Functional characterization of ATP1A1 and DEspR variants associated with essentia

项目简介（由申请人提供）：项目概述原发性高血压是一个主要的公共卫生问题，因为它的发病率很高，并且是发达国家主要死亡和发病原因的主要危险因素-冠状动脉疾病，中风，慢性肾脏疾病和周围血管疾病。由于其多基因性和环境因素带来的复杂性，对其潜在的遗传机制的阐明是至关重要的。我们报道了来自撒丁岛北部的病例对照高血压队列中，ATP1A1 （α 1 Na， k - atp酶，P<0.000005）和DEspR（双重内皮素-1/血管生长因子信号肽受体，P<0.03）与高血压/正常血压的关联。我们检测到这两个基因座与男性高血压/正常血压的关联比女性更强，这表明5'调控区可能存在潜在的变异。后续研究发现，ATP1A1和DEspR启动子区域的4T缺失/插入多态性（4Tdelins）和C/T （rs6535847）多态性分别与男性人群高血压易感性降低相关。C/T （rs6535847）多态性修饰了存在于DEspR启动子区域的CATAAAA序列，产生了一个新的TATAAAA- box，这表明可能对DEspR转录有影响。这些结果为我们目前的应用奠定了基础，我们计划研究ATP1A1 4tins和DEspR rs6535847 T等位基因作为潜在的功能变异，解释了撒丁岛男性人群中这两个保护性等位基因所带来的高血压易感性风险降低。因此，以下具体目标被优先考虑：目的1：我们将使用SEAP（人类胎盘碱性磷酸酶的分泌形式）作为报告分子来监测组织培养细胞中携带4tins （p4Tins）或4tdel （p4Tdel）等位基因的ATP1A1启动子区域和携带rs6535847 C （pCATAAAA）或rs6535847 T等位基因（pTATAAAA）的DEspR启动子区域的转录活性，以监测其活性。目的2：我们将通过无线遥测法测量幼龄（4月龄）和老年（16月龄）DEspR、ATP1A1和野生型小鼠的血压，研究DEspR和ATP1A1单倍性不全对血压的影响。这将在生物学背景下评估不同的DEspR和ATP1A1表达水平对血压的可能影响。完成所提出的具体目标将阐明在撒丁岛北部遗传隔离人群中赋予性别特异性高血压保护的DEspR和ATP1A1功能多态性。此外，这些结果可以为一般人群高血压遗传易感性的研究方向提供关键的见解。高血压是心脏病、中风和肾衰竭的主要危险因素。尽管破译高血压及其靶器官相关并发症的遗传机制的努力越来越多，但高血压的遗传基础仍有待充分阐明。我们的研究将有助于在撒丁岛北部人群中建立与高血压发展相关的两个遗传因素。这一信息将为进一步研究它们在普通人群中各自在高血压中的作用提供一个框架，并有助于改进对原发性高血压及其靶器官并发症的干预和预防策略。

DESCRIPTION (provided by applicant): Project Summary Essential hypertension is a major public health concern due to its high prevalence and its role as a leading risk factor for leading causes of death and morbidity in the developed world - coronary artery disease, stroke, chronic renal disease and peripheral vascular disease. Elucidation of underlying genetic mechanism is critical but remains elusive due to its polygenic nature and added complexity brought on by environmental factors. We reported the association of ATP1A1 (alpha 1 Na,K-ATPase, P<0.000005) and DEspR (dual endothelin-1/vascular growth factor signal peptide receptor, P<0.03) with hypertension/normotension in a case-control hypertension cohort from northern Sardinia. We detected stronger association of both loci with hypertension/normotension in males than in females pointing to the 5'-regulatory region as harboring putative variants underlying their associations. Follow up studies identified a 4T deletion/insertion polymorphism (4Tdelins) and a C/T (rs6535847) polymorphism within the ATP1A1 and DEspR promoter regions respectively associated with decreased susceptibility to hypertension in the male population. The C/T (rs6535847) polymorphism modifies a CATAAAA sequence present in DEspR promoter region to generate a new TATAAAA- box, suggesting a putative effect on DEspR transcription. These results form the basis for our current application in which we plan to investigate the ATP1A1 4T ins and DEspR rs6535847 T alleles as potential functional variants accounting for the decreased risk of hypertension susceptibility conferred by these two protective alleles in the male Sardinian population. Thus, the following specific aims are prioritized: AIM 1: We will test the transcriptional activity of ATP1A1 promoter region carrying either the 4T ins (p4Tins) or the 4T del (p4Tdel) alleles and DEspR promoter region harboring either the rs6535847 C (pCATAAAA) or the rs6535847 T alleles (pTATAAAA) in tissue culture cells using SEAP (a secreted form of human placental alkaline phosphatase) as a reporter molecule to monitor activity. AIM 2: We will investigate the effect of DEspR and ATP1A1 haploinsufficiency on blood pressure by measuring blood pressure by radiotelemetry in young (4 months of age) and aging (16 months of age) DEspR, ATP1A1 and wild-type mice. This will assess in a biological context the putative effects of differential DEspR and ATP1A1 expression levels on blood pressure. Accomplishing the proposed specific aims will elucidate DEspR and ATP1A1 functional polymorphisms that confer sex- specific protection to essential hypertension in a genetically isolated population from northern Sardinia. Moreover, these results could shed key insight into setting the direction for the investigation of genetic susceptibility to hypertension in the general population. PUBLIC HEALTH RELEVANCE: Project Narrative Hypertension is a leading risk factor for heart disease, stroke and renal failure. Despite increasing efforts to decipher the genetic mechanisms of hypertension and its target organ associated complications, the genetic underpinnings of hypertension remain to be fully elucidated. Our research will help to establish two genetic factors associated with development of hypertension in a northern Sardinian population. This information will provide a framework to investigate further their respective roles in hypertension in the general population and help to improve intervention and prevention strategies for essential hypertension and its target organ complications.