Non-Coding Variants of Angiotensinogen Gene and Hypertension

 描述(申请人提供)：高血压是心肌梗死、心力衰竭、血管疾病、中风和肾功能衰竭的严重危险因素。肾素-血管紧张素系统(RAS)在血压调节中起着重要作用。最近的基因组相关研究表明，位于人血管紧张素原基因内含子I的A/G SNP(Rs2004776)与高血压相关。含有1164A等位基因的HAGT基因的核苷酸序列与HNF-3结合位点的同源性高于1164G。HNF3家族属于“先锋”转录因子，其与启动子和增强子的结合使其他组织特异性转录因子能够获得染色质。HAGT基因启动子和内含子I上的SNP可分为两种主要单倍型：单倍型I(含1164A)和单倍型II(含1164G)。与单倍型-II相比，单倍型-I中的核苷酸变体与转录因子结合更强，并且与单倍型-II相比，含有单倍型-I的报告结构具有更高的启动子活性。HPRT基因单倍型-I或单倍型II的转基因小鼠通过HPRT基因座的敲入方法获得。初步研究表明：(A)与单倍型-II相比，含有单倍型-I的转基因小鼠HAGT启动子中的CpG二核苷酸甲基化程度较低，可被转录因子访问，(B)与单倍型-II相比，含有单倍型-I的雄性TG小鼠的肝、肾和脂肪中HAGT基因的基础表达和GR诱导表达增加，(C)与单倍型-II相比，含有单倍型-I的转基因动物的肝和肾染色质与HNF3β、C/EBPβ、STAT-3和GR结合得更强，(D)含有hren基因和hagt基因单倍型-I的双TG小鼠与单倍型-II相比，血压升高，而单倍型-II在高碳水化合物：高脂肪：高盐饮食的西方饮食中进一步增加。在本研究中，将通过DNA甲基化分析、染色体构象捕获(3C)分析、芯片分析和瞬时转染实验来分析1164位SNP和内含子I中的其他顺式作用DNA元件对hagt基因转录调控的作用。利用含有hren基因的雄性/雌性双转基因小鼠和Hagt基因的单倍型-I或单倍型-II，分析西方饮食对Hagt基因表达、血压调节和终末器官损伤的影响。

 DESCRIPTION (provided by applicant): Hypertension is a serious risk factor for myocardial infarction, heart failure, vascular disease, stroke, and renal failure. The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure (BP). Recent genome wise association studies (GWAS) have shown that an A/G SNP (rs2004776) located at +1164 in intron-I of the human angiotensinogen (hAGT) gene is associated with hypertension. The nucleotide sequence of hAGT gene containing +1164A allele has stronger homology with HNF-3 binding site as compared to +1164G. HNF3 family belongs to "pioneer" transcription factors whose binding to promoters and enhancers enables chromatin access for other tissue-specific transcription factors. SNPs in the promoter and intron I of the hAGT gene can be divided in 2 major haplotypes: haplotype-I (containing +1164A) and II (containing +1164G). Nucleotide variants in haplotype-I bind more strongly to transcription factors as compared to haplotype-II and reporter construct containing haplotype-I has increased promoter activity as compared to haplotype-II on transient transfection. Transgenic mice containing either haplotype-I or II of the hAGT gene were generated by knock-in approach at the HPRT locus. Preliminary studies have shown that: (a) CpG dinucleotides in the hAGT promoter of TG mice containing haplotype- I are hypo-methylated and accessible to transcription factors as compared to haplotype-II, (b) male TG mice containing haplotype-I have increased basal and GR induced expression of the hAGT gene in liver, kidney and fat as compared to haplotype-II, (c) chromatin from liver and kidney of transgenic animals containing haplotype-I binds more strongly to HNF3β, C/EBPβ, STAT-3 and GR as compared to haplotype-II, (d) double TG mice containing hREN gene and haplotype-I of the hAGT gene have increased BP as compared to haplotype-II which is further increased by Western diet containing high carbohydrate: high fat: high salt diet. In the present application, th role of SNP at +1164 and other cis-acting DNA elements in intron-I on transcriptional regulation of the hAGT gene will be analyzed by DNA methylation assay, chromosome conformation capture (3C) assay, ChIP assay and transient transfection assay. The effect of Western diet on the expression of the hAGT gene, blood pressure regulation, and end organ damage will be analyzed using male/female double transgenic mice containing hREN gene and either haplotype-I or haplotype-II of the hAGT gene.