区域 防止盐敏感型高血压的发展到1-2 MBP。我们已经创建了12个 来自Chr 13株系5的亚同源株系重叠。这些菌株将受到挑战， 高盐饮食和顺次表型对血压和蛋白尿缩小区域负责 将血压由含有110至159个基因的10 MBP降至少于2 MBP的区域 含有10-20个基因。2)利用测序和基因表达分析，确定下列哪项的优先顺序 位置候选基因(预计1-5个)将在功能研究中进一步评估。我们将比较 目的区域的所有基因的cdna序列，这些基因存在于动物的几个组织中。 防止高血压的亚同基因大鼠与易感SS品系比较以确定序列变异 这可能会改变蛋白质的功能。同时，我们将进行实时PCR表达研究，以查看 寻找亚基因大鼠与易感SS株之间的差异表达基因。最后， 我们将对SS大鼠和同源菌株的整个感兴趣区域进行测序，以确定潜在的 差异表达基因5‘或3’区或高度保守的因果序列变异 侧翼区域。3)测试哪些优先候选基因会改变SS中的血压 使用转基因技术的遗传背景。如果基因的表达下调，或者有 SS大鼠中的一种失活突变，我们将测试该基因的表达是否全局上调 慢病毒转基因可降低SS大鼠的血压。或者，我们将在基因中过度表达该基因 在SS中发现基因表达或蛋白质活性上调的亚基因菌株 老鼠。具有改变血压的潜在因果突变的候选基因的最终功能验证 筛选将依赖于在控制下表达正确等位基因的BAG转基因品系的创建 在允许的遗传背景(SS或亚同源菌株)中使用原核 注射。该区域影响血压的候选基因(S)的特征可能识别新的 有助于控制动脉压的途径和新治疗方法的发展 用于治疗高血压。

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.