Functional characterization of ATP1A1 and DEspR variants associated with essentia

项目简介(申请人提供)：高血压是一个主要的公共卫生问题，因为它的高患病率及其作为发达国家主要死亡和发病原因的主要风险因素的作用--冠状动脉疾病、中风、慢性肾脏疾病和外周血管疾病。阐明潜在的遗传机制是至关重要的，但由于其多基因性质和环境因素带来的额外复杂性，仍然难以捉摸。我们报道了来自撒丁岛北部的病例对照高血压队列中ATP1A1(α1 Na，K-ATPase，P&lt；0.000005)和Despr(双重内皮素-1/血管生长因子信号肽受体，P&lt；0.03)与高血压/正常血压的关系。我们发现，在男性中，这两个基因座与高血压/血压正常之间的关联比在女性中更强，指出5‘-调节区蕴含着潜在的关联变异体。随访研究发现，ATP1A1和Despr启动子区域的4T缺失/插入多态(4Tdelins)和C/T多态(Rs6535847)分别与男性人群高血压易感性降低相关。C/T(Rs6535847)多态修饰Despr启动子区域的CATAAAA序列，产生新的TATAAAA-box，提示可能对Despr转录产生影响。这些结果为我们目前的应用奠定了基础，我们计划研究ATP1A1 4T INS和Despr rs6535847 T等位基因作为潜在的功能变异，说明这两个保护等位基因在男性撒丁岛人群中降低高血压易感性的风险。目的1：以分泌型人胎盘碱性磷酸酶(SEAP)为报告分子，在组织培养细胞中检测携带4T ins(P4Tins)或4T del(P4Tdel)等位基因的ATP1A1启动子区域和含有rs6535847 C(PCATAAAA)或rs6535847 T等位基因(PTATAAAA)的Despr启动子区域的转录活性。目的：研究Despr和ATP1A1单倍体功能不全对青年(4月龄)和老年(16月龄)Despr、ATP1A1和野生型小鼠血压的影响。这将在生物学背景下评估Despr和ATP1A1不同表达水平对血压的假定影响。实现所提出的特定目标将阐明Despr和ATP1A1功能多态，在与撒丁岛北部遗传隔离的人群中，Despr和ATP1A1功能多态为原发性高血压提供性别特异性保护。此外，这些结果可能为确定普通人群高血压遗传易感性的研究方向提供关键的见解。公共卫生相关性：项目叙述高血压是心脏病、中风和肾功能衰竭的主要危险因素。尽管人们越来越努力地破译高血压及其靶器官相关并发症的遗传机制，但高血压的遗传学基础仍未完全阐明。我们的研究将有助于确定在撒丁岛北部人群中与高血压发展相关的两个遗传因素。这一信息将提供一个框架，进一步研究它们在普通人群中各自在高血压中的作用，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.